Search results

Title: Cas6 processes tight and relaxed repeat RNA via multiple mechanisms: A hypothesis..
Creator: Sefcikova, Jana, Roth, Mitchell, Yu, Ge, Li, Hong
Abstract/Description: RNA molecules are flexible yet foldable. Proteins must cope with this structural duality when forming biologically active complexes with RNA. Recent studies of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs)-mediated RNA immunity illustrate some remarkable mechanisms with which proteins interact with RNA. Currently known structures of CRISPR-Cas6 endoribonucleases bound with RNA suggest a conserved protein recognition mechanism mediated by RNA stem-loops. However, a...
Show moreRNA molecules are flexible yet foldable. Proteins must cope with this structural duality when forming biologically active complexes with RNA. Recent studies of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs)-mediated RNA immunity illustrate some remarkable mechanisms with which proteins interact with RNA. Currently known structures of CRISPR-Cas6 endoribonucleases bound with RNA suggest a conserved protein recognition mechanism mediated by RNA stem-loops. However, a survey of CRISPR RNA reveals that many repeats either lack a productive stem-loop (Relaxed) or possess stable but inhibitory structures (Tight), which raises the question of how the enzyme processes structurally diverse RNA. In reviewing recent literature, we propose a bivalent trapping and an unwinding mechanism for CRISPR-Cas6 to interact with the Relaxed and the Tight repeat RNA, respectively. Both mechanisms aim to create an identical RNA conformation at the cleavage site for accurate processing.
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Date Issued: 2017-06-01
Identifier: FSU_pmch_28493337, 10.1002/bies.201700019, PMC5699886, 28493337, 28493337
Format: Citation

Title: California reanalysis downscaling at 10 km using an ocean-atmosphere coupled regional model system.
Creator: Li, Haiqin, Kanamitsu, Masao, Hong, Song-You
Abstract/Description: A fully coupled regional downscaling system for both the Regional Spectral Model (RSM) for atmosphere and the Regional Ocean Modeling System (ROMS) for the ocean was developed for the purpose of downscaling observed analysis or global model outputs. The two models share the same grid and resolution with efficient parallelization through the use of dual message passing interfaces. Coupled downscaling was performed using historical Simple Ocean Data Assimilation (SODA) oceanic reanalysis and...
Show moreA fully coupled regional downscaling system for both the Regional Spectral Model (RSM) for atmosphere and the Regional Ocean Modeling System (ROMS) for the ocean was developed for the purpose of downscaling observed analysis or global model outputs. The two models share the same grid and resolution with efficient parallelization through the use of dual message passing interfaces. Coupled downscaling was performed using historical Simple Ocean Data Assimilation (SODA) oceanic reanalysis and NCEP/DOE (R-2) atmospheric reanalysis in order to study the impact of coupling on the regional scale atmospheric analysis. The results were subsequently compared with the uncoupled downscaling forced by the prescribed observed sea surface temperature (SST). An evaluation of the SST and ocean current from the coupled experiment yielded realistic small-scale oceanic features that are nearly absent in the oceanic reanalysis. Upwelling over the California coast is well resolved and comparable to findings obtained from high-resolution observations. The coupling impact on the atmospheric circulation mainly modulates the near surface atmospheric variables when compared to the simulation conducted without coupling. The duration of the Catalina Eddy detected in the coupled experiment increased by about 6.5% when compared to that in the uncoupled experiment. The offshore land breeze is enhanced by about 10%, whereas the change in the onshore sea breeze is very small during the summer.
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Date Issued: 2012
Identifier: FSU_migr_coaps_pubs-0059, 10.1029/2011JD017372
Format: Citation

Title: The Impact of DNA Topology and Guide Length on Target Selection by a Cytosine-Specific Cas9.
Creator: Tsui, Tsz Kin Martin, Hand, Travis H, Duboy, Emily C, Li, Hong
Abstract/Description: Cas9 is an RNA-guided DNA cleavage enzyme being actively developed for genome editing and gene regulation. To be cleaved by Cas9, a double stranded DNA, or the protospacer, must be complementary to the guide region, typically 20-nucleotides in length, of the Cas9-bound guide RNA, and adjacent to a short Cas9-specific element called Protospacer Adjacent Motif (PAM). Understanding the correct juxtaposition of the protospacer- and PAM-interaction with Cas9 will enable development of versatile...
Show moreCas9 is an RNA-guided DNA cleavage enzyme being actively developed for genome editing and gene regulation. To be cleaved by Cas9, a double stranded DNA, or the protospacer, must be complementary to the guide region, typically 20-nucleotides in length, of the Cas9-bound guide RNA, and adjacent to a short Cas9-specific element called Protospacer Adjacent Motif (PAM). Understanding the correct juxtaposition of the protospacer- and PAM-interaction with Cas9 will enable development of versatile and safe Cas9-based technology. We report identification and biochemical characterization of Cas9 from Acidothermus cellulolyticus (AceCas9). AceCas9 depends on a 5'-NNNCC-3' PAM and is more efficient in cleaving negative supercoils than relaxed DNA. Kinetic as well as in vivo activity assays reveal that AceCas9 achieves optimal activity when combined with a guide RNA containing a 24-nucleotide complementarity region. The cytosine-specific, DNA topology-sensitive, and extended guide-dependent properties of AceCas9 may be explored for specific genome editing applications.
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Date Issued: 2017-06-16
Identifier: FSU_pmch_28277645, 10.1021/acssynbio.7b00050, PMC5706465, 28277645, 28277645
Format: Citation

Title: Isolating and Crystallizing the Permuted HD Domain of CRISPR.
Creator: Hubert, Joshua, Li, Hong, Ramia, Nancy, Department of Chemistry and Biochemistry
Abstract/Description: CRISPR-cas systems have been found to confer RNA guided immunity in prokaryotes comparable to the eukaryotic RNA interference. These Clustered Regularly Interspaced Short Palindromic Repeats, as their name entails, are repeated sequences varying from 25 to 45 nucleotides in length separated by variable spacers. The CRISPR system has been found in many different bacteria and Archaea. Associated with the CRISPR locus are cas genes, which are thought to encode for nucleases, helicases, and...
Show moreCRISPR-cas systems have been found to confer RNA guided immunity in prokaryotes comparable to the eukaryotic RNA interference. These Clustered Regularly Interspaced Short Palindromic Repeats, as their name entails, are repeated sequences varying from 25 to 45 nucleotides in length separated by variable spacers. The CRISPR system has been found in many different bacteria and Archaea. Associated with the CRISPR locus are cas genes, which are thought to encode for nucleases, helicases, and polymerases involved in the CRISPR defense mechanism. The mechanism involved with the defense occurs when the CRISPR locus is transcribed into a long RNA that will be processed into short sequences used as a guide to target and cleave the invader genome sequences through base pairing. Together, the CRISPR-cas systems are able to protect the bacteria or archaea from invading DNA or RNA. Cas 10 is the signature protein of type III CRISPR systems and is characterized by a permuted Histidine-Aspartic acid (HD) domain predicted to possess a nuclease activity. The presence of the permuted HD and the presence of a nucleotidyl cylcase-like have guided the belief that the permuted HD domain may be the activity site. After several attempts, it was found that the HD domain protein from both PF1129 and TTHB147 is insoluble in water and is consistently lost in the pellet during centrifugation. However, there is still a small concentration of the domain collected in the elution, showing that some of the HD domain can be purified to the last step. The max concentration collected of the permuted HD domain from the TTHB147 was found to be 2.45 mg/mL.
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Date Issued: 2014
Identifier: FSU_migr_uhm-0317
Format: Thesis

Title: Loss of function of the Cik1/Kar3 motor complex results in chromosomes with syntelic attachment that are sensed by the tension checkpoint.
Creator: Jin, Fengzhi, Liu, Hong, Li, Ping, Yu, Hong-Guo, Wang, Yanchang
Abstract/Description: The attachment of sister kinetochores by microtubules emanating from opposite spindle poles establishes chromosome bipolar attachment, which generates tension on chromosomes and is essential for sister-chromatid segregation. Syntelic attachment occurs when both sister kinetochores are attached by microtubules from the same spindle pole and this attachment is unable to generate tension on chromosomes, but a reliable method to induce syntelic attachments is not available in budding yeast. The...
Show moreThe attachment of sister kinetochores by microtubules emanating from opposite spindle poles establishes chromosome bipolar attachment, which generates tension on chromosomes and is essential for sister-chromatid segregation. Syntelic attachment occurs when both sister kinetochores are attached by microtubules from the same spindle pole and this attachment is unable to generate tension on chromosomes, but a reliable method to induce syntelic attachments is not available in budding yeast. The spindle checkpoint can sense the lack of tension on chromosomes as well as detached kinetochores to prevent anaphase onset. In budding yeast Saccharomyces cerevisiae, tension checkpoint proteins Aurora/Ipl1 kinase and centromere-localized Sgo1 are required to sense the absence of tension but are dispensable for the checkpoint response to detached kinetochores. We have found that the loss of function of a motor protein complex Cik1/Kar3 in budding yeast leads to syntelic attachments. Inactivation of either the spindle or tension checkpoint enables premature anaphase entry in cells with dysfunctional Cik1/Kar3, resulting in co-segregation of sister chromatids. Moreover, the abolished Kar3-kinetochore interaction in cik1 mutants suggests that the Cik1/Kar3 complex mediates chromosome movement along microtubules, which could facilitate bipolar attachment. Therefore, we can induce syntelic attachments in budding yeast by inactivating the Cik1/Kar3 complex, and this approach will be very useful to study the checkpoint response to syntelic attachments.
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Date Issued: 2012-02-01
Identifier: FSU_pmch_22319456, 10.1371/journal.pgen.1002492, PMC3271067, 22319456, 22319456, PGENETICS-D-11-01695
Format: Citation

Title: Magnetic Ground States And Magnetodielectric Effect In Rcr(bo3)(2) (r = Y And Ho).
Creator: Sinclair, R., Zhou, H. D., Lee, M., Choi, S., Li, G., Hong, T., Calder, S.
Abstract/Description: The layered perovskites RCr(BO3)(2) (R = Y and Ho) with magnetic triangular lattices were studied by performing ac/dc susceptibility, specific heat, elastic and inelastic neutron scattering, and dielectric constant measurements. The results show (i) both samples' Cr3+ spins order in a canted antiferromagnetic structure with TN around 8-9 K, while the Ho3+ ions do not order down to T = 1.5 K in HoCr(BO3)(2); (ii) when a critical magnetic field HC around 2-3 T is applied below TN, the Cr3+...
Show moreThe layered perovskites RCr(BO3)(2) (R = Y and Ho) with magnetic triangular lattices were studied by performing ac/dc susceptibility, specific heat, elastic and inelastic neutron scattering, and dielectric constant measurements. The results show (i) both samples' Cr3+ spins order in a canted antiferromagnetic structure with TN around 8-9 K, while the Ho3+ ions do not order down to T = 1.5 K in HoCr(BO3)(2); (ii) when a critical magnetic field HC around 2-3 T is applied below TN, the Cr3+ spins in the Y compound and both the Cr3+ and Ho3+ spins in the Ho compound order in a ferromagnetic state; (iii) both samples exhibit dielectric constant anomalies around the transition temperature and critical field, but the Ho compound displays a much stronger magnetodielectric response. We speculate that this is due to the magnetostriction, which depends on both the Cr3+ and the Ho3+ ions' ordering in the Ho compound. Moreover, by using linear spin-wave theory to simulate the inelastic neutron scattering data, we estimated the Y compound's intralayer and interlayer exchange strengths as ferromagnetic J(1) = -0.12 meV and antiferromagnetic J(2) = 0.014 meV, respectively. The competition between different kinds of superexchange interactions results in the ferromagnetic intralayer interaction.
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Date Issued: 2017-05-08
Identifier: FSU_libsubv1_wos_000401223700002, 10.1103/PhysRevB.95.174410
Format: Citation

Title: Assessing the Radiative Effects of Global Ice Clouds Based on CloudSat and CALIPSO Measurements.
Creator: Hong, Yulan, Liu, Guosheng, Li, J.-L. F.
Abstract/Description: Although it is well established that cirrus warms Earth, the radiative effect of the entire spectrum of ice clouds is not well understood. In this study, the role of all ice clouds in Earth's radiation budget is investigated by performing radiative transfer modeling using ice cloud properties retrieved from CloudSat and CALIPSO measurements as inputs. Results show that, for the 2008 period, the warming effect (similar to 21.8 +/- 5.4 W m(-2)) induced by ice clouds trapping longwave radiation...
Show moreAlthough it is well established that cirrus warms Earth, the radiative effect of the entire spectrum of ice clouds is not well understood. In this study, the role of all ice clouds in Earth's radiation budget is investigated by performing radiative transfer modeling using ice cloud properties retrieved from CloudSat and CALIPSO measurements as inputs. Results show that, for the 2008 period, the warming effect (similar to 21.8 +/- 5.4 W m(-2)) induced by ice clouds trapping longwave radiation exceeds their cooling effect (similar to-16.7 +/- 1.7 W m(-2)) caused by shortwave reflection, resulting in a net warming effect (similar to 5.1 +/- 3.8 W m(-2)) globally on the earthatmosphere system. The net warming is over 15 W m(-2) in the tropical deep convective regions, whereas cooling occurs in the midlatitudes, which is less than 10 W m(-2) in magnitude. Seasonal variations of ice cloud radiative effects are evident in the midlatitudes where the net effect changes from warming during winter to cooling during summer, whereas warming occurs all year-round in the tropics. Ice cloud optical depth t is shown to be an important factor in determining the sign and magnitude of the net radiative effect. Ice clouds with tau < 4.6 display a warming effect with the largest contributions from those with tau approximate to 1.0. In addition, ice clouds cause vertically differential heating and cooling of the atmosphere, particularly with strong heating in the upper troposphere over the tropics. At Earth's surface, ice clouds produce a cooling effect no matter how small the tau value is.
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Date Issued: 2016-11
Identifier: FSU_libsubv1_wos_000386205900006, 10.1175/JCLI-D-15-0799.1
Format: Citation

Title: Bipartite recognition of target RNAs activates DNA cleavage by the Type III-B CRISPR-Cas system.
Creator: Elmore, Joshua R, Sheppard, Nolan F, Ramia, Nancy, Deighan, Trace, Li, Hong, Terns, Rebecca M, Terns, Michael P
Abstract/Description: CRISPR-Cas systems eliminate nucleic acid invaders in bacteria and archaea. The effector complex of the Type III-B Cmr system cleaves invader RNAs recognized by the CRISPR RNA (crRNA ) of the complex. Here we show that invader RNAs also activate the Cmr complex to cleave DNA. As has been observed for other Type III systems, Cmr eliminates plasmid invaders in Pyrococcus furiosus by a mechanism that depends on transcription of the crRNA target sequence within the plasmid. Notably, we found that...
Show moreCRISPR-Cas systems eliminate nucleic acid invaders in bacteria and archaea. The effector complex of the Type III-B Cmr system cleaves invader RNAs recognized by the CRISPR RNA (crRNA ) of the complex. Here we show that invader RNAs also activate the Cmr complex to cleave DNA. As has been observed for other Type III systems, Cmr eliminates plasmid invaders in Pyrococcus furiosus by a mechanism that depends on transcription of the crRNA target sequence within the plasmid. Notably, we found that the target RNA per se induces DNA cleavage by the Cmr complex in vitro. DNA cleavage activity does not depend on cleavage of the target RNA but notably does require the presence of a short sequence adjacent to the target sequence within the activating target RNA (rPAM [RNA protospacer-adjacent motif]). The activated complex does not require a target sequence (or a PAM) in the DNA substrate. Plasmid elimination by the P. furiosus Cmr system also does not require the Csx1 (CRISPR-associated Rossman fold [CARF] superfamily) protein. Plasmid silencing depends on the HD nuclease and Palm domains of the Cmr2 (Cas10 superfamily) protein. The results establish the Cmr complex as a novel DNA nuclease activated by invader RNAs containing a crRNA target sequence and a rPAM.
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Date Issued: 2016-02-15
Identifier: FSU_pmch_26848045, 10.1101/gad.272153.115, PMC4762429, 26848045, 26848045, gad.272153.115
Format: Citation

Title: A Non-Stem-Loop CRISPR RNA Is Processed by Dual Binding Cas6.
Creator: Shao, Yaming, Richter, Hagen, Sun, Shengfang, Sharma, Kundan, Urlaub, Henning, Randau, Lennart, Li, Hong
Abstract/Description: A subclass of recently discovered CRISPR repeat RNA in bacteria contains minimally recognizable structural features that facilitate an unknown mechanism of recognition and processing by the Cas6 family of endoribonucleases. Cocrystal structures of Cas6 from Methanococcus maripaludis (MmCas6b) bound with its repeat RNA revealed a dual site binding structure and a cleavage site conformation poised for phosphodiester bond breakage. Two non-interacting MmCas6b bind to two separate AAYAA motifs...
Show moreA subclass of recently discovered CRISPR repeat RNA in bacteria contains minimally recognizable structural features that facilitate an unknown mechanism of recognition and processing by the Cas6 family of endoribonucleases. Cocrystal structures of Cas6 from Methanococcus maripaludis (MmCas6b) bound with its repeat RNA revealed a dual site binding structure and a cleavage site conformation poised for phosphodiester bond breakage. Two non-interacting MmCas6b bind to two separate AAYAA motifs within the same repeat, one distal and one adjacent to the cleavage site. This bound structure potentially competes with a stable but non-productive RNA structure. At the cleavage site, MmCas6b supplies a base pair mimic to stabilize a short 2 base pair stem immediately upstream of the scissile phosphate. Complementary biochemical analyses support the dual-AAYAA binding model and a critical role of the protein-RNA base pair mimic. Our results reveal a previously unknown method of processing non-stem-loop CRISPR RNA by Cas6.
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Date Issued: 2016-04-05
Identifier: FSU_pmch_26996962, 10.1016/j.str.2016.02.009, PMC4823167, 26996962, 26996962, S0969-2126(16)00068-X
Format: Citation

Title: Cleavage of the SUN-domain protein Mps3 at its N-terminus regulates centrosome disjunction in budding yeast meiosis.
Creator: Li, Ping, Jin, Hui, Koch, Bailey A, Abblett, Rebecca L, Han, Xuemei, Yates, John R, Yu, Hong-Guo
Abstract/Description: Centrosomes organize microtubules and are essential for spindle formation and chromosome segregation during cell division. Duplicated centrosomes are physically linked, but how this linkage is dissolved remains unclear. Yeast centrosomes are tethered by a nuclear-envelope-attached structure called the half-bridge, whose components have mammalian homologues. We report here that cleavage of the half-bridge protein Mps3 promotes accurate centrosome disjunction in budding yeast. Mps3 is a single...
Show moreCentrosomes organize microtubules and are essential for spindle formation and chromosome segregation during cell division. Duplicated centrosomes are physically linked, but how this linkage is dissolved remains unclear. Yeast centrosomes are tethered by a nuclear-envelope-attached structure called the half-bridge, whose components have mammalian homologues. We report here that cleavage of the half-bridge protein Mps3 promotes accurate centrosome disjunction in budding yeast. Mps3 is a single-pass SUN-domain protein anchored at the inner nuclear membrane and concentrated at the nuclear side of the half-bridge. Using the unique feature in yeast meiosis that centrosomes are linked for hours before their separation, we have revealed that Mps3 is cleaved at its nucleus-localized N-terminal domain, the process of which is regulated by its phosphorylation at serine 70. Cleavage of Mps3 takes place at the yeast centrosome and requires proteasome activity. We show that noncleavable Mps3 (Mps3-nc) inhibits centrosome separation during yeast meiosis. In addition, overexpression of mps3-nc in vegetative yeast cells also inhibits centrosome separation and is lethal. Our findings provide a genetic mechanism for the regulation of SUN-domain protein-mediated activities, including centrosome separation, by irreversible protein cleavage at the nuclear periphery.
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Date Issued: 2017-06-13
Identifier: FSU_pmch_28609436, 10.1371/journal.pgen.1006830, PMC5487077, 28609436, 28609436, PGENETICS-D-17-00030
Format: Citation

Title: 3D Molecular Cytological and Genetic Analysis of the SUN-Domain (Sad1-Unc-84) Proteins in Maize (Zea Mays, L.) Meiosis; Discovery of a Novel Plant Sun Family, Including SUN3, a Candidate Gene for the Desynaptic1 (dy1) Mutant.
Creator: Murphy, Shaun Patrick, Bass, Hank Henry W., Hurt, Myra M., Fadool, James, Li, Hong, Epstein, Lloyd, Program in Molecular Biophysics, Florida State University
Abstract/Description: Meiosis is the process by which sexually reproducing organisms reduce their genomes from diploid (2n) to haploid (n) during the formation of gametes. It requires that homologous chromosomes pair, synapse, recombine, and finally segregate. These widely conserved processes are under genetic control, yet the exact details of many of the underlying molecular mechanisms remain under active investigation. The initial pairing and subsequent synapsis events are immediately preceded by the clustering...
Show moreMeiosis is the process by which sexually reproducing organisms reduce their genomes from diploid (2n) to haploid (n) during the formation of gametes. It requires that homologous chromosomes pair, synapse, recombine, and finally segregate. These widely conserved processes are under genetic control, yet the exact details of many of the underlying molecular mechanisms remain under active investigation. The initial pairing and subsequent synapsis events are immediately preceded by the clustering of telomeres on the nuclear envelope in a widely conserved structure referred to as the bouquet arrangement of meiotic chromosomes. In animals and plants, genes required for genome reduction at meiosis I have been characterized and show a high degree of conservation between kingdoms and species within them. Higher plants (most notably maize) have provided an excellent large-genome model system for the study of the cytology of homologous chromosome behavior and therefore have allowed an in depth dissection of the meiotic process in eukaryotes. At the cellular level, meiotic chromosome behavior is accompanied by changes in the architecture of the cell nucleus, particularly with respect to the interaction of telomeres with the nuclear periphery. This dissertation presents the work involving the analysis of a classic meiotic mutant of maize, desynaptic (dy1). The dy1 mutant is characterized by a precocious telomere-nuclear envelope detachment phenotype at mid prophase, resulting in chromosome breaks, anaphase bridges, micronuclei, and defective pollen development. In this study, we observed new phenotypes as early as the telomere bouquet stage of meiotic prophase in dy1 lines of maize. Using linkage and translocation mapping techniques, the dy1 mutation was mapped to the long arm of chromosome 3, where a candidate gene with homology to a nuclear envelope-associated SUN domain protein gene was identified. SUN (Sad1p/Unc-84) domain proteins function with other proteins to form a physical link between the nucleoskeleton and the cytoskeleton. These bridges transfer forces across the nuclear envelope and are increasingly recognized to play roles in nuclear positioning, nuclear migration, cell cycle-dependent breakdown and reformation of the nuclear envelope, telomere-led nuclear reorganization during meiosis, and karyogamy. Using bioinformatic and molecular approaches, we characterized the family of maize SUN-domain proteins, starting with a screen of maize genomic sequence data. We characterized five different maize ZmSUN genes (ZmSUN1-5), which fell into two structural classes likely of ancient origin. Orthologs of these genes and prevalent in the plant kingdom as they are also found in other monocots, eudicots, and even mosses. The first class described here designated canonical C-terminal SUN-domain (CCSD, ZmSUN1 and ZmSUN2), includes structural homologs of the animal and fungal SUN-domain protein genes. The second class, the plant-prevalent mid-SUN 3 transmembrane (PM3, ZmSUN3-5), includes a novel but conserved structural variant SUN-domain protein gene class. Analysis of the expression levels for these genes revealed very low expression in multiple tissue types, with the exception of ZmSUN5 which showed a pollen=preferred expression profile. Cloning and Peptide antibodies specific for ZmSUN3, and ZmSUN4 were used in western-blot and cell-staining assays to show that they are expressed and show concentrated staining at the nuclear periphery. In order to characterize the CCSD class of SUNproteins, we obtained new reagents and performed immunolocalization analyses coupled with high resolution 3D deconvolution microscopy. We identified a novel structure at the maize nuclear periphery we refer to as the "Nuclear SUN Belt", NSB, which was present in multiple somatic cell types as well as meiotic nuclei. During meiosis, the NSB was present at the onset and well into the leptotene stage of meiotic prophase. Surprisingly at the bouquet stage the NSB appeared to be localized opposite of the nucleolus in a crescent shape, occupying a small region (<1/3) of the surface of the nuclear periphery, often co-localizing with meiotic telomeres. During late prophase, the NSB returned temporarily until the release of the telomeres from the NE and subsequent NE breakdown prior to metaphase. The NSB later returned in post-meiotic nuclei including uninucleate cells, and prophase II nuclei. Using peptide antibodies specific for the CCSD class, we detected a severe disruption of SUN proteins at the nuclear envelope in a line of maize defective in meiotic telomere tethering and chromosome synapsis (desynaptic1, dy1) as well as a line defective in the transition from a prophase microtubule array to a metaphase spindle (divergent1, dv1) (SHAMINA et al. 2000b; STAIGER and CANDE 1990a). The findings presented in this dissertation provide valuable new information regarding the spatial distribution and dynamics of maize SUN proteins at the NE and for an initial interpretation of the phenotypes of historical meiotic mutants of maize.
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Date Issued: 2011
Identifier: FSU_migr_etd-5060
Format: Thesis

Title: Structural and Functional Studies of a RNA-Guided RNA Modification Enzyme: Box H/ACA RNP.
Creator: Liang, Bo, Li, Hong, Alabugin, Igor, Taylor, Kenneth A., Bass, Hank W., Bertram, Richard, Program in Molecular Biophysics, Florida State University
Abstract/Description: As an RNA-guided RNA modification enzyme, the box H/ACA RNP recruits the guide RNA to recognize the substrate RNA, whereas the protein partners carry out the catalysis. Most intriguingly, box H/ACA RNPs share the same four conserved proteins, Cbf5, Nop10, L7Ae and Gar1, and are able to utilize more than 100 guide RNAs to guide posttranscriptional modifications in most stable RNAs. With respect to structural studies, archaeal box H/ACA ribonucleoproteins (RNPs) are probably one of the most...
Show moreAs an RNA-guided RNA modification enzyme, the box H/ACA RNP recruits the guide RNA to recognize the substrate RNA, whereas the protein partners carry out the catalysis. Most intriguingly, box H/ACA RNPs share the same four conserved proteins, Cbf5, Nop10, L7Ae and Gar1, and are able to utilize more than 100 guide RNAs to guide posttranscriptional modifications in most stable RNAs. With respect to structural studies, archaeal box H/ACA ribonucleoproteins (RNPs) are probably one of the most extensively characterized ribonucleoprotein particles to this day. The dissertation presented here describes this work's contribution to the understanding of the nature of the RNA-guided RNA modification enzyme and of the most complex pseudouridine synthases. In chapter 2, we explored the structural basis of an archaeal box H/ACA protein complex comprised of three of the four essential proteins, Cbf5, Nop10 and Gar1. It was the first time we obtained molecular insights into these three proteins and the implications of a severe disease called dyskeratosis congenita (DC). We have also identified a DC mutation cluster site within a modeled dyskerin (Cbf5 homolog in humans) structure. In chapter 3, we further characterized the three-dimensional structure of a catalytically deficient archaeal box H/ACA RNP complex, including the guide RNA, the substrate RNA, Cbf5, Nop10 and Gar1. We devised a non-intrusive 2-aminopurine (2-AP) fluorescence assay which allowed us to determine the precise placement of the target uridine at the active site requires a conformational change of the guide-substrate RNA duplex by L7Ae. In chapter 4, we further examined the structural basis for accurate placement of substrate by accessory proteins using the 2-AP fluorescence assay. Our results revealed that each of the three accessory proteins, Nop10, L7Ae and Gar1, as well as an active site residue, have distinct effects on substrate conformations, suggesting the cooperative network of box H/ACA RNP. In chapter 5, we described a substrate-bound functional archaeal box H/ACA RNP that revealed detailed information about the active site. The substrate RNA containing 5-fluoruridine at the modification position is fully docked and rearranged in a manner similar to those of stand-alone pseudouridine synthases. The complementary biochemical studies further revealed the importance of a conserved protein loop and a guide-substrate RNA pocket in the binding to the substrate. With further comparison of available structures of stand-alone pseudouridine synthases-RNA complexes, we are able to summarize the common mechanism among all pseudouridine synthases, perhaps also a theme in other widespread RNA-guided enzymes. The accomplishments of this work greatly enhance our understanding of the enzymatic architecture of box H/ACA RNPs, unravel the many intriguing features of the most complex pseudouridine synthases, and shed light on the nature of the RNA-guided RNA modification and the assembly architecture of the telomerase holoenzyme.
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Date Issued: 2009
Identifier: FSU_migr_etd-7183
Format: Thesis

Title: Biochemical Characterization of the RNA Splicing Endonuclease.
Creator: Calvin, Kate, Li, Hong, Hurt, Myra, Cross, Timothy, Logan, Timothy, Miller, Brian, Program in Molecular Biophysics, Florida State University
Abstract/Description: In eukaryotes and archaea 5-25% of transfer RNA (tRNA) precursors contain intervening sequences, or introns, that interrupt the molecules' functional regions. Because functional tRNA molecules are necessary for protein synthesis, removing these introns is essential to sustain life. tRNA introns are removed in a two-to-three step process mediated by three different proteins. The RNA splicing endonuclease acts first to cleave two phosphodiester bonds at the intron boundaries within the folded...
Show moreIn eukaryotes and archaea 5-25% of transfer RNA (tRNA) precursors contain intervening sequences, or introns, that interrupt the molecules' functional regions. Because functional tRNA molecules are necessary for protein synthesis, removing these introns is essential to sustain life. tRNA introns are removed in a two-to-three step process mediated by three different proteins. The RNA splicing endonuclease acts first to cleave two phosphodiester bonds at the intron boundaries within the folded precursor RNAs. The endonuclease performs this function upon nuclear tRNA introns and all archaeal introns. It is well-established that in all organisms the endonuclease step in the splicing pathway is completely conserved, with evidence for the conservation of cleavage chemistry being provided by biochemical studies. However, no detailed information was previously available regarding the endonuclease's specific mechanisms. This research addresses two key aspects of the splicing endonuclease mechanism, namely, substrate recognition and catalysis. Chapter 2 explores the structural elements in a phenotypical archaeal splicing endonuclease and its RNA substrate required for recognition and catalysis. These assays explicitly demonstrate the enzyme and substrate elements involved in recognition and binding. They also support previous findings regarding a conserved triad hypothesized to be catalytic and lay the foundation for the more in-depth studies in Chapter 3. Chapter 3 presents a series of kinetics experiments investigating this conserved triad in which kinetic parameters KM and k2 are obtained. The primary substrate recognition elements in the endonuclease are strictly conserved. However, splicing endonucleases in different organisms are found to have different subunit compositions and substrate specificities. No biochemical studies to date have shed light on how this occurs. Chapter 4 presents studies exploring how the enzyme's quaternary structure affects substrate recognition and cleavage. These studies are continued in Chapter 5, where it is demonstrated that enzyme assembly alone can dictate both substrate specificity and activity. Taken in total, the work presented in this Dissertation provides significant insight regarding how the endonuclease precisely recognizes intron-exon junctions and accelerates the cleavage reaction. It also sheds considerable light on how enzyme subunit composition and quaternary structure relate to the mechanism of RNA recognition.
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Date Issued: 2007
Identifier: FSU_migr_etd-4514
Format: Thesis

Title: Reading Between the Filaments: Structural Characterization of Two Different F-Actin Cross-Linking Proteins by Electron Microscopy.
Creator: Hampton, Cheri M., Taylor, Kenneth A., Ouimet, Charles, Chase, P. Bryant, Fajer, Piotr, Li, Hong, Program in Molecular Biophysics, Florida State University
Abstract/Description: We use a lipid monolayer system to prepare paracrystalline rafts of F-actin with two different cross-linking proteins, α-actinin and villin. α-Actinin cross-links F-actin into relatively loose networks throughout cells, while villin forms dense, tight bundles of filaments in specialized structures known as microvilli. Our monolayer system allows us to examine each of these cross-linkers as they interact with F-actin in a simplified two-component system. In order to analyze the data from these...
Show moreWe use a lipid monolayer system to prepare paracrystalline rafts of F-actin with two different cross-linking proteins, α-actinin and villin. α-Actinin cross-links F-actin into relatively loose networks throughout cells, while villin forms dense, tight bundles of filaments in specialized structures known as microvilli. Our monolayer system allows us to examine each of these cross-linkers as they interact with F-actin in a simplified two-component system. In order to analyze the data from these arrays, we have hybridized methods from 2-D crystal analysis with a single-particle approach to refine strategies for correspondence analysis and classification of images. For the α-actinin:F-actin rafts, we use this strategy to characterize the highly variable cross-links as 2-D projection images. The villin:F-actin rafts require further refinement of the hybrid strategy by application to 3-D volumes from electron tomography. Both protein arrays yield unique insights to the architectural arrangement of cross-linking proteins between filaments. In the α-actinin:F-actin rafts we use correspondence analysis to demonstrate that otherwise polar arrays of F-actin can have insertions of filaments of reversed polarity within them, and that these polarity differences do not influence the cross-links. We developed a method for left-right independent classification of the α-actinin cross-links to recover the high variability in the cross-link angular distribution by increasing the signal-to-noise ratio of the class averages. These averages are combined to recreate the original cross-link as it appears in a process we call "mapping-back." Measurements reveal that the length of cross-links can vary. From the classification of cross-links we demonstrate and model the novel occurrence of α-actinin bound to successive cross-over repeats on the same actin filament which we have termed "monofilament" binding. We further illustrate that the length variation of these monofilament-bound α-actinins are quantized to 55 Å, the distance between two adjacent actin monomers in the filament. The villin:F-actin rafts displayed homogeneous cross-linking. Docking of the homologous gelsolin atomic coordinates into the three-dimensional volumes reveals that villin does not interact with F-actin in the same manner as gelsolin. This data supports multiplicity of binding modes even in highly homologous proteins, and suggests a new mode of F-actin-binding for the villin protein.
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Date Issued: 2006
Identifier: FSU_migr_etd-4301
Format: Thesis

Title: Pairing Correlations and Phase Transitions in Mesoscopic Systems.
Creator: Sumaryada, Tony Ibnu, Volya, Alexander, Li, Hong, Piekarewicz, Jorge, Rogachev, Grigory, Dobrosavljevic, Vladimir, Department of Physics, Florida State University
Abstract/Description: Pairing correlations and phase transitions in mesoscopic or small systems are studied through out this dissertation. We start our discussion by showing the importance of short range correlations and their role in forming bound Cooper pairs. For a model Hamiltonian, we solved the Schr¨odinger equation in the harmonic oscillator basis analytically, the concept of self consistency is used to get the whole energy spectrum. Using variational methods applied to a trial wave function, we derived the...
Show morePairing correlations and phase transitions in mesoscopic or small systems are studied through out this dissertation. We start our discussion by showing the importance of short range correlations and their role in forming bound Cooper pairs. For a model Hamiltonian, we solved the Schr¨odinger equation in the harmonic oscillator basis analytically, the concept of self consistency is used to get the whole energy spectrum. Using variational methods applied to a trial wave function, we derived the BCS equations, which again should be solved self consistently with particle number to produce the total energy. Some examples of BCS calculations in realistic case like in the Sn isotopes are shown. Various approximations such as one level, two levels and five levels systems are discussed. In the five levels model calculations, we compare our results with the previous works by other authors. We also find a good agreement with the experimental data. We extend our BCS calculations by adding the three body interaction term. This additional term is unlikely to improve our results compared to the experiment. In a separate work, using numerical and analytical methods implemented for different models we conduct a systematic study of thermodynamic properties of pairing correlations in mesoscopic nuclear systems. Various quantities are calculated and analyzed using the exact solution of pairing. An in-depth comparison of canonical, grand canonical, and microcanonical ensemble is conducted. The nature of the pairing phase transition in a small system is of particular interest. We discuss the onset of discontinuities in the thermodynamic variables, fluctuations, and evolution of zeros of the canonical and grand canonical partition functions in the complex plane. The behavior of the Invariant Correlational Entropy is also studied in the transitional region of interest. The change in the character of the phase transition due to the presence of magnetic field is discussed along with studies of superconducting thermodynamics.
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Date Issued: 2007
Identifier: FSU_migr_etd-0406
Format: Thesis

Title: Computational Analysis of the U2 Snrna-Intron Duplex.
Creator: Xu, Darui, Greenbaum, Nancy L., Li, Hong, Alabugin, Igor, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: Pairing of a consensus sequence of the precursor (pre)-mRNA intron with a short region of the U2 small nuclear (sn)RNA during assembly of the eukaryotic spliceosome results in formation of a complementary helix of seven base pairs with a single unpaired adenosine, whose 2' OH initiates the nucleophilic attack at the pre-mRNA 5' splice site during the first step of splicing. The structure of the spliceosomal branch site solved by Newby and Greenbaum showed that a highly conserved pseudouridine...
Show morePairing of a consensus sequence of the precursor (pre)-mRNA intron with a short region of the U2 small nuclear (sn)RNA during assembly of the eukaryotic spliceosome results in formation of a complementary helix of seven base pairs with a single unpaired adenosine, whose 2' OH initiates the nucleophilic attack at the pre-mRNA 5' splice site during the first step of splicing. The structure of the spliceosomal branch site solved by Newby and Greenbaum showed that a highly conserved pseudouridine residue in U2 snRNA induces a dramatically altered structure compared with that of its unmodified counterpart. In this study, both modified and unmodified U2 snRNA-intron duplexes were analyzed using computer simulations including preliminary molecular dynamics (MD) simulations, electrostatic potential, surface area, and solvation free energy calculations. The preliminary MD simulations produce stable trajectories of the RNA duplexes in solution. The surface electrostatic potentials were calculated using finite difference Poisson-Boltzmann algorithm and a hybrid boundary element and finite difference Poisson-Boltzmann approach. Results show a region of exceptionally negative potential near the 2' OH of the branch site adenosine. The two RNA duplexes have similar solvent accessible surface areas, whereas the surface accessible area of the 2' OH of the branch site adenosine of the modified RNA duplex is considerably smaller than that of the unmodified RNA duplex. The solvation free energy calculation indicates that the unmodified RNA duplex is favored over the modified RNA duplex.
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Date Issued: 2003
Identifier: FSU_migr_etd-0414
Format: Thesis

Title: Use of Inorganic Quantum Dot-Cationic Liposome Hybrids for the Delivery and Expression of Calcium-Sequestering Parvalbumin into Mammalian Cell Cultures.
Creator: Ellis, Charles Christian, Strouse, Geoffrey, Logan, Timothy, Li, Hong, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: Left Ventricular Diastolic Dysfunction is one of the main causes of Heart Failure. It is caused by a defect in the relaxation of cardiac muscle usually as the result of failure of the heart cells to remove cytoplasmic Ca2+ following muscle contraction. This defect is corrected by the presence of Ca2+ sequestering Parvalbumin Major Isoform I (Parvalbumin), a naturally occurring soluble protein in skeletal muscle, which then binds free Ca2+, resulting in increased rates of diastolic relaxation....
Show moreLeft Ventricular Diastolic Dysfunction is one of the main causes of Heart Failure. It is caused by a defect in the relaxation of cardiac muscle usually as the result of failure of the heart cells to remove cytoplasmic Ca2+ following muscle contraction. This defect is corrected by the presence of Ca2+ sequestering Parvalbumin Major Isoform I (Parvalbumin), a naturally occurring soluble protein in skeletal muscle, which then binds free Ca2+, resulting in increased rates of diastolic relaxation. Since Parvalbumin does not naturally occur in cardiac tissue, ectopic expression through gene therapy provides a vehicle to deliver the gene needed to express this therapeutic protein. This has been accomplished by others using viral vectors but due to the problems associated with viral delivery, non-viral delivery methods are becoming more popular. Cationic Liposomes are a commonly used non-viral method of gene delivery and due to their physical and chemical properties inorganic nano-particles are attracting much interest in the field as well. It is the aim of this research to investigate whether cationic liposomes containing organic-phase fluorescent CdSe/ZnS quantum dots can be used as an efficient method of gene delivery into mammalian cells with built-in optical tracers. Organic-phase CdSe/ZnS was synthesized, purified and encapsulated into liposomes using various ratios of 1,2 – dioleoyl – 3 – trimethylammonium – propane (DOTAP), 1,2 – dioleoyl – sn – glycero – 3 – phosphoethanolamine (DOPE), Cholesterol and 3β – [N – (N', N' – dimethylaminoethan) – carbamoyl] cholesterol (DC-Chol) and used to deliver circular plasmid DNA coding for a Parvalbumin-mCherry fusion protein into Chinese Hamster Ovary (CHO) cells. We are able to show that using this system of cationic liposome-quantum dot hybrids we are able to deliver and express the target gene.
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Date Issued: 2010
Identifier: FSU_migr_etd-0571
Format: Thesis

Title: Biochemical Characterization of Human Matrix Metalloproteinases and Their Newly Designed Inhibitors Related to Stroke.
Creator: Cao, Qiang, Sang, Qing-Xiang Amy, Wang, Yan-Chang, Li, Hong, Alabugin, Igor, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: Matrix metalloproteinases (MMPs), a family of enzymes known for their proteolytic activities on processing extracellular matrix substrates, may play an integral role in blood-brain barrier opening following an ischemic stroke. Several matrix metalloproteinases are proposed to play vital roles in early or late stages of blood brain barrier opening. Matrix metalloproteinase inhibitor (MMPI) has been showing beneficial effect in the treatment of the blood brain opening related to stroke. As old...
Show moreMatrix metalloproteinases (MMPs), a family of enzymes known for their proteolytic activities on processing extracellular matrix substrates, may play an integral role in blood-brain barrier opening following an ischemic stroke. Several matrix metalloproteinases are proposed to play vital roles in early or late stages of blood brain barrier opening. Matrix metalloproteinase inhibitor (MMPI) has been showing beneficial effect in the treatment of the blood brain opening related to stroke. As old generation matrix metalloproteinase inhibitor failed in oncology clinical trials, our collaborators, Drs. Martin A. Schwartz and Yonghao Jin, have designed and synthesized new biologically friendly mercaptosulfonamide inhibitors. Characterization and selection of effective matrix metalloproteinase inhibitors were performed by evaluating their stability, potency, and selectivity by enzymatic kinetics. According to dissociation constant related to enzyme and inhibitor binding, our data indicates that those inhibitors are capable of inhibiting MMP-2, -9, and membrane-type 1 MMP (MT1-MMP) effectively and selectively. Selected inhibitors were studied with cell wound healing assays in human microvascular endothelial cell model to investigate selected MMPI activities and impact on cell behavior. By blocking MMP activities in cell culture, our inhibitors were able to reduce human brain microvascular endothelial cell wound healing process. Protein expression patterns in cell culture were investigated with proteomics after inhibitor treatment. The reduced expressions of several proteins, which are related to cell division, cell adhesion and cell death, have been discovered. It is also verified the blocking function of our inhibitor in human brain microvascular endothelial cell wound healing assay. Overall, our newly designed matrix metalloproteinase inhibitor efficiently inhibits matrix metalloproteinase which carries intermediate or deep S1' pocket at protein and cellular level. Upon application of matrix metalloproteinase inhibitor, it has been implicated that blocking of matrix metalloproteinase activities are involved in the decreasing of other cell function modulators. The most potent and specific inhibitors have been selected as promising compounds, which have been further tested in animal models to evaluate their efficacy in the prevention of blood brain barrier opening associated with stroke by our collaborator Dr. Gary A. Rosenberg. This study is the first enzymological and cellular analysis of mercaptosulfonamide inhibitors.
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Date Issued: 2010
Identifier: FSU_migr_etd-4624
Format: Thesis

Title: Mercaptosulfonamides: Potential Modulators of Human Mesenchymal Stem Cell Behavior.
Creator: Roycik, Mark Druen, Sang, Qing-Xiang, Overton, J. Michael, Li, Hong, Schlenoff, Joseph B., Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: Residing in a host of tissues, including skin, muscle, liver, heart, and even hair follicles, non-embryonic stem cells, which are gaining a popularity rivaling that of their embryonic precursors, are capable of combating a number of pathological disorders. Owing to their ability to regenerate specialized tissues associated with each of the three germ layers and being derived from the marrow of human bone, adult mesenchymal stem cells (hMSCs) have been viewed with increased promise, and have...
Show moreResiding in a host of tissues, including skin, muscle, liver, heart, and even hair follicles, non-embryonic stem cells, which are gaining a popularity rivaling that of their embryonic precursors, are capable of combating a number of pathological disorders. Owing to their ability to regenerate specialized tissues associated with each of the three germ layers and being derived from the marrow of human bone, adult mesenchymal stem cells (hMSCs) have been viewed with increased promise, and have been suggested as putative therapeutics for an array of pathologies including Parkinson's disease, cardiovascular disease, arthritis, and even diabetes. However, hMSC cytotherapy has been limited due to inconsistencies observed between their in vitro and in vivo behaviors that include alterations to self-renewal properties and varying propensities to differentiate, necessitating the elucidation of the mechanisms controlling their overall growth and fate. Despite promiscuous expression profiles, the presence of various extracellular protein products, proteolytic enzymes, and adhesion molecules has clearly implicated potential roles for matrix remodeling pathways and associated interactions—features leading to the involvement of a family of enzymes known as matrix metalloproteinases (MMPs). As the primary enzymes responsible for extracellular matrix maintenance, MMPs are also essential in numerous fundamental cellular processes such as proliferation, differentiation, and apoptosis. Combined with evidences indicating their expression in hMSCs, MMPs were studied for their potential control in the proliferation and differentiation of hMSCs. Facilitating this assessment was a subset of small, synthetic mercaptosulfonamide matrix metalloproteinase inhibitors (MMPIs), which, employing a thiol zinc-binding group, reversibly coordinate the catalytic Zn2+ in the active site of MMPs to block their proteolytic activities. Characterized for their inhibitory potential and biological compatibility in the first two parts of this discussion, the mercaptosulfonamide MMPIs were primarily utilized as 'tools' to delineate any potential MMP involvement in hMSC behavior. Although the use of these compounds led to the finding that MMP activity was not an integral component of hMSC behavior, several mercaptosulfonamides and their non-inhibitory complements were unexpectedly shown to enhance the adipocytic differentiation of these cells, ultimately suggesting a novel role for these compounds—potential modulators of stem cell differentiation.
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Date Issued: 2010
Identifier: FSU_migr_etd-4613
Format: Thesis

Title: Backbone Dynamics in an Intramolecular Prolylpeptide SH3 Complex from Diphtheria Toxin Repressor, DtxR.
Creator: Bhattacharya, Nilakshee, Logan, Timothy M., Zhou, Huan-Xian, Li, Hong, Steinbock, Oliver, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: Diphtheria toxin repressor is a regulatory protein from Corynebacterium diphtheriae, the causal agent of Diphtheria. The diphtheria toxin repressor (DtxR) contains an SH3-like domain that forms an intramolecular complex with a proline-rich (Pr) peptide segment that serves to stabilize the inactive state of the repressor. During activation of DtxR by transition metals, this intramolecular complex must dissociate as the SH3 domain and Pr segment form different interactions in the active...
Show moreDiphtheria toxin repressor is a regulatory protein from Corynebacterium diphtheriae, the causal agent of Diphtheria. The diphtheria toxin repressor (DtxR) contains an SH3-like domain that forms an intramolecular complex with a proline-rich (Pr) peptide segment that serves to stabilize the inactive state of the repressor. During activation of DtxR by transition metals, this intramolecular complex must dissociate as the SH3 domain and Pr segment form different interactions in the active repressor. In this study we investigate the dynamics of this intramolecular complex using backbone amide nuclear spin relaxation rates determined experimentally using NMR spectroscopy and computed from molecular dynamics trajectories. The SH3 domain in the unbound and bound states showed typical dynamics in that the secondary structures were fairly ordered with high generalized order parameters and low effective correlation times while residues in the loops connecting b-strands exhibited reduced generalized order parameters and required additional motional terms to adequately model the relaxation rates. Residues forming the Pr segment also exhibited low order parameters with internal rotational correlation times on the order of 0.6 â 1 ns. Further analysis showed that the SH3 domain was rich in ms motions while the Pr segment was rich in motions on the 100s ms timescale. Molecular dynamics trajectories of PrSH3 and SH3 indicated structural rearrangements that might contribute to the observed relaxation rates and, together with the observed relaxation rate data, suggested that the Pr segment exhibits a binding â unbinding equilibrium. The intramolecular complex resisted any significant change in the binding affinity between the Pr segment and the SH3 domain due to mutations in the Pr segment. The results of this study provide key insights into the nature of the intramolecular complex and provide a better understanding of the biological role of the SH3 domain in regulating DtxR activity.
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Date Issued: 2007
Identifier: FSU_migr_etd-1408
Format: Thesis

Title: Understanding Structural Mechanisms of Endolytic RNA Cleavage Enzymes.
Creator: Mitchell, Michelle Hall, Li, Hong, Gilmer, Penny J., Miller, Brian, Ellington, W. Ross, Stefanovic, Branko, Program in Molecular Biophysics, Florida State University
Abstract/Description: The RNA splicing and processing endonuclease from Nanoarchaeum equitans (NEQ) belongs to the recently identified (ab)2 family of splicing endonucleases that require two different subunits for splicing activity. N. Equitans splicing endonuclease consists of the catalytic subunit (NEQ205) and the structural subunit (NEQ261). Here we report the crystal structure of the functional NEQ enzyme at 2.1 Angstroms resolution containing both subunits, as well as that of the NEQ261 subunit alone at 2.2...
Show moreThe RNA splicing and processing endonuclease from Nanoarchaeum equitans (NEQ) belongs to the recently identified (ab)2 family of splicing endonucleases that require two different subunits for splicing activity. N. Equitans splicing endonuclease consists of the catalytic subunit (NEQ205) and the structural subunit (NEQ261). Here we report the crystal structure of the functional NEQ enzyme at 2.1 Angstroms resolution containing both subunits, as well as that of the NEQ261 subunit alone at 2.2 Angstroms resolution. The functional enzyme resembles previously known a2 and a4 endonucleases but forms a heterotetramer; a dimer of two heterodimers of the catalytic subunit (NEQ205) and the structural subunit (NEQ261). Surprisingly, NEQ261 alone forms a homodimer, similar to the previously known homodimer of the catalytic subunit. The homodimers of isolated subunits are inhibitory to heterodimerization as illustrated by a covalently linked catalytic homodimer that had no RNA cleavage activity upon mixing with the structural subunit. Detailed structural comparison reveals a more favorable hetero- than homo-dimerization interface, thereby suggesting a possible regulation mechanism of enzyme assembly through available subunits. Finally, the uniquely flexible active site of the NEQ endonuclease provides a possible explanation for its broader substrate specificity.
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Date Issued: 2009
Identifier: FSU_migr_etd-2380
Format: Thesis

Title: Structural Studies of Box C/D (SNO)RNP Particle, an RNA Guided Modification Enzyme.
Creator: Oruganti, Sri vidya, Li, Hong, Keller, Laura R., Blaber, Michael, Steinbock, Oliver, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: Small nucleolar RNAs (snoRNAs) are localized in the nucleolus as small ribonucleoprotein particle (snoRNP) that are RNA-protein complexes and catalyze site specific modifications on the ribosomal RNA. When compared to many protein enzymes, the snoRNPs are unique in that they use the RNA component of the snoRNP for substrate recognition by base pairing through its antisense sequence and the protein component for catalyzing the modification. Hence, each snoRNP particle has the same catalytic...
Show moreSmall nucleolar RNAs (snoRNAs) are localized in the nucleolus as small ribonucleoprotein particle (snoRNP) that are RNA-protein complexes and catalyze site specific modifications on the ribosomal RNA. When compared to many protein enzymes, the snoRNPs are unique in that they use the RNA component of the snoRNP for substrate recognition by base pairing through its antisense sequence and the protein component for catalyzing the modification. Hence, each snoRNP particle has the same catalytic unit but different substrate recognition unit. One member of snoRNP, called the "box C/D snoRNP" catalyzes 2'-O-ribose methylation in the most conserved and functionally important regions of the ribosome. The box C/D snoRNP consists of a bipartite box C/D snoRNA and four core proteins in eukaryotes (Fibrillarin, Nop56p, Nop58p, and Snu13p) and three core proteins in archaea (Fibrillarin, Nop56/58p and L7Ae). The box C/D RNA acts as a guide for substrate recognition by its antisense sequence to the substrate while the proteins distribute on the bipartite box C/D RNA to catalyze the 2'-O-ribose modification. In eukaryotes, the proteins are differentially distributed on the bipartite RNA with Snu13p, Nop58p and Fibrillarin on the C/D motif and Nop56p and fibrillarin on the C'/D' motif. In archaea, a symmetric distribution of proteins on the C/D and C'/D' motifs is observed with L7Ae, Nop56/58p and fibrillarin on both the motifs. In the past decade a number of RNPs with RNA guided mechanisms for catalysis have been identified. With little information available, insights on any RNP assembly and catalysis would provide a global understanding of the protein-protein, protein-RNA and RNA-RNA interactions in a RNP particle. The present study focuses on understanding the box C/D snoRNP assembly and catalysis using biochemical and crystallographic techniques. To understand the ability of Snu13p to initiate snoRNP assmebly and its differential specificity for the box C/D bipartite RNA, a crystal structure of free Snu13p was obtained. By comparing the structure of Snu13p with its eukaryal and archaeal homologs whose structures with RNA are available, similar structural features are observed thus confirming its ability to induce RNA conformational change. In addition the high resolution structure revealed a structural divergence between the eukaryotic and the archaeal homologs that may account for their different RNA specificities. Finally, structure of the core protein complex of an archaeal box C/D snoRNP, Nop56/58-Fibrillarin complex, has been obtained. We report two crystal structures of the Nop56/58-fibrillarin complex from Pyrococcus furiosus: one with full-length protein (3.6 Å) and the other in which the C-terminal KKE/D tail of Nop56/58 is truncated (2.7 Å). In both structures, a Nop56/58-fibrillarin homodimer is formed by interactions of the coiled-coil domains of Nop56/58, confirming the generality of this previously observed arrangement. However, the conformation of Nop56/58 in the new structures differs substantially from that in the earlier structure, resulting in repositioning of fibrillarin and the cognate catalytic sites within the complex. These studies provide an attractive model of box C/D snoRNP assembly that comprises "one complex, two active site" model. The structural features facilitating this model depend on protein-protein interactions as well as the plasticity in proteins and RNA. Finally, efforts to confirm this assembly model by X-ray crystallographic techniques are described.
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Date Issued: 2007
Identifier: FSU_migr_etd-2361
Format: Thesis

Title: Adeno-Associated Virus-2 and Its Primary Cellular Receptor-Cryo-EM Structure of a Heparin Complex.
Creator: O’Donnell, Jason, Li, Hong, Chapman, Michael, Taylor, Kenneth, Safron, Sanford, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: Adeno-associated virus serotype 2 (AAV-2) is a leading candidate vector for gene therapy. Cell entry starts with attachment to a primary receptor, Heparan Sulfate Proteoglycan (HSPG) before binding to a co-receptor. Here, cryo-electron microscopy provides direct visualization of the virus–HSPG interactions. Single particle analysis was performed on AAV-2 complexed with a 17 kDa heparin fragment at 8.3 Å resolution. Heparin density covers the shoulder of spikes surrounding viral 3-fold...
Show moreAdeno-associated virus serotype 2 (AAV-2) is a leading candidate vector for gene therapy. Cell entry starts with attachment to a primary receptor, Heparan Sulfate Proteoglycan (HSPG) before binding to a co-receptor. Here, cryo-electron microscopy provides direct visualization of the virus–HSPG interactions. Single particle analysis was performed on AAV-2 complexed with a 17 kDa heparin fragment at 8.3 Å resolution. Heparin density covers the shoulder of spikes surrounding viral 3-fold symmetry axes. Previously implicated, positively charged residues R448/585, R451/588 and R350/487 from another subunit cluster at the center of the heparin footprint. The footprint is much more extensive than apparent through mutagenesis, including R347/484, K395/532 and K390/527 that are more conserved, but whose roles have been controversial. It also includes much of a region proposed as a co-receptor site, because prior studies had not revealed heparin interactions. Heparin density bridges over the viral 3-fold axes, indicating multi-valent attachment to symmetry-related binding sites.
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Date Issued: 2009
Identifier: FSU_migr_etd-2459
Format: Thesis

Title: Tandem Claisen Mislow-Evans Rearrangement in Formation of the A-G Ring System in Pinnatoxin A.
Creator: Pelc, Matthew J., Zakarian, Armen, Dudley, Greg, Li, Hong, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: Pinnatoxin A, a marine-derived macrocycle originating from a bivalve within the shellfish Pinna muricata and Pinna attenuata, was first isolated in 1995 by Uemura and has been a target of several synthetic studies due to its intriguing structure and bioactivity. Key features of this natural product are 14 chiral centers, a 6,7-spiroimine ring, 5,6-bicyclo ketal ring, and a 6,5,6-trispiroketal ring. The most notable features of the A-G 6,7-spirobicyclic ring structure is its chiral, quaternary...
Show morePinnatoxin A, a marine-derived macrocycle originating from a bivalve within the shellfish Pinna muricata and Pinna attenuata, was first isolated in 1995 by Uemura and has been a target of several synthetic studies due to its intriguing structure and bioactivity. Key features of this natural product are 14 chiral centers, a 6,7-spiroimine ring, 5,6-bicyclo ketal ring, and a 6,5,6-trispiroketal ring. The most notable features of the A-G 6,7-spirobicyclic ring structure is its chiral, quaternary center at its center, and the imine moiety incorporated into the A ring which has been found to be stable under mildly acidic conditions. The quaternary chiral center in synthetic chemistry is also noted to be one of the most difficult to form with high diastereoselectivity. Herein a stereoselective synthesis of the A-G ring of Pinnatoxin A is described. The synthesis relies on a key step involving a cascade [3,3]-sigmatropic Claisen rearrangement followed by a Mislow-Evans rearrangement that forms the central quaternary chiral center with excellent diastereoselectivity in one step. This tandem rearrangement not only builds the A-G rings chiral core but also succeeds in functionalizing the G ring for further modification towards Pinnatoxin A's total synthesis. In addition to the synthesis, a second key â-alkyl cross coupling is described using a palladium catalyzed Suzuki-Miyaura protocol in preparation for the tandem rearrangement. Also presented is evidence for the need to obtain the correct stereochemistry in incorporation of the E-F rings for formation of the imine to occur. Overall the A-G ring of Pinnatoxin A is synthesized with its longest linear sequence of 23 steps.
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Date Issued: 2006
Identifier: FSU_migr_etd-2048
Format: Thesis

Title: Biophysical and Biochemical Investigation of an Archaeal Box C/D SRNP: RNA-Protein Interactions of a Kink Turn RNA within the Functional Enzyme.
Creator: Moore, Terrie Luong, Li, Hong, Epstein, Lloyd M., Logan, Timothy M., Dorsey, John G., Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: Box C/D snoRNPs catylze the specific 2'O-methylation of rRNA in important regions the ribosome, although the role of the modifications is unclear. Eukaryotic box C/D snoRNPs consists of a box C/D RNA and four proteins, Fibrillarin, Nop56, Nop58, and 15.5kD. Archaeal homologs are simiplified containing three proteins, L7Ae, Nop5p, and Fibrillarin with a box C/D RNA. The box C/D sequences are proposed to form the recently recognized kink turn structure which is found in many types of RNA. Most...
Show moreBox C/D snoRNPs catylze the specific 2'O-methylation of rRNA in important regions the ribosome, although the role of the modifications is unclear. Eukaryotic box C/D snoRNPs consists of a box C/D RNA and four proteins, Fibrillarin, Nop56, Nop58, and 15.5kD. Archaeal homologs are simiplified containing three proteins, L7Ae, Nop5p, and Fibrillarin with a box C/D RNA. The box C/D sequences are proposed to form the recently recognized kink turn structure which is found in many types of RNA. Most are associated with proteins and protein binding may nucleate the assembly of other proteins onto the RNA. Dissecting the structure and biochemical properties of box C/D snoRNPs may not only help in understanding the function of the modifications, but may also give insight into the role of kink turn RNAs in RNP assembly. An archaeal box C/D RNA embedded within the intron of pre-tRNATrp from Archaeglobus Fulgidus(AF) that guides two modifications in the tRNA was used as the model for the investigation of three complexes: L7Ae-box C/D RNA, L7Ae-box C'/D' RNA, and the entire box C/D sRNP. Extensive crystallization trials resulted in crystals for each complex. A crystal structure of the box C/D RNA-L7Ae complex was determined to 2.7Å and shows the box C/D sequences do form a kink turn. Detailed structural comparisons of the AF L7Ae-box C/D RNA complex with previously determined crystal structures of L7Ae homologs in complex with functionally distinct kink turn RNAs revealed a conserved RNA-protein interface suggesting a conformational "adaptability" of the kink turn RNAs in binding L7Ae homologs. NMR characterization of L7Ae-box C/D RNA and L7Ae-box C'/D' RNA interactions suggests a structural change in the RNAs upon binding L7Ae and the RNAs may be dynamic structures that do not form stable kink turns alone. The underlying differences in primary and secondary structures of the kink turns may lead to different tertiary structures and dynamic behavior in kink turn RNAs that may confer specificity of L7Ae homologs for different kink turn RNAs. These analyses provide a structural basis for interpreting the functional roles of the box C/D sequences in directing specific assembly of box C/D sRNPs.
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Date Issued: 2005
Identifier: FSU_migr_etd-2320
Format: Thesis

Title: Functional and Structural Study of Crispr Processing Endonuclease Cas6.
Creator: Wang, Ruiying, Li, Hong, Epstein, Lloyd M., Miller, Brian G., Roper, Michael, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) interference is a newly discovered small-RNA based immune system used by bacteria and archaea. One of the essential steps in this interference is to process long CRISPR RNAs into small CRISPR RNAs (crRNAs). Cas6 has been found to perform the CRISPR RNA processing endonuclease activity in archaea, and our goal is to study the molecular basis of Cas6's function as the endonuclease to bind and cleave the repeat RNAs. The work...
Show moreCRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) interference is a newly discovered small-RNA based immune system used by bacteria and archaea. One of the essential steps in this interference is to process long CRISPR RNAs into small CRISPR RNAs (crRNAs). Cas6 has been found to perform the CRISPR RNA processing endonuclease activity in archaea, and our goal is to study the molecular basis of Cas6's function as the endonuclease to bind and cleave the repeat RNAs. The work described in this manuscript mostly focuses on study of the structures of Cas6 protein and Cas6-RNA complexes. The main objectives of this dissertation have been outlined. In chapter 2, our goal is to understand the CRISPR processing and the enzyme involved in this small RNA generating process. A high resolution crystal structure of free Cas6 from Pyrococcus furiosus has been obtained. This structure, together with the cleavage and binding activity assays, reveals the characteristics of this endoribonuclease. In chapter 3, we want to understand the molecular mechanism of the binding and cleavage activity of Cas6, the complex structure of Cas6 and its substrate RNA was solved. Our findings suggest that Cas6 processes CRISPR RNA by a beads-on-a-string model. And Cas6 is a single-stranded RNA specific endonuclease. In chapter 4, we apply the crystallization on various repeat RNAs and Cas6 protein's non-catalytic homologue (Cas6nc) to understand how RAMP proteins, the most abundant family of proteins involved in CRISPR interference, interact with various CRISPR repeat RNAs. The structure and the complementary biochemical analysis suggest a wrap-around model of RAMP-RNA interactions, and it also sheds light on how RAMP proteins are able to specifically interact with different repeat RNA substrates.
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Date Issued: 2010
Identifier: FSU_migr_etd-4631
Format: Thesis

Title: Structural and Mechanistic Studies of Ribonucleoprotein Particle Pseudouridine Synthase.
Creator: Zhou, Jing, Li, Hong, Schlenoff, Joseph B., Striegel, Andre, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: Box H/ACA ribonucleoprotein particle (RNP), an RNA-guided RNA modification enzyme, is comprised of four conserved proteins (Cbf5, Nop10, Gar1 and L7Ae) and one guide RNA. This complex recognizes the substrates by the guide RNA and catalyzes the chemical modification by the protein partners. In archaea and eukaryotes, box H/ACA RNP is responsible for pseudouridylation in most stable RNAs, using more than 100 guide RNAs. In this thesis, we present the structural studies of this most complex...
Show moreBox H/ACA ribonucleoprotein particle (RNP), an RNA-guided RNA modification enzyme, is comprised of four conserved proteins (Cbf5, Nop10, Gar1 and L7Ae) and one guide RNA. This complex recognizes the substrates by the guide RNA and catalyzes the chemical modification by the protein partners. In archaea and eukaryotes, box H/ACA RNP is responsible for pseudouridylation in most stable RNAs, using more than 100 guide RNAs. In this thesis, we present the structural studies of this most complex pseudouridine synthase. Based on this complicated system, we extend our understanding of the catalytic mechanism of pseudouridylation. In Chapter 2, we present a crystal structure of the substrate binding archaeal box H/ACA RNP complex which shows the detailed information of the active site. The 5-fluorouridine on the target position of substrate RNA is fully docked and rearranged into (5S,6R)-5-fluoro-6-hydroxy-pseudouridine (f5oh6Ψ) similar to other known structures of non RNA-guide pseudouridine synthases. This structure also reveals the substrate RNA binding details and the functions of conserved protein partners. In Chapter 3, we apply the crystallization on various substrate analogs with the functional H/ACA RNP complex. We also combine the activity assays and structures, provide a complete view of the enzyme active site along the reaction pathway, and suggest pre-organization as an important catalytic strategy. In Chapter 4, we focus on the specificity of PUA domain of Cbf5 for the absolutely conserved ACA sequence on guide RNA. We discovered that the high specificity enables the Cbf5 to catch the short RNA substrate containing ACA at the 3'end during crystallization. The activity assay shows that ACA trinucleotides block the Cbf5 activity on the small hairpin RNA substrate in a non RNA-guide manner.
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Date Issued: 2010
Identifier: FSU_migr_etd-7263
Format: Thesis

Title: Computational Approaches to the Description of Long Time Scale Biomolecular Events.
Creator: Carbone, Irina, Yang, Wei, Ye, Ming, Bruschweiler, Rafael, Li, Hong, Fenley, Marcia, Program in Molecular Biophysics, Florida State University
Abstract/Description: Molecular modeling of proteins and DNA is an attractive goal because it allows to gain insight into dynamic behavior of molecules on atomistic level. Such studies have a great potential to complement existing experimental techniques in investigating mechanisms of biomolecular phenomena. However, due to large size and ruggedness of free energy landscapes of biopolymers, simulations of long-time scale events often suffer from the pseudoergodicity problem, which manifests as inability to explore...
Show moreMolecular modeling of proteins and DNA is an attractive goal because it allows to gain insight into dynamic behavior of molecules on atomistic level. Such studies have a great potential to complement existing experimental techniques in investigating mechanisms of biomolecular phenomena. However, due to large size and ruggedness of free energy landscapes of biopolymers, simulations of long-time scale events often suffer from the pseudoergodicity problem, which manifests as inability to explore configurational space of interest within available computation time. The studies presented here reflect efforts to resolve this problem both by exploring advantages and limitations of the existing simulation techniques in studying behavior of specific biomolecular systems, and by testing new simulation methodologies. Part I is concerned with the mechanism of 8-oxoguanine DNA lesion recognition by the bacterial DNA repair enzyme MutM. Two qualitative studies using the Targeted Molecular Dynamics technique explore possible molecular interactions in the MutM/DNA complex associated with the enzyme's sliding along the ds-DNA and the extrusion of the interrogated base into the MutM's catalytic pocket. The findings suggest that MutM may require rocking motion of the bases encountered in its sliding along the DNA. The rocking motion of the oxoguanine is likely to be restricted due to repulsive electrostatic interactions of its O8 atom with the DNA phosphate backbone and may result in braking of the sliding motion of the MutM. This led to the proposal of the braking recognition mechanism in which recognition occurs due to arrest of the sliding process at the lesion site, which makes possible the otherwise slower process of extruding the base into the catalytic pocket of the MutM for excision. The second study suggests that binding between the conserved Arg 112 residue of MutM and the cytosine estranged during the oxoguanine extrusion may be important to prevent partial oxoguanine extrusion from becoming an alternative sliding pathway. Chapter 1.4 describes an attempt to investigate a possibility of long-range lesion recognition by MutM by computing free energy of MutM/DNA complexes. The Orthogonal Space Random Walk technique used for the free energy calculations in this experiment represents one of recent advancements in the generalized ensemble simulation methodology. Failure to obtain converged free energy estimates for the MutM/DNA complexes led to the discussion of possible limitations of this technique and to the proposal that MutM binding to the DNA in the vicinity of a lesion may require a global conformational reorganization of the DNA in comparison to lesion-free MutM/DNA complexes. Part II presents three small model systems studies of the efficiency of generalized ensemble simulation techniques and reflects a part of the recent methodological development in this field. The generalized ensemble techniques utilize sampling of the configrational space with modified probabilities in order to overcome the barrier crossing problem. Chapters 2.2 and 2.4 are concerned with history-dependant methods of formulating a priory unknown efficient probability modifications. In these studies the use of the Wang-Landau recursion approach in the metadynamics technique and the hybrid Wang-Landau recursion / adaptive reweighing approach for the Simulated Scaling technique are tested. The chapter 2.3 is concerned with an attempt of resolving the diffusion sampling problem associated with the generalized ensemble methods by implementing the Self-Guided Langevin dynamics approach for the Essential Energy Space modality of the metadynamics technique. All three studies demonstrated superior sampling efficiency of the proposed method enhancements for the selected model systems.
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Date Issued: 2009
Identifier: FSU_migr_etd-4226
Format: Thesis

Title: Development and Implementation of an NMR-Compatible 3-D Perfusion Bioreactor to Study HMSC Microenvironment.
Creator: Crowe, Jason J., Ma, Teng, Goldsby, Kenneth, Logan, Timothy, Grant, Samuel, Li, Hong, Program in Molecular Biophysics, Florida State University
Abstract/Description: Tissue engineering is one of the fastest growing biotechnology industries. The overarching research goals of the tissue engineering industry are highly intertwined with the field of medicine. The tissue engineering field seeks to create a variety of technologies that can be used in a clinical setting for tissue repair therapy. These technologies include everything from cell-based trophic factor therapies to engineered tissue grafts, and potentially engineered tissues. At the forefront of...
Show moreTissue engineering is one of the fastest growing biotechnology industries. The overarching research goals of the tissue engineering industry are highly intertwined with the field of medicine. The tissue engineering field seeks to create a variety of technologies that can be used in a clinical setting for tissue repair therapy. These technologies include everything from cell-based trophic factor therapies to engineered tissue grafts, and potentially engineered tissues. At the forefront of tissue engineering research is the extensive use of adult stem cells. Adult stem cells are located throughout the body and have the ability to differentiate into a variety of different tissue types. This plasticity enables adult stem cells to be used in a wide range of tissue engineering research. Despite all of the advancements made in the tissue engineering field, some obstacles still need to be overcome before tissue engineering can be used extensively as a clinical therapy for tissue damage and repair. One of the largest obstacles is the creation of tissue engineering technologies that mimic the existing human body conditions as close as possible. The creation of fully biomimetic tissue engineering technologies will increase the quality and functionality of the engineered grafts and therapies. The research presented will discuss the development and implementation of a biomimetic perfusion bioreactor. The reactor was designed to incorporate a number of environmental factors that play a large role in controlling cellular behaviors into a culture system capable of supporting the growth of a human mesenchymal stem cell construct. Furthermore, the reactor was constructed to allow the tissue construct to be studied in a non-invasive manner. This characteristic provides a way to keep the integrity of the cell construct throughout the experimentation process so it remains functional and viable until it is potentially needed for tissue therapy.
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Date Issued: 2009
Identifier: FSU_migr_etd-2986
Format: Thesis

Title: Increasing the Yield of Thy-1 EGFP Expression in Insect and Mammalian Cells.
Creator: Kasprzak, Agnieszka, Logan, Timothy M., Li, Hong, Dorsey, John, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: Thy-1 is being pursued as a model system for NMR studies of structure and dynamics of intact glycoproteins. However, the low expression yield of the Thy-1 EGFP glycoprotein in insect and mammalian cells has been an obstacle for these studies. The objective of the following studies is to increase the yield of purified Thy-1 EGFP with correct post translational modifications and homogeneity in the three glycosylation sites. To improve the yield in the baculovirus based insect cell expression...
Show moreThy-1 is being pursued as a model system for NMR studies of structure and dynamics of intact glycoproteins. However, the low expression yield of the Thy-1 EGFP glycoprotein in insect and mammalian cells has been an obstacle for these studies. The objective of the following studies is to increase the yield of purified Thy-1 EGFP with correct post translational modifications and homogeneity in the three glycosylation sites. To improve the yield in the baculovirus based insect cell expression systems we investigated the effect of viral titer, multiplicity of infection (MOI), cell density, and length of expression. The MOI and cell density need to be optimized to give the highest yield of recombinant protein. It was found that the titer of a virus needs to be at least 1X108 pfu/ml and that more than two amplifications decrease both titer and expression levels. The optimal MOI used in for virus amplification was found to be between 0.01 to 0.05, while for expression, the optimal MOI is between 1-5. Optimal cell density for amplification of virus is 2.5 X106 cells/ml, while expression cell density is optimal at 3.0 X106 cells/ml. Optimized time of expressing Thy-1 EGFP is 72 hours post infection. Using these optimized conditions, the maximum yield of Thy-1 was 500 μg per liter of culture. Mammalian cell expression in Lec-1 cells provides a more homogenous glycosylation pattern on Thy-1 EGFP than observed from wild type CHO cells. In lec-1 cells the factors investigated to optimize the expression yield were media volume, time of expression and cell confluency. The optimal conditions for expression were found to be 48 hours in 50 mls of serum free media with 2mM butyric acid and 20mM HEPES. The cell confluency should be 100% before initiating expression. Using these optimized conditions the maximum protein yield was 2 mg of Thy-1 per liter of expression media. In a final set of experiments we sought to inhibit the activity of proteases that co-purify with Thy-1 through most of the purification steps. We screened for the effect of protease inhibitors such as E-64, leupeptin, aprotinin, and PMSF to minimize the degradation during purification. Protease activity was analyzed via zymogram assay and PMSF yielded the highest inhibition at a 0.5 mM concentration. The other protease inhibitors were less or completely ineffective.
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Date Issued: 2005
Identifier: FSU_migr_etd-3327
Format: Thesis

Title: Identifying Weak and Strong Binding States of Myosin V on F-Actin in the ADP·Pi Condition.
Creator: Dai, Aguang, Taylor, Kenneth A., Wang, Xiaoqiang, Li, Hong, Keller, Thomas C. S., Stagg, Scott, Program in Molecular Biophysics, Florida State University
Abstract/Description: Myosin V, as a member of the myosin family, can "walk" along F-actin to transport many "cargos", such as mRNA and secretary vesicles, to its destination. This study is part of a research project that aims to understand the interaction between myosin V with F-actin through direct visualization of biochemical states of myosin V that bind weakly to F-actin and are thus conformationally heterogeneous. Because of its processivity, it's a good model to illustrate how myosin interacts with F-actin...
Show moreMyosin V, as a member of the myosin family, can "walk" along F-actin to transport many "cargos", such as mRNA and secretary vesicles, to its destination. This study is part of a research project that aims to understand the interaction between myosin V with F-actin through direct visualization of biochemical states of myosin V that bind weakly to F-actin and are thus conformationally heterogeneous. Because of its processivity, it's a good model to illustrate how myosin interacts with F-actin to fulfill its function (Chapter 1). Specimens of full-length myosin V with F-actin were cryogenically vitrified to maintain the close-to-nature conformation. ADP·Pi was added into the solution to keep myosin V in the inhibited state in which the heads are folded back onto the cargo binding domain thereby preventing both heads from binding to a single actin filament at the same time. Electron Tomography rather than single-particle method was used because the relatively low affinity of inhibited myosin V to F-actin which makes the decoration far away from saturation. With subsequent sub-volume processing, ET can provide molecular-level information for relatively heterogeneous and/or sparse macromolecule complexes (Chapter 2). New strategies and methods for processing tomographic sub-volumes with heterogeneity were designed to extract homogenous and meaningful class-averages. F-actin repeats with or without myosin V decoration were extracted to do the sub-volume averaging. Multivariate Data Analysis (MDA) and cluster analysis were used to deal with the heterogeneity issue. More homogeneous repeats were clustered into the same classes and corresponding class-averages were generated to improve signal-to-noise ratio (SNR). Repeats with myosin V decoration were later identified and grouped together to get the conformational information. Focused classification was used to further separate different conformations of the bound myosin V (Chapter 3). The new data processing methods present much conformational information of inhibited myosin V on F-actin. The enumeration of bound myosin V shows that inhibited myosin V mainly is bound to F-actin with only one head. Analysis of the binding angle of the lever-arm with respect to the F-actin filament shows the lever-arm angle of most bound myosin V is not close to 90º, which means that most bound myosin might be not in the transition state. This could be due to Pi release after myosin V binds to F-actin such that Myosin V is in the rigor-like state, even though ADP·Pi is present in the solution. However, for some bound myosin V, the lever-arm angle is really close to 90º, which means the lever-arm is in the "up" position or that the myosin head is binding to actin weakly in a previously unidentified orientation. One explanation is that even though the binding to F-actin could accelerate the release of Pi, some bound myosin V might be still frozen with ADP·Pi during fast-freezing. In addition, copies of double-head bound myosin V were found. The existence of double-head binding requires that the two heads be bound to actin in different conformations; this could be because of random Brownian motion of the second head and subsequent collision. Further quasi-atomic models were built and docked into class-averages to determine the possible nucleotide condition and provide structural information beyond the resolution of the density map. The improved procedure, with quantitative analysis and simulated data verification provides integrated and detailed structural information of inhibited myosin V on F-actin (Chapter 4). Here we identified and characterized the conformation of inhibited myosin V bound to the F-actin filament. We found most inhibited myosin V bound to F-actin with one head. Also we found the lever-arm of most myosin V could be in a rigor-like position rather than the transition position. In addition to single head binding, we found some copies of double-head binding, one head in rigor-like binding state and the other in the transition state. It's the first time to obtain the molecular-level structural information of inhibited myosin V on F-actin, which could fix the missing structural gap for the myosin V ATPase cycle, thus helping us better understand the mechanism of myosin (Chapter 4).
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Date Issued: 2014
Identifier: FSU_migr_etd-8969
Format: Thesis

Title: Biochemical and Structural Characterization of Bacterial Effector Complexes in Viral Defense.
Creator: Ramia, Nancy, Li, Hong, Hurt, Myra, Stagg, Scott, Keller, Thomas C. S., Zhu, Fanxiu, Program in Molecular Biophysics, Florida State University
Abstract/Description: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) offer an adaptive immune system that protects bacteria and archaea from nucleic acid invaders through an RNA-mediated nucleic acid cleavage mechanism. Our knowledge of CRISPR nucleic acid cleavage mechanisms is limited to three examples of widely different ribonucleoprotein particles that target either DNA or RNA. CRISPR Type I and II silencing complexes have been well studied and shown to target DNA using via a complex named...
Show moreClustered Regularly Interspaced Short Palindromic Repeats (CRISPR) offer an adaptive immune system that protects bacteria and archaea from nucleic acid invaders through an RNA-mediated nucleic acid cleavage mechanism. Our knowledge of CRISPR nucleic acid cleavage mechanisms is limited to three examples of widely different ribonucleoprotein particles that target either DNA or RNA. CRISPR Type I and II silencing complexes have been well studied and shown to target DNA using via a complex named Cascade and the single protein Cas9 respectively. Type III systems can target both DNA and RNA but the mechanism is still not well understood. The work presented here focuses on the silencing complexes in Type III CRISPR systems and shed some light on the activity of the largest subunit of these complexes. Staphylococcus epidermidis belongs to the Type III-A CRISPR system and has been shown to interfere with invading DNA in vivo. The Type III-A CRISPR system is characterized by the presence of Csm1, a Cas10 family of proteins member, that has a permuted histidine-aspartate (HD) and a nucleotidyl cyclase-like domain, both of which contain sequence features characteristic of nucleases. In chapter 2, we show in vitro that a recombinant S. epidermidis Csm1 cleaves single-stranded DNA exonucleolytically in the 3'-5' direction and in a divalent-metal dependent manner. We further showed that its DNA cleavage activity resides in the GGDD motif of the cyclase-like domain rather than the HD domain. Our data suggest that Csm1 might work in the context of an effector complex to degrade invading DNA. Type III-B Cmr complex from Pyrococcus furiosis has been shown to target RNA. Cmr2 is the largest subunit of the complex, and belongs to the Cas10 family; its domains organization is comparable to Csm1. In chapter 3, we present a structural and functional study of Cmr2 showing that Cmr2 is not the catalytic site of the Cmr complex for the RNA-guided RNA cleavage. However, exciting results of a DNA cleavage activity by Cmr2 suggest a possible dual silencing of DNA and RNA by type III-B systems. Taken in total, the work presented in this dissertation provides insights into the silencing mechanism of the effector complexes in viral defense and highlights the role of their largest subunit, the signature protein Cas10, in type III CRISPR systems.
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Date Issued: 2013
Identifier: FSU_migr_etd-7983
Format: Thesis

Title: Structure and Function of the Cmr Ribonucleoprotein Complex in Crispr RNA-Mediated RNA Cleavage.
Creator: Cocozaki, Alexis, Li, Hong, Travis, Joseph, Stagg, Scott, Horabin, Jamila, Deng, Wu-min, Program in Molecular Biophysics, Florida State University
Abstract/Description: The Clustered Regularly Interspaced Short Palindromic Repeat loci found in most archaea and some bacteria contain DNA sequences (spacers) that originate from genetic invaders like viruses, transposons, and plasmids. The CRISPR clusters are transcribed into RNA and then processed into short guide CRISPR RNAs (crRNA) that are incorporated into ribonucleoprotein complexes to recognize invaders through complementary base-pairing. The Cmr complex of Pyrococcus furiosus, is an example of a...
Show moreThe Clustered Regularly Interspaced Short Palindromic Repeat loci found in most archaea and some bacteria contain DNA sequences (spacers) that originate from genetic invaders like viruses, transposons, and plasmids. The CRISPR clusters are transcribed into RNA and then processed into short guide CRISPR RNAs (crRNA) that are incorporated into ribonucleoprotein complexes to recognize invaders through complementary base-pairing. The Cmr complex of Pyrococcus furiosus, is an example of a ribonucleoprotein effector complex that uses crRNAs to recognize and cleave target RNA. This complex is composed of six subunits Cmr1-6 that can use diverse crRNAs of 39nt or 45nt lengths to recognize and destroy diverse target RNA sequences. The RNA cleavage activity of the Cmr complex follows a ruler mechanism by which the cleavage occurs at the 14th nucleotide form the 3' end of the crRNA in a metal dependent manner. Although the biological function of the Cmr complex is now well understood, the mechanistic details of its activity such as how the complex assembles and the roles of the different subunits, particularly the identity of the catalytic site, remain unknown. This work uses biochemical assays of RNA cleavage, in-vitro assembly studies, and structural studies to address those questions. I first addressed the role of the largest subunit of the Cmr complex Cmr2 alone and in complex with another subunit, Cmr3. Initial predictions suggested that Cmr2 may harbor the active site of the complex. However, through structural and mutagenesis studies I showed that Cmr2 does not play a direct role in the RNA-cleavage catalysis of the Cmr complex. The interaction between Cmr2 and Cmr3 results in a highly positively charged region between the two proteins that contains the nucleotide-binding site of Cmr2, as determined by solving the structure of Cmr2-Cmr3. Although Cmr3 contains multiple conserved structural elements and potentially catalytic residues, mutagenesis showed that it does not play a role in cleaving target RNAs either. To further characterize the assembly of the Cmr complex and determine the roles of its subunits, I worked in collaboration with Michael Spilman from the Scott Stagg laboratory. We obtained a low-resolution structure of the crRNA- and target RNA-bound Cmr1-6 complex. The results show that the complex has a helical architecture comprised of a Cmr2-3 foot, a Cmr4-5 twisted ladder composed of 3 Cmr4-5 steps, and a Cmr6-Cmr1 head. The crRNA-target RNA duplex binds vertically along the length of the Cmr complex. The results corroborated the previous findings on the roles of Cmr2 and Cmr3 as they showed that the two proteins are involved in specific crRNA binding. While the nature of the complex's active site remains elusive, the results provide structural support for the ruler mechanism of catalysis of the Cmr complex. The helical architecture of the complex revealed an unexpected similarity to the type I CRISPR effector complexes and suggested potential functional and structural similarities among all CRISPR effector complexes.
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Date Issued: 2013
Identifier: FSU_migr_etd-7753
Format: Thesis

Title: Structural Mechanism for Ordered Assembly of Box C/D Small Ribonucleoprotein Particles.
Creator: Brown, Karen Raquel, Li, Hong, Greenbaum, Nancy L., Safron, Sanford A., Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: Box C/D RNAs are non-coding small RNAs that guide methylation and processing of ribosomal RNAs. All snoRNAs are associated with specific proteins in snoRNP particles. The four core proteins identified in yeast are Nop1p (nucleolar protein 1 or Fibrillarin), Nop58p (also known as Nop5p), Nop56p, and Snu13p (small nuclear protein 13). In the archaeal organism Archaeoglobus fulgidus (AF), these four proteins correspond to AF Fibrillarin, AF Nop5p(homologue of Nop56p/Nop58p), and AF L7Ae....
Show moreBox C/D RNAs are non-coding small RNAs that guide methylation and processing of ribosomal RNAs. All snoRNAs are associated with specific proteins in snoRNP particles. The four core proteins identified in yeast are Nop1p (nucleolar protein 1 or Fibrillarin), Nop58p (also known as Nop5p), Nop56p, and Snu13p (small nuclear protein 13). In the archaeal organism Archaeoglobus fulgidus (AF), these four proteins correspond to AF Fibrillarin, AF Nop5p(homologue of Nop56p/Nop58p), and AF L7Ae. Previous biochemical and crystallographic studies have identified the network and the order of interactions among the four components however, no insight has been obtained on structural changes that take place during the step-wise assembly of the particles. Steady-state fluorescence resonance energy transfer (FRET) was used to monitor the conformational changes of a model box C/D RNA during sRNP assembly and substrate binding. Significant RNA conformational changes were observed at each assembly step that was dependent on structural integrities of both the proteins and the box C/D RNA. Steady-state FRET experiments show direct evidence that the kink-turn structure formed by the box C/D RNA is induced by the binding of L7Ae. The conformational change due to the binding of a Nop5p/Fibrillarin complex requires dimerization of the complex, as binding of the double mutant Nop5p/Fibrillarin complex at a 100:1 protein:RNA molar ratio failed to display the same level of decrease in the FRET efficiency as observed for the wild-type protein. Steady-state FRET results on the assembly of sRNPs using the box C/D (A/C mutant) RNA show a minimal disruption on the RNA conformational change upon Nop5p/Fibrillarin binding, despite the fact that L7Ae binds to only one box C/D motif. These protein-induced RNA conformational changes explain the obligated order of assembly of the box C/D sRNP particles and support an "induced-fit" model for box C/D sRNP assembly. These findings also raised a testable hypothesis that the box C/D RNA conformation stabilized by bound proteins facilitates specific recognition of the target RNA.
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Date Issued: 2005
Identifier: FSU_migr_etd-2951
Format: Thesis

Title: Determination and Application of the Proton Chemical Shift Tensor by Solid State Nuclear Magnetic Resonance Spectroscopy.
Creator: Truong, Milton L., Cross, Timothy A., Bertram, Richard, Brüschweiler, Rafael, Li, Hong, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: Orientational restraints resulting from a wide range of anisotropic nuclear spin interactions such as chemical shifts, heteronuclear dipolar interactions, and quadrupolar interactions, have been widely used for determining the structures of peptides and proteins from aligned samples. The aligned samples have a unique orientation with respect to the magnetic field axis, such that the information from the orientation dependent nuclear spin interactions within a peptide plane allows for the...
Show moreOrientational restraints resulting from a wide range of anisotropic nuclear spin interactions such as chemical shifts, heteronuclear dipolar interactions, and quadrupolar interactions, have been widely used for determining the structures of peptides and proteins from aligned samples. The aligned samples have a unique orientation with respect to the magnetic field axis, such that the information from the orientation dependent nuclear spin interactions within a peptide plane allows for the characterization of the peptide plane orientation with respect to the alignment axis. By obtaining the orientations of all the peptide planes with respect to the same alignment axis, a model can be generated for the three-dimensional backbone conformation. Proton (1H) chemical shifts from solid state nuclear magnetic resonance (NMR) have been underutilized as an orientational restraint for backbone conformations in peptides and proteins. This is largely due to discrepancies in the model for the chemical shift tensors. A two dimension heteronuclear correlation (HETCOR) sequence was developed to obtain accurate chemical shift tensors to form a useable model of the orientations of the principal axes of the amide 1H chemical shift. Further improvements to HETCOR were made to form the two dimensional pulse sequences, WIM-HETCOR and WIM-HETCOR Echo. Both WIM-HETCOR and WIM-HETCOR Echo pulse sequences incorporate a windowless isotropic mixing (WIM) sequence for cross polarization. WIM-HETCOR Echo was used to obtain the amide 1H CSA tensor element magnitudes for a representative dipeptide, alanyl-15N-leucine (AL). The orientation of the amide 1H CSA tensor was obtained by comparing simulated chemical shift correlation spectra with the experimental WIM-HETCOR Echo spectra. The variability and application of the 1H CSA tensor was explored using membrane peptides: gramicidin A and piscidin 1.
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Date Issued: 2010
Identifier: FSU_migr_etd-1507
Format: Thesis

Title: Functional and Structural Study of CRISPR Processing Endonuclease Cas6.
Creator: Wang, Ruiying, Li, Hong, Epstein, Lloyd M., Miller, Brian G., Roper, Michael, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) interference is a newly discovered small-RNA based immune system used by bacteria and archaea. One of the essential steps in this interference is to process long CRISPR RNAs into small CRISPR RNAs (crRNAs). Cas6 has been found to perform the CRISPR RNA processing endonuclease activity in archaea, and our goal is to study the molecular basis of Cas6's function as the endonuclease to bind and cleave the repeat RNAs. The work...
Show moreCRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) interference is a newly discovered small-RNA based immune system used by bacteria and archaea. One of the essential steps in this interference is to process long CRISPR RNAs into small CRISPR RNAs (crRNAs). Cas6 has been found to perform the CRISPR RNA processing endonuclease activity in archaea, and our goal is to study the molecular basis of Cas6's function as the endonuclease to bind and cleave the repeat RNAs. The work described in this manuscript mostly focuses on study of the structures of Cas6 protein and Cas6-RNA complexes. The main objectives of this dissertation have been outlined. In chapter 2, our goal is to understand the CRISPR processing and the enzyme involved in this small RNA generating process. A high resolution crystal structure of free Cas6 from Pyrococcus furiosus has been obtained. This structure, together with the cleavage and binding activity assays, reveals the characteristics of this endoribonuclease. In chapter 3, we want to understand the molecular mechanism of the binding and cleavage activity of Cas6, the complex structure of Cas6 and its substrate RNA was solved. Our findings suggest that Cas6 processes CRISPR RNA by a beads-on-a-string model. And Cas6 is a single-stranded RNA specific endonuclease. In chapter 4, we apply the crystallization on various repeat RNAs and Cas6 protein's non-catalytic homologue (Cas6nc) to understand how RAMP proteins, the most abundant family of proteins involved in CRISPR interference, interact with various CRISPR repeat RNAs. The structure and the complementary biochemical analysis suggest a wrap-around model of RAMP-RNA interactions, and it also sheds light on how RAMP proteins are able to specifically interact with different repeat RNA substrates.
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Date Issued: 2010
Identifier: FSU_migr_etd-7248
Format: Thesis

Title: Metabolic and Functional Plasticity in Bacteria Revealed with Genetic Selections for Triosephosphate Isomerase Activity and Bromoacetate Resistance.
Creator: Desai, Kevin, Miller, Brian, Bass, Hank, Li, Hong, Zhu, Lei, Stroupe, M. Elizabeth, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: Modern protein catalysts are often viewed as possessing exquisite specificities for their cognate physiological substrates. In contrast, primordial catalysts are thought to have possessed much broader substrate specificities, a characteristic that likely afforded the survival of their host organisms under a plethora of diverse environmental conditions. Recent experimental work suggests that present day enzymes often retain the ability to recognize and transform a variety of natural and...
Show moreModern protein catalysts are often viewed as possessing exquisite specificities for their cognate physiological substrates. In contrast, primordial catalysts are thought to have possessed much broader substrate specificities, a characteristic that likely afforded the survival of their host organisms under a plethora of diverse environmental conditions. Recent experimental work suggests that present day enzymes often retain the ability to recognize and transform a variety of natural and unnatural compounds that are structurally distinct from their target substrate. The widespread existence of such promiscuity could prove generally useful both in the natural and directed evolution of new proteins. To probe the persistence of enzyme promiscuity in modern proteomes we studied the model organism Escherichia coli due to its rapid growth, ease of genetic manipulation and many years of prior research on this organism which have generated abundant knowledge on its metabolism. The first exploration into uncovering enzyme promiscuity, described in chapter two, examines the proton transfer reaction catalyzed by triosephosphate isomerase (TIM). Triosephosphate isomerase catalyzes the interconversion of D-glyceraldehyde 3-phosphate and dihydroxyacetone phosphate, an essential step in glycolytic and gluconeogenic metabolism. To uncover promiscuous isomerases embedded within the E. coli genome, we searched for genes capable of restoring growth of a TIM-deficient bacterium under gluconeogenic conditions. Rather than discovering an isomerase, we selected yghZ, a gene encoding for a member of the aldo-keto reductase superfamily. Here we show that YghZ catalyzes the stereospecific, NADPH-dependent reduction of L-glyceraldehyde 3-phosphate, the enantiomer of the TIM substrate. This transformation provides an alternate pathway to the formation of dihydroxyacetone phosphate. In chapter three we show that Gpr co-purifies with a b-type heme cofactor. Gpr associates with heme in a 1:1 stoichiometry to form a complex that is characterized by a Kd value of 5.8 ± 0.2 µM in the absence of NADPH and a Kd value of 11 ± 1.3 µM in the presence of saturating NADPH. The absorbance spectrum of reconstituted Gpr indicates that heme is bound in a hexacoordinate low-spin state under both oxidizing and reducing conditions. The physiological function of heme association with Gpr is unclear, as the L-glyceraldehyde 3-phosphate reductase activity of Gpr does not require the presence of the cofactor. Bioinformatics analysis reveals that Gpr clusters with a family of putative monooxygenases in several organisms, suggesting that Gpr may act as a heme-dependent monooxygenase. The discovery that Gpr associates with heme is interesting because Gpr shares 35% amino acid identity with the mammalian voltage-gated K+ channel β-subunit, an NADPH-dependent oxidoreductase that endows certain voltage-gated K+ channels with hemoprotein-like, O2-sensing properties. To date the molecular origin of O2 sensing by voltage-gated K+ channels is unknown and the results presented herein suggest a role for heme in this process. In chapter four we probe the network of genes within E. coli that can provide resistance to the nonnatural toxin bromoacetate. Microbial niches contain toxic chemicals that are capable of forcing organisms into periods of intense natural selection to afford survival. Elucidating the mechanisms by which microbes evade environmental threats has direct relevance for understanding and combating the rise of antibiotic resistance. In this study we used a toxic small-molecule, bromoacetate, to model the selective pressures imposed by antibiotics and anthropogenic toxins. We report the results of genetic selection experiments that identify nine genes from Escherichia coli whose overexpression affords survival following exposure to a lethal concentration of bromoacetate. Eight of these genes encode putative transporters or transmembrane proteins, while one encodes the essential peptidoglycan biosynthetic enzyme, UDP-N-acetylglucosamine enolpyruvoyl transferase (MurA). Biochemical studies demonstrate that the primary physiological target of bromoacetate is MurA, which becomes irreversibly inactivated via alkylation of a critical active-site cysteine. Genetic experiments also identify 63 single-gene mutants of E. coli that display increased susceptibility to bromoacetate. One hypersensitive bacterium lacks yliJ, a gene that encodes a glutathione transferase capable of catalyzing the detoxification of bromoacetate with a kcat/Km value of 5.4 × 103 M-1 s-1. The catalytic proficiency of YliJ, which exceeds 5 orders of magnitude, is particularly noteworthy considering the enzyme is unlikely to have previously encountered bromoacetate. In total, our results indicate that nearly 2% of the E. coli proteome contributes to, or can be recruited to provide, bromoacetate resistance. This illustrates the wealth of intrinsic survival mechanisms that can be exploited by bacteria when they are challenged with toxins. The work described here illuminates the vast metabolic and functional plasticity of protein function harbored within bacteria. Their ability to recruit latent and weakly active proteins for novel functions enables survival under diverse nutritional and environmental challenges.
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Date Issued: 2010
Identifier: FSU_migr_etd-4608
Format: Thesis

Title: Predictive Sampling of Protein Conformational Changes.
Creator: Li, Xubin, Yang, Wei, Taylor, Kenneth A., Steinbock, Oliver, Li, Hong, Cross, Timothy A., Florida State University, College of Arts and Sciences, Department of Chemistry and...
Show moreLi, Xubin, Yang, Wei, Taylor, Kenneth A., Steinbock, Oliver, Li, Hong, Cross, Timothy A., Florida State University, College of Arts and Sciences, Department of Chemistry and Biochemistry
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Abstract/Description: In aqueous solution, solute conformational transitions are governed by intimate interplays of the fluctuations of solute–solute, solute–water, and water–water interactions. To more effectively sample conformational transitions in aqueous solution, we devised a predictive sampling method: the generalized orthogonal space tempering (gOST) algorithm. Specifically, in the Hamiltonian perturbation part, a solvent-accessible-surface-area-dependent term is introduced to implicitly perturb near...
Show moreIn aqueous solution, solute conformational transitions are governed by intimate interplays of the fluctuations of solute–solute, solute–water, and water–water interactions. To more effectively sample conformational transitions in aqueous solution, we devised a predictive sampling method: the generalized orthogonal space tempering (gOST) algorithm. Specifically, in the Hamiltonian perturbation part, a solvent-accessible-surface-area-dependent term is introduced to implicitly perturb near-solute water–water fluctuations; more importantly in the orthogonal space response part, the generalized force order parameter is generalized as a two-dimension order parameter set, in which essential solute–solvent and solute–solute components are separately treated. The gOST algorithm is evaluated through a molecular dynamics simulation study on the explicitly solvated deca-alanine peptide. On the basis of a fully automated sampling protocol, the gOST simulation enabled repetitive folding and unfolding of the solvated peptide within a single continuous trajectory and allowed for detailed constructions of deca-alanine folding/unfolding free energy surfaces. In addition, by employing the gOST method we enabled efficient molecular dynamics simulation of repetitive breaking and reforming of salt bridge structures within a minimalist salt-bridge model, the Asp-Arg dipeptide and thereby were able to map its detailed free energy landscape in aqueous solution. Our results reveal the critical role of local solvent structures in modulating salt-bridge partner interactions and imply the importance of water fluctuations on conformational dynamics that involves solvent accessible salt bridge formations. Based on the gOST method, we have developed a solvation force orthogonal space tempering (SFOST) algorithm, in which several major changes were made from the original gOST method. Due to compensating fluctuations of essential solute-solvent and solute-solute interactions, only essential solute-solvent interactions are perturbed in the SFOST algorithm. Importantly, the above treatment enabled us to incorporate a high order orthogonal space sampling strategy. Specifically, to enlarge fluctuations of essential solute-solvent interactions, a third order treatment was introduced to accelerate the coupled responses caused by fluctuations of essential solute-solvent interactions, which come from synchronous fluctuations of essential solute-solute interactions and solvent-solvent interactions. The SFOST algorithm was evaluated through a molecular dynamics simulation study on the explicitly solvated deca-alanine peptide. More importantly, the SFOST simulation explicitly revealed the compensating fluctuations between the essential solute-solvent interactions and the solvent-solvent interactions, suggesting that solvent cooperative fluctuations intimately interplay with deca-alanine conformational transitions. In addition, the SFOST algorithm was also employed to study ion conduction through gramicidin A (gA). By enlarging fluctuations of the ion-environment interactions, the SFOST simulation enabled several round trips of ion permeation through the channel and allowed detailed construction of free energy surfaces along the conduction. The calculated observables agree very well with experiment. We also found that fluctuations of channel orientations play an essential role in ion conduction. Furthermore, by employing the SFOST algorithm we enabled predictive sampling of the conformational ensemble of the p53 transcriptional activation domain 1 (TAD1). Strikingly, a helical structure resembling the MDM2-bound form was found in our SFOST simulation, indicating the pre-existing nature of the structure. Detailed studies of free energy surfaces revealed that the most popular state is not a fully disordered form but a partially helical state. Upon binding to MDM2, the hydrophobic interactions at the interface shift the conformational equilibrium to favor the total helical structure. In addition to the predictive sampling methods, we developed a Gaussian kernel Monte Carlo (GKMC) method to smoothly approximate multidimensional free energy surfaces of biomolecular processes. By taking a discrete probability distribution of sampled collective variables as an input, a biased Monte Carlo simulation is performed to efficiently resample the distribution in the collective variable space, leading to a smooth analytical estimate of the free energy surface. The GKMC method is evaluated by resampling data of a generalized orthogonal space tempering simulation of deca-alanine peptide, aiming to construct smooth one-dimensional and two-dimensional free energy surfaces along certain collective variables. As demonstrated in these model studies, the GKMC method can robustly construct smooth multidimensional free energy surfaces with super resolutions, which preserve probability distributions of target molecular processes. Constructing smooth free energy surfaces plays a vital role in interpreting simulation data to understand molecular processes of interest.
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Date Issued: 2016
Identifier: FSU_FA2016_Li_fsu_0071E_13616
Format: Thesis

Title: Cleavage Of The Sun-domain Protein Mps3 At Its N-terminus Regulates Centrosome Disjunction In Budding Yeast Meiosis.
Creator: Li, Ping, Jin, Hui, Koch, Bailey A., Abblett, Rebecca L., Han, Xuemei, Yates, John R., Yu, Hong-Guo
Abstract/Description: Centrosomes organize microtubules and are essential for spindle formation and chromosome segregation during cell division. Duplicated centrosomes are physically linked, but how this linkage is dissolved remains unclear. Yeast centrosomes are tethered by a nuclear-envelope-attached structure called the half-bridge, whose components have mammalian homologues. We report here that cleavage of the half-bridge protein Mps3 promotes accurate centrosome disjunction in budding yeast. Mps3 is a single...
Show moreCentrosomes organize microtubules and are essential for spindle formation and chromosome segregation during cell division. Duplicated centrosomes are physically linked, but how this linkage is dissolved remains unclear. Yeast centrosomes are tethered by a nuclear-envelope-attached structure called the half-bridge, whose components have mammalian homologues. We report here that cleavage of the half-bridge protein Mps3 promotes accurate centrosome disjunction in budding yeast. Mps3 is a single-pass SUN-domain protein anchored at the inner nuclear membrane and concentrated at the nuclear side of the half-bridge. Using the unique feature in yeast meiosis that centrosomes are linked for hours before their separation, we have revealed that Mps3 is cleaved at its nucleus-localized Nterminal domain, the process of which is regulated by its phosphorylation at serine 70. Cleavage of Mps3 takes place at the yeast centrosome and requires proteasome activity. We show that noncleavable Mps3 (Mps3-nc) inhibits centrosome separation during yeast meiosis. In addition, overexpression of mps3-nc in vegetative yeast cells also inhibits centrosome separation and is lethal. Our findings provide a genetic mechanism for the regulation of SUN-domain protein-mediated activities, including centrosome separation, by irreversible protein cleavage at the nuclear periphery.
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Date Issued: 2017-06
Identifier: FSU_libsubv1_wos_000404512600017, 10.1371/journal.pgen.1006830
Format: Citation

Title: Studies on Human Glucokinase Regulation.
Creator: Martinez, Juliana Andrea, Miller, Brian G., Ellington, W. Ross, Logan, Timothy M., Li, Hong, Florida State University, College of Arts and Sciences, Department of Chemistry and...
Show moreMartinez, Juliana Andrea, Miller, Brian G., Ellington, W. Ross, Logan, Timothy M., Li, Hong, Florida State University, College of Arts and Sciences, Department of Chemistry and Biochemistry
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Abstract/Description: Over the last decades, the enzyme Glucokinase (GCK) has interested biochemists due to its physiological and kinetic characteristics. GCK is vital for glucose homeostasis, as it controls the levels of insulin and glycogen synthesis in the human body by catalyzing the phosphorylation of glucose. GCK displays a positive kinetic cooperativity towards glucose despite being a monomeric enzyme with only one binding site for this substrate. Previous crystallography and NMR experiments showed that...
Show moreOver the last decades, the enzyme Glucokinase (GCK) has interested biochemists due to its physiological and kinetic characteristics. GCK is vital for glucose homeostasis, as it controls the levels of insulin and glycogen synthesis in the human body by catalyzing the phosphorylation of glucose. GCK displays a positive kinetic cooperativity towards glucose despite being a monomeric enzyme with only one binding site for this substrate. Previous crystallography and NMR experiments showed that cooperativity in GCK originates from order-disorder transitions within the enzyme’s intrinsically disordered small domain that happen in the same timescale as catalysis. A very detailed description of protein structure has been gained from both techniques in the presence of glucose. Unfortunately, the dynamic nature of the unbound GCK state prevents these techniques from revealing its structure. The first two chapters of this dissertation are focused on biochemical and biophysical investigations into the cooperative regulation of GCK. First, we investigated the role of a connecting region between the two protein domains on GCK cooperativity. Using steady state kinetics and equilibrium fluorescence we found the connecting region must possess a minimal length to enable catalytic turnover. Also, we found that the sequence identity of the connecting region is directly linked to GCK cooperativity. Second, a non-natural fluorescent amino acid (fUAA) was site specifically incorporated into a single tryptophan version of GCK to perform Förster resonance energy transfer (FRET) experiments. Steady state FRET, lifetime, and FRET-lifetime measurements on wild-type GCK demonstrate that the enzyme in the unliganded-state exists in at least three uniformly distributed conformations originating from a broad conformational ensemble. A slightly more abundant state is postulated to correspond to the "super open" conformation previously observed in the crystal structure of the unliganded GCK. Upon glucose binding, the conformational ensemble is narrowed to two conformations, one of which is dominant and postulated to be the closed conformation of the enzyme. Similar experiments conducted on GCK variants that activate GCK by abolishing cooperativity, support the hypothesis that these variants function via two distinct mechanisms, one (α-type) that produces a shift in the conformational ensemble to resemble the wild-type glucose bound state, while the other (β-type) does not substantially perturb the conformational ensemble. GCK is specifically regulated in the liver by the Glucokinase Regulatory Protein (GKRP), which binds and inhibits GCK. GKRP inhibition is in turn modulated by sugar phosphates; fructose 1-phosphate weakens the GCK-GKRP binding while sugar 6-phosphates (fructose 6-phosphate or sorbitol 6-phospate) strengthen the binding. As an alternative therapeutic approach to treat diabetes, Amgen has optimized a group of piperazines that bind to GKRP and disrupt the GCK-GKRP complex. Interestingly, the binding sites of sugar phosphates and piperazines are 30Å removed from the GCK-GKRP binding interface. In the third part of this dissertation, we aim to understand the mechanism for the ligand-mediated control of the GCK-GKRP interaction. Using mutagenesis, GCK inhibition assays, stopped-flow and fluorescence techniques we were able to propose a model in which GKRP exists in two conformations with different affinities towards GCK, and GKRP ligands modulate the distribution between the two conformations. The difference in GCK affinity is due to the N-terminus-mediated repositioning of two arginines located at the GCK-GKRP binding interface; in the high-affinity GKRP state, the two arginines are positioned to electrostatically interact with GCK, while in the low-affinity state they are stabilized in different rotameric positions. According to our model, sugar 6-phosphates shifts the GKRP population towards the high-affinity state, while piperazine binding produces the low-affinity state.
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Date Issued: 2016
Identifier: FSU_FA2016_Martinez_fsu_0071E_13576
Format: Thesis

Title: The Structure Study of Myosin Filament, Z-Disc and HIV Envelope Protein.
Creator: Hu, Zhongjun, Taylor, Kenneth A., Liu, Xiuwen, Li, Hong, Chase, P. Bryant, Stagg, Scott M., Florida State University, College of Arts and Sciences, Institute of Molecular...
Show moreHu, Zhongjun, Taylor, Kenneth A., Liu, Xiuwen, Li, Hong, Chase, P. Bryant, Stagg, Scott M., Florida State University, College of Arts and Sciences, Institute of Molecular Biophysics
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Abstract/Description: We describe a cryo-electron microscopy 3D image reconstruction of relaxed myosin II-containing thick filaments from the flight muscle of the giant waterbug Lethocerus indicus. The relaxed thick filament structure is a key element of muscle physiology because it facilitates the re-extension process following contraction. Conversely, the myosin heads must disrupt their relaxed arrangement in order to drive contraction. Previous models predicted that Lethocerus myosin in thick filament was...
Show moreWe describe a cryo-electron microscopy 3D image reconstruction of relaxed myosin II-containing thick filaments from the flight muscle of the giant waterbug Lethocerus indicus. The relaxed thick filament structure is a key element of muscle physiology because it facilitates the re-extension process following contraction. Conversely, the myosin heads must disrupt their relaxed arrangement in order to drive contraction. Previous models predicted that Lethocerus myosin in thick filament was unique in having an intermolecular head-head interaction, as opposed to the intramolecular head-head interaction observed in all other species. In contrast to the predicted model, we find an intramolecular head-head interaction, which is similar to that of other thick filaments but oriented in a distinctly different way. The arrangement of myosin's long α-helical coiled-coil rod domain has been hypothesized as either curved layers or helical sub-filaments. Our reconstruction is the first report having sufficient resolution to track the rod α-helices in their native environment at resolutions ~5.5Å, and shows that for Lethocerus, the layer arrangement is correct. Threading separate paths through the forest of myosin coiled-coils are four non-myosin peptides. We suggest that the unusual position of the heads and the rod arrangement separated by non-myosin peptides is an adaptation for mechanical signal transduction whereby applied tension disrupts the myosin heads as a component of stretch activation (Chapter 2). We improved the map resolution to 4.3Å of relaxed myosin filament and built an atomic model for the entire myosin molecule. In addition to the asymmetry of the heads, the entire coiled-coil lacks symmetry. The coiled-coil is broken in two places, once near the head-rod junction and again at the Skip 4 location. Unique interactions between the regulatory light chain and the myosin rods appear to stabilize the unique orientation of the myosin heads (Chapter 3). The Z-disk is a complex structure comprising over 40 proteins that are involved in the transmission of force developed during muscle contraction and in important signaling pathways that govern muscle homeostasis. In the Z-disk antiparallel thin filaments from adjacent sarcomeres are crosslinked by α-actinin. The structure of the Z-disk lattice varies greatly throughout the animal kingdom. In vertebrates the thin filaments form a tetragonal lattice, whereas invertebrate flight muscle has a hexagonal lattice. A detailed description at a high resolution of the Z-disk lattice is needed in order to better understand muscle function and disease. The molecular architecture of the Z-disk lattice in honey bee (Apis mellifera) is known from plastic embedded thin sections to a resolution of 7 nm, which is not sufficient to adequately dock known protein crystal structures. We have chemically isolated intact honey bee Z-disks from honey bee indirect flight muscle, thus obviating the need of plastic sectioning. We have employed cryo electron tomography and image processing to investigate the arrangement of proteins within the hexagonal lattice of the Apis Z-disk (Chapter 4). HIV envelope protein (Env), which consists of three gp120 and three gp41 molecules, recognizes the CD4 protein in T cells and facilitates fusion of HIV and T cell membranes. Env neutralization is a key step to prevent HIV infection. We investigate the binding effect of PG16 Fab to HIV-MN Env spikes in situ, by using cryo-electron tomography combined with subvolume alignment and classification to identify antibody-bound and antibody-free conformations of Env. By not biasing the spike selection, we find that Env spikes in situ do not have a distinctly symmetric trimeric structure, but can vary between a symmetric trimer and a T-shaped trimer. PG16 Fab changes the equilibrium towards a more symmetric trimer structure despite binding with a stoichiometry of one Fab per Env trimer (Chapter 5).
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Date Issued: 2017
Identifier: FSU_2017SP_Hu_fsu_0071E_13667
Format: Thesis

Title: Molecular Determinants of Recognition Associated with Assembly of the U2-Dependent Eukaryotic Spliceosome and the Self-Splicing ai5γ Group II Intron.
Creator: Popović, Milena, Greenbaum, Nancy L., Logan, Timothy M., Quine, Jack R., Li, Hong, Brüschweiler, Rafael P., Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: RNA-RNA interactions involved in recognition associated with ribozyme catalysis are essential for ribozyme function. Research described in this dissertation focuses on RNA-RNA interactions at, and in the vicinity of two splice sites of two splicing systems: the U2-dependent spliceosome and a yeast mitochondrial group II intron ai5gamma;. In the U2-dependent spliceosomes, an RNA complex is formed by the U2 and U6 snRNAs, creating a network of indispensable helices, which are believed to be the...
Show moreRNA-RNA interactions involved in recognition associated with ribozyme catalysis are essential for ribozyme function. Research described in this dissertation focuses on RNA-RNA interactions at, and in the vicinity of two splice sites of two splicing systems: the U2-dependent spliceosome and a yeast mitochondrial group II intron ai5gamma;. In the U2-dependent spliceosomes, an RNA complex is formed by the U2 and U6 snRNAs, creating a network of indispensable helices, which are believed to be the active components of the spliceosome. Helix III has been shown to be essential in mammals. The goal of our study was to analyze structural evidence for formation of U2-U6 Helix III in an in vitro protein-free system, including the possibility of interaction with the intron strand. The questions addressed were: a) which pairing of the three available strands dominates; b) alternatively, if the three strands form a complex, is there an RNA triple helix in solution? NMR studies of the three strand complex representing the U2-intron-U6 pairing showed formation of the U2-intron duplex and the U2-U6 duplex, but no interaction of the U6 snRNA with the U2-intron duplex in the region of the putative Helix III. NMR studies of the extended Helix III samples corroborate this finding and confirm that the U6 snRNA immediately upstream of the ACAGAGA sequence does not interact with the U2-intron duplex in the region of the proposed Helix III. Group II introns, large ribozymes and mobile genetic elements found in prokaryotes and eukaryotic organelles, share common structural and catalytic features with the spliceosome in eukaryotes. At the functional core of group II introns is the pairing of the EBS1-IBS1 sequences (exon binding sequence one and intron binding sequence one, respectively), which is essential for preserving fidelity of the splice site. The EBS1 guide sequence is a part of an 11-nucleotide loop at the terminus of the ID3 stem loop, which is a subdomain of Domain one (D1), the largest of the group II intron domains. The goal of the latter part of this dissertation was to investigate the structural features of the ID3 stem loop and the ID3-IBS1 complex. We investigated the effects of the ID3 stem loop structure on the EBS1-IBS1 pairing. A question addressed was whether the large 11-nucleotide loop forms a stable structure. We tested whether the EBS1-IBS1 pairing forms in solution and what structural changes the ID3 loop undergoes upon formation of the EBS1-IBS1 pairing. We wanted to determine the effects of ID3 structure on the availability of bases of EBS1 for base pairing and thus for the 5' splice site selection. Solution NMR structure of the ID3 stem loop shows a structured stem and a fairly structured base of the loop, as well as an unstructured or dynamic loop, involving residues of the EBS1 sequence. NMR spectroscopic study of the ID3-IBS1 complex in solution indicates that the unstructured region of the ID3 loop becomes structured upon interaction with the IBS1 sequence, in an apparent induced-fit mechanism, by which both the guide sequence and the target become structured upon interaction. An important observation here is that the double stranded EBS1-IBS1 region ends at the 5' splice site, placing it at the single/double stranded junction, which may play an important role in recognition and/or accessibility of the 5' splice site. The placement of the EBS1 sequence in the specific structural context of the ID3 loop may be an important feature, which aids the recognition of the 5' splice site. We show here that by virtue of being placed within the loop of a certain size, the two potential base pairs downstream of the 5' splice site, which could form in a free duplex, do not form in the context of the loop. These findings are important because we show that positioning of a guide sequence within a loop determines the availability of bases for pairing and controls the extent of base pairing and thus the position of the 5' splice site.
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Date Issued: 2012
Identifier: FSU_migr_etd-5910
Format: Thesis

Title: Electrostatics of Nucleic Acids and Hydration Properties of the Pseudouridin Dependent Spliceosomal Branch Site Helix.
Creator: Xu, Darui, Greenbaum, Nancy L., Fenley, Marcia O., Fenley, Marcia O., Li, Hong, Alabugin, Igor, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: Nucleic acids are the fundamental building blocks of the genetic systems of all living organisms. The structural conformation and function of nucleic acids are determined by the balance among a lot of factors. This work examines two of the key factors: electrostatics and hydration. Because electrostatic interactions are strong and long-ranged in nature, they play an important role in many aspects of the structural and functional properties of polyelectrolytes, including nucleic acids....
Show moreNucleic acids are the fundamental building blocks of the genetic systems of all living organisms. The structural conformation and function of nucleic acids are determined by the balance among a lot of factors. This work examines two of the key factors: electrostatics and hydration. Because electrostatic interactions are strong and long-ranged in nature, they play an important role in many aspects of the structural and functional properties of polyelectrolytes, including nucleic acids. Currently, the most popular approach for analyzing electrostatics of nucleic acids is the continuum model based on the Poisson-Boltzmann equation (PBE), which is typically solved numerically using the finite difference algorithm. In the first part of this work, the electrostatic features of the most common and basic structural form of nucleic acids, the Watson-Crick (WC) DNA double helices in A- and Bconformations, are examined using a newly developed hybrid boundary element and finite difference PBE algorithm ( Boschitsch, A.H. and Fenley, M.O., J. Comput. Chem., 2004, 25:935-55). The hybrid PBE algorithm is an improvement of the current finite difference algorithm and can produce accurate surface electrostatic potentials of nucleic acids in a simulated aqueous salt environment. The high resolution surface electrostatic potential maps and the numerical results obtained by the hybrid PBE solver illustrated many fine and distinct electrostatic features of A- and B-DNA. It shows that the major groove of A-DNA is much more electronegative than the minor groove. The closely packed backbone atoms are mainly responsible for creating the enhanced negativity. The minor groove of B-DNA is more negative than the major groove, and the base atoms contribute to the sequence specific features in both the major and minor groove. DNA with A/B junction has both profoundly negative major and minor grooves. Next, the electrostatic characteristics of the most common non-WC base pair in RNA, the G'U wobble base pairs are studied in details. Results show that the major groove of an isolated G'U wobble base pair has a region of broad and pronounced electronegativiy, compared with WC base pairs. However, when incorporated in RNA helices, the presence of the enhanced electronegativity depends on the stacking pattern and groove geometry of the G'U wobble base pairs. Finally, the hydration patterns of the pseudouridine dependent spliceosomal branch site helix are examined using molecular dynamics simulations with explicit solvent representation. The branch site helix, formed by the base pairing between the pre-mRNA intron and U2 snRNA, is part of the critical RNA:RNA interaction network in the spliceosome. The branch site helix positions the branch site adenosine, the nucleophile in the first step of splicing, for reaction. NMR structures illustrated that the highly conserved pseudouridine in U2 snRNA induces the extrahelical position of the branch site adenosine (Newby, M.I. and Greenbaum N.L., Nat Struct Biol., 2002, 9:958-65). The hydration analysis obtained in this work located two tightly bound water molecules in the pseudouridine dependent branch site helix. One is near the backbone of the conserved pseudouridine, forming water mediated hydrogen bonding between pseudouridine NH1 and the backbone. This bridging interaction presumably will enhance the conformational stability of the helix, and has been identified for pseudouridine in tRNA. The other hydration site is near the branch site adenosine, contributing to the formation of the base tripe, which stabilize the extrahelical position of the branch site adenosine. In comparison, the unmodified counterpart of the branch site helix, where the pseudouridine is repalced with uridine, shows only one hydration site. It is located between the stacked branch site adenosine and its neigboring base, stabilizing the stacked conformation of the branch site adenosine.
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Date Issued: 2007
Identifier: FSU_migr_etd-7080
Format: Thesis

Title: The Influence of Adamalysin 19 and Polyelectrolyte Multilayers on Human Vascular Smooth Muscle Cells.
Creator: Newcomer, Robert Glenn, Sang, Qing-Xiang Amy, Keller, Thomas C. S., Li, Hong, Steinbock, Oliver, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: Under normal physiological conditions, mature human coronary artery smooth muscle cells (hCASMCs) exhibit a 'contractile' phenotype marked by low rates of proliferation, migration, and protein synthesis, but these cells possess the remarkable ability to dedifferentiate into a separate 'synthetic' phenotype when stimulated by conditions of pathologic stress. Therapeutic interventions to relieve blockages in arteries can damage the innermost layer of endothelial cells in treated vessels,...
Show moreUnder normal physiological conditions, mature human coronary artery smooth muscle cells (hCASMCs) exhibit a 'contractile' phenotype marked by low rates of proliferation, migration, and protein synthesis, but these cells possess the remarkable ability to dedifferentiate into a separate 'synthetic' phenotype when stimulated by conditions of pathologic stress. Therapeutic interventions to relieve blockages in arteries can damage the innermost layer of endothelial cells in treated vessels, triggering this pathologic phenotypic transition in hCASMCs, and stent implantation can serve to exacerbate vessel damage. The work presented here describes a two-pronged investigation into hCASMC behavior, examining first the potential contributions of adamalysin-19 (A Disintegrin And Metalloproteinase-19, ADAM19) to hCASMC invasion and migration, and second the effects of a series of thin polyelectrolyte multilayer (PEMU) films in terms of their ability to induce a variety of effects in these cells, including some changes that are consistent with hCASMC phenotypic modulation. Antibody inhibition of the ADAM19 catalytic domain is shown to decrease the invasion of hCASMCs through both type I collagen and fibronectin, and a combination of functionblocking antibodies targeting the '1 integrin and the disintegrin domain of ADAM19 similarly inhibited hCASMC invasion through type I collagen. Separate antibodies targeting either the catalytic domain or the disintegrin domain of ADAM19 each inhibited hCASMC migration in response to monolayer injury. Transient hCASMC transfection with a glutamine to alanine mutation (E346A), deactivating the catalytic activity of ADAM19, again resulted in diminished invasion through type I collagen, but wild type (WT) transfections had no effect on invasion, and neither E346A nor WT transfections affected hCASMC migration. Coimmunopreciptation studies with hCASMC lysates suggest that a processed (56 kDa) form of ADAM19 interacts with a processed (62 kDa) form of the '1 integrin. A variety of thin PEMU films are shown here to exhibit bioactive properties that induce unique responses from cultured hCASMCs. Hydrophobic surfaces terminating with either Nafion or poly(styrenesulfonic acid) (PSS) exhibit changes consistent with contractile or synthetic phenotypes, respectively, while a hydrophilic, zwitterionic copolymer of poly(acrylic acid) (PAA) and 3-[2-(acrylamido)-ethyl dimethylammonio] propane sulfonate (PAA-co-PAEDAPS) suppresses cytoskeletal formation, inhibiting proliferation while also exhibiting a remarkable resistance to cell attachment. Two-dimensional gel electrophoresis (2DE) and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOFMS) revealed differential expression of both heat shock protein 90 (HSP90) and actin on these surfaces. Fluorescent immunocytochemical staining revealed differences in the cellular localization of both HSP90 and several isoforms of actin on different PEMU surfaces, in addition to unique patterns of expression for ADAM19 and the intermediate filament protein vimentin. Western blotting confirmed that the expression of HSP90 was increased on PSS when compared to glass, and that the expression of '-smooth muscle actin ('-SM actin) was diminished on PSS when compared to Nafion. Furthermore, the ionophore A23187 was shown to induce contraction in hCASMC monolayers on Nafion-terminated surfaces. These results demonstrate that novel PEMU culture systems might eventually be tailored to provide simple, reproducible models suitable for the study of pathologic hCASMC phenotypic modulation, and may also suggest potential in vivo applications for implantable devices, while the demonstrated reductions in invasion and migration following ADAM19 inhibition suggest a possible role for this enzyme in a scheme targeting neointima formation. The primary strength of a multilayer coating strategy is the treatment of cells that are most proximal to a lesion, exerting non-systemic effects in the most localized fashion possible. Furthermore, control of the PEMU deposition parameters also affords the opportunity to fine-tune these surfaces, so as to direct or deter the growth and attachment of selected cells, or in the case of hCASMCs, potentially influencing the resultant phenotype of attaching cells.
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Date Issued: 2007
Identifier: FSU_migr_etd-7051
Format: Thesis

Title: Innovating Two-Dimensional Liquid Chromatography.
Creator: Vanmiddlesworth, Bradley J. (Bradley James), Dorsey, John G., Ruse, Michael, Cooper, William T., Roper, Michael G., Li, Hong, Department of Chemistry and Biochemistry, Florida...
Show moreVanmiddlesworth, Bradley J. (Bradley James), Dorsey, John G., Ruse, Michael, Cooper, William T., Roper, Michael G., Li, Hong, Department of Chemistry and Biochemistry, Florida State University
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Abstract/Description: Liquid chromatography is ubiquitous, but two-dimensional liquid chromatography is rare. The difference is in the difficulty of method development, as once floated variables have increased pertinence. Three issues for method development in two-dimensional liquid chromatography have been described previously: 1) To preserve the resolution produced in the first dimension, an eluting analyte peak from the first dimension must be sampled at least four times across the band width. 2) The mobile...
Show moreLiquid chromatography is ubiquitous, but two-dimensional liquid chromatography is rare. The difference is in the difficulty of method development, as once floated variables have increased pertinence. Three issues for method development in two-dimensional liquid chromatography have been described previously: 1) To preserve the resolution produced in the first dimension, an eluting analyte peak from the first dimension must be sampled at least four times across the band width. 2) The mobile phase of the first dimension becomes the injection solvent for the second dimension. 3) Each additional dimension adds further dilution of the analyte band. It is the goal of this research to fundamentally describe and mitigate the hurdles of two-dimensional liquid chromatography while concurrently adding to the overall practice of chromatography. Three investigations have been undertaken. The first describes the elucidation of pressure as the limiting factor in column reequilibration post-gradient for the goal of reducing the time of analysis of a second dimension. The second quantifies the effect of band shape distortion due to injection solvent mismatch with the goal of making predictions for column selection and organic modifier choice. The third is a proof-of-concept for a novel approach to separations where two force vectors are applied simultaneously to produce a two dimensional separation in less time and therefore, less dilution. Taken separately, these three are of general use to fundamental separation science, but collected they are of specific use to reduce the difficulty of method development of two-dimensional liquid chromatography.
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Date Issued: 2011
Identifier: FSU_migr_etd-5244
Format: Thesis

Title: Metabolite Identification by Nuclear Magnetic Resonance Spectroscopy.
Creator: Bingol, Ahmet Kerem, Logan, Timothy M., Brüschweiler, Rafael, Zhou, Huan-Xiang, Li, Hong, Srivastava, Anuj, Program in Molecular Biophysics, Florida State University
Abstract/Description: The metabolic makeup of a biological system is a key determinant of its biological state providing detailed insights into its function. Identification and quantification of the metabolites in a system form critical components of metabolomics. Nuclear magnetic resonance (NMR) spectroscopy is a unique tool for this purpose providing a wealth of atomic-detail information without requiring extensive fractionation of samples. So far, a majority of NMR metabolomics studies have been performed by...
Show moreThe metabolic makeup of a biological system is a key determinant of its biological state providing detailed insights into its function. Identification and quantification of the metabolites in a system form critical components of metabolomics. Nuclear magnetic resonance (NMR) spectroscopy is a unique tool for this purpose providing a wealth of atomic-detail information without requiring extensive fractionation of samples. So far, a majority of NMR metabolomics studies have been performed by using 1D NMR techniques because of the short duration of the experiments. The drawback of 1D NMR is the high occurrence of peak overlaps that impairs metabolite identification and quantification. The use of multidimensional NMR techniques can resolve peak overlaps and provide connectivity information of atoms within molecules, thereby outweighing the longer measurement times. In this thesis, we introduce novel approaches to identify metabolites by using multidimensional NMR spectroscopy. Our main approach consists of two major steps. In the first step, the metabolite mixture is deconvoluted into its individual components and in the second step; each individual component is analyzed by using its NMR spectrum. In order to achieve fast, robust and (semi-)automated deconvolution, DeCoDeC technique is introduced and applied to a variety of 1H and 13C TOCSY based NMR spectra. Deconvoluted TOCSY traces are directly queried in metabolite databanks for identification. Since many metabolites are not present in metabolite databanks, we developed a strategy to extract their carbon backbone structures (topology), which is a prerequiste for de novo structure determination. This led to the determination of 112 topologies of unique metabolites in E. coli from a single sample that constitutes the "topolome" of a cell. The topolome is dominated by carbon topologies of carbohydrates (34.8%) and amino acids (45.5%) that can constitute building blocks of more complex structures. Furthermore, since databanks are designed to query 1D NMR spectrum, querying of TOCSY traces against 1D NMR spectra in databanks resulted in imperfect matches. To overcome this, we created a customized 13C TOCSY database, which substantially improved the accuracy of database query of 13C TOCSY traces. Together these new tools open up the prospect to enable routine yet accurate analysis of an increasingly complex and diverse range of molecular solutions including metabolomics samples.
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Date Issued: 2013
Identifier: FSU_migr_etd-7298
Format: Thesis

Title: Molecular Characterization of Tea Catechin Treated Human Prostate Cancer Cell Lines.
Creator: Suh, Yewseok, Sang, Qing-Xiang Amy, III, Thomas C.S. Keller, Schlenoff, Joseph B., Li, Hong, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: Prostate cancer is the most prevalent cancer diagnosed among men in the United States. The major green tea polyphenol epigallocatechin-3 gallate (EGCG) has been shown to exert remarkable preventive effects against various types of cancer including prostate cancer. Recent human clinical study proved that EGCG can prevent progression of high grade prostatic intraepithelial neoplasia (PIN) to prostate cancer. Cellular studies show that EGCG exhibits antiproliferative and apoptotic effects in...
Show moreProstate cancer is the most prevalent cancer diagnosed among men in the United States. The major green tea polyphenol epigallocatechin-3 gallate (EGCG) has been shown to exert remarkable preventive effects against various types of cancer including prostate cancer. Recent human clinical study proved that EGCG can prevent progression of high grade prostatic intraepithelial neoplasia (PIN) to prostate cancer. Cellular studies show that EGCG exhibits antiproliferative and apoptotic effects in androgen-responsive LNCaP and androgen-unresponsive DU145, PC3 prostate cancer cell lines. Previously, we have established a new type of prostate cancer line, androgen repressed carcinoma of prostate (ARCaP). ARCaP cells are highly invasive and metastatic and this cell line showed unique response to androgen since the hormone repressed the proliferation. In this study, we show that androgen-repressed ARCaP prostate cancer cell line, which represents more advanced and aggressive type of prostate cancer, is resistant to EGCG treatment. In Western blot analyses, EGCG treated ARCaP cell line showed increase in phosphorylation of NF-κB and decrease in activation of p38 MAPK and Bax/Bcl-2 ratio. The levels of p21/CIP1/WAF1, cyclin-dependent kinases (CDKs) 2, 4, 6, activated forms of Akt and c-Jun NH2-terminal protein kinase (JNK) remain unchanged in EGCG treated ARCaP cells whereas decrease in active Akt, active JNK, and CDKs 2, 4, 6, and increased level of p21/CIP1/WAF1 were observed in LNCaP cells upon EGCG treatment. Moreover, EGCG treatment confers stronger adherence to types I, II, IV collagen extracellular matrix proteins on ARCaP cells. On the contrary, LNCaP cells lost the adhesion significantly to all extracellular matrix proteins tested, including collagens, fibronectin, laminin, vitronectin, and tenascin. Most importantly, ARCaP cells formed more colonies on soft agar in our anchorage-independent assay when treated with EGCG whereas the colony forming ability of LNCaP cells was totally abolished under the same condition. This study suggests that the use of tea catechin EGCG as anticancer agent may not be effective for treating patients with androgen repressed subtype of prostate cancer. This is the first study of apoptosis in ARCaP cell line. The GeneChip microarray analysis revealed several genes that were differentially expressed when treated with EGCG. Among those, matrix metalloproteinases (MMPs) 1 and 3 were significantly up regulated in LNCaP cells upon EGCG treatment. Both RNA transcription and protein secretion/activation of these MMPs were observed by GeneChip assay, reverse transcription-polymerase chain reaction (RT-PCR) and by enzyme linked immunosorbent assay (ELISA) which can detect proMMP1 and total MMP3 in cell culture media. This feature is very unique in that (1) the MMPs are generally known to be involved in tumor invasion and metastasis not the cell death, and (2) the other EGCG sensitive prostate cancer cell lines, DU145 and PC3, did not display such characteristics. EGCG did not affect the expression of these MMPs in ARCaP cells also. Using GeneChip analysis, we found several genes whose expressions were oppositely regulated in LNCaP and ARCaP cells upon EGCG treatment. These include early growth response -1 (EGR1), growth arrest and DNA damage inducible gene 45 (GADD45). The expression level of EGR1 and GADD45 were decreased in ARCaP cells but the level was increased in LNCaP cells after EGCG treatment. These results suggest that the proapoptotic EGR1 and GADD45 may play a role in EGCG induced apoptosis in LNCaP cells and thus may explain, at least in part, the resistance of ARCaP cells against such apoptotic stimuli. The role of these proteins in EGCG induced apoptosis is not known. The decreased level of topoisomerase II in EGCG treated LNCaP cells is also exciting. Topoisomerases are necessary in DNA replication and thus for survival of the organism. Since only LNCaP cells, but not ARCaP cells, displayed reduced expression of topoisomerase II during EGCG induced apoptosis and since ARCaP cells underwent apoptosis when treated with topoisomerase inhibitor etoposide, the function of this enzyme might be involved in life or death decision of ARCaP and LNCaP cells. Elucidating the molecular effects of these proteins and the mechanisms of how these proteins function in ARCaP and LNCaP cell lines would help understanding the prostate cancer and may help with future design of cancer chemopreventive and chemotherapeutic agents.
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Date Issued: 2006
Identifier: FSU_migr_etd-0402
Format: Thesis

Title: Isotopic Enrichment and Receptor-Binding Analysis of Insect and Lec1 Cell Expressed Avian Thy-1.
Creator: Walton, Wendy Jean, Logan, Timothy M., Hyson, Richard L., Keller, Thomas C. S., Li, Hong, Marshall, Alan, Program in Molecular Biophysics, Florida State University
Abstract/Description: Over half of the proteins found in nature are anticipated to be glycosylated, yet less than 2% of the protein structures in the protein data bank are glycoproteins. Clearly, we are behind in our structural understanding of glycoproteins. One reason for the lack of structural information for glycoproteins is due to their oligosaccharide heterogeneity. The two main methods for determining detailed three-dimensional protein structures are X-ray crystallography and nuclear magnetic resonance....
Show moreOver half of the proteins found in nature are anticipated to be glycosylated, yet less than 2% of the protein structures in the protein data bank are glycoproteins. Clearly, we are behind in our structural understanding of glycoproteins. One reason for the lack of structural information for glycoproteins is due to their oligosaccharide heterogeneity. The two main methods for determining detailed three-dimensional protein structures are X-ray crystallography and nuclear magnetic resonance. Oligosaccharide heterogeneity hinders crystallization for X-ray crystallography analysis and complicates resonance assignment of nuclear magnetic resonance data. Another challenge is expressing high enough yields from eukaryotic cells for X-ray crystallography and nuclear magnetic resonance analysis. Additionally, unlike bacteria, eukaryotic cell lines typically cannot synthesize amino acids from glucose and ammonium chloride. Thus, eukaryotic media must contain amino acids for cell growth and protein production. Structural analysis of glycoproteins by nuclear magnetic resonance requires that the amino acids are labeled with 13C- and 15N and that the oligosaccharides are labeled with 13C. Media containing 13C- and 15N-labeled amino acids is extremely expensive. This dissertation explains methods that address each of these challenges utilizing the glycoprotein Thy-1. An economic approach for labeling recombinant protein expressed from insect cells for NMR is explained. A method for labeling oligosaccharides on glycoproteins expressed from mammalian cells is also explained. Changes in previously established expression and purification protocols for Thy-1 derived from Lec1 cells, which produce relatively homogeneous glycoforms, have increased yields to levels suitable for nuclear magnetic resonance studies. In addition, the biological relevancy of recombinant Thy-1, which is expressed with truncated oligosaccharides and without it's glycosylphosphatidylinositol anchor, was confirmed by receptor-binding analysis.
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Date Issued: 2006
Identifier: FSU_migr_etd-1272
Format: Thesis

Title: Probing Protein Conformation and Protein-Protein Interaction by Hydrogen Deuterium Exchange Coupled with FT-ICR MS.
Creator: Tao, Yeqing, Marshall, Alan G. (Alan George), Blaber, Michael, Dorsey, John G., Li, Hong, Florida State University, College of Arts and Sciences, Department of Chemistry and...
Show moreTao, Yeqing, Marshall, Alan G. (Alan George), Blaber, Michael, Dorsey, John G., Li, Hong, Florida State University, College of Arts and Sciences, Department of Chemistry and Biochemistry
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Abstract/Description: With the development of ionization techniques such as electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI), mass spectrometry expands its subject of study from small volatile molecules to non-volatile macromolecules, such as proteins, nucleic acids, lipids, and other biomolecules. Advancement in the mass analyzer in terms of mass resolving power and mass accuracy further enables mass spectrometry to study highly complexed biological samples and therefore...
Show moreWith the development of ionization techniques such as electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI), mass spectrometry expands its subject of study from small volatile molecules to non-volatile macromolecules, such as proteins, nucleic acids, lipids, and other biomolecules. Advancement in the mass analyzer in terms of mass resolving power and mass accuracy further enables mass spectrometry to study highly complexed biological samples and therefore becomes an indispensable tool for many biological applications. Among them, structure biology focuses on elucidating the higher order structures of biomacromolecules such as proteins and nucleic acids, because the structures of these molecules are usually closely related to their biological functions. When high-resolution Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) is combined with solution phase hydrogen/deuterium exchange (HDX), it becomes a powerful tool for investigating protein structures and conformational changes induced by protein-protein, or protein-ligand interactions. HDX-MS becomes a powerful technique for structural biology and can complement traditional tools such as X-ray crystallography, NMR, and cryo-EM to answer biological questions. In Chapter 2, back exchange level for our current HDX-MS system is evaluated. Back exchange is the major problem for many HDX-MS systems. This chapter describes the experiments performed to identify the sources of back exchange and to test one possible way to reduce it. Heated metal capillary with different temperatures are tested, and the results show back exchange is not related to the capillary temperature, but rather the length of retention time. A shorter gradient is proposed and tested and exhibit less back exchange. Subzero column temperature is also tested as a possible approach to reduce back exchange. Subzero temperatures -5 C, -8 C, and -9 C are tested, and the results show back exchange level decreases as the temperature decreases. Chapter 3 describes the investigation on the assembly of the assimilatory NADPH-sulfite reductase (SiR), which is a structurally complex oxidoreductase that catalyzes the six-electron reduction of sulfite to organic sulfide. SiR is composed of two subunits, a flavin-binding flavoprotein (SiRFP) and an iron-containing hemoprotein (SiRHP). By use of hydrogen/deuterium exchange and biochemical analysis, SiR holoenzyme is shown to assemble through the N-terminus of SiRHP and the NADPH-binding domain of SiRFP. A novel form of the hemoprotein that occurs in the absence of its cofactors is also discovered. Apo-SiRHP forms a homotetramer, also dependent on its N-terminus that is unable to assemble with SiRFP. From these results, it is proposed that homo tetramerization of apo-SiRHP serves as a quality control mechanism to prevent the formation of inactive holoenzyme in the case of limiting cellular siroheme. Chapter 4 describes the investigation conducted on the archaeal MCM helicase for understanding its structural and mechanistic aspects of DNA unwinding. Although interactions of the encircled DNA strand within the central channel provide an accepted mode for translocation, interactions with the excluded strand on the exterior surface have mostly been ignored with regard to DNA unwinding. By applying HDX monitored by FT-ICR, binding sites for ssDNA is revealed, using multiple substrates targeting both the encircled and excluded strand interactions. Interacting residues on both the interior and exterior of SsoMCM are identified. Based on identified contacts, positively charged residues within the external waist region were mutated and shown to generally lower DNA unwinding without negatively affecting the ATP hydrolysis. The combined data globally identifies binding sites for ssDNA during SsoMCM unwinding. Chapter 5 is focused on AIMP3/P18-LmnA interaction. Recent reports reveal AIMP3 specifically mediates degradation of mature Lamin A (LmnA), a major component of the nuclear envelope matrix. HDX experiments for AIMP3 and LmnA C-terminus are conducted, and the results show that the AIMP3-LmnA interaction involves one of the two putative binding sites and an adjacent novel interface on AIMP3, and LmnA binds AIMP3 via its extreme C-terminus. Together these findings provide a structural insight for understanding the interaction between AIMP3 and LmnA in AIMP3-induced LmnA degradation. In Chapter 6, lysyl-tRNA synthetase (KRS) binding to the 67-kDa laminin receptor (67LR) is elucidated. 67LR is a membrane protein that promotes cell migration by inducing laminin-dependent degradation of extracellular matrix in cancer metastasis. Lysyl-tRNA synthetase (KRS) is normally associated with gene translation, and a recent study revealed its novel functions associated with 67LR-dependent cell migration and metastasis. In this HDX-based study, the binding surfaces between KRS and 37LRP are identified, and the conformational changes occur during their translocation from cytosol to the cell membrane. These results provide important insights into the KRS-37LRP interaction and pave the way for designing anti-metastasis therapeutics targeting this interaction.
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Date Issued: 2016
Identifier: FSU_FA2016_Tao_fsu_0071E_13544
Format: Thesis

Title: Methods for Epitope Characterization of Adeno-Associated Virus Type-2 Through Antibody Neutralization Escape Mutants.
Creator: Mitchell, Daniel Anthony, Tang, Hengli, Chapman, Michael, Li, Hong, Roux, Kenneth, Reeves, Robert, Program in Molecular Biophysics, Florida State University
Abstract/Description: The Adeno-Associated Virus has moved to the forefront as a vector for human gene therapy. Vectors have been constructed with AAV to repair many genetic deficiencies such as cystic fibrosis and hemophilia and have demonstrated remarkable success. For the application of a gene therapy vector, the particle must be afforded maximal capability to deliver the therapeutic gene without immunological elimination. Unfortunately, AAV is endemic in the human population and as a result, a large proportion...
Show moreThe Adeno-Associated Virus has moved to the forefront as a vector for human gene therapy. Vectors have been constructed with AAV to repair many genetic deficiencies such as cystic fibrosis and hemophilia and have demonstrated remarkable success. For the application of a gene therapy vector, the particle must be afforded maximal capability to deliver the therapeutic gene without immunological elimination. Unfortunately, AAV is endemic in the human population and as a result, a large proportion of individuals harbor immunity to AAV leading to rapid elimination upon subsequent exposure. One of the major obstacles for the development of AAV as a vector is the absence of robust epitope data. If such data was in hand, gene therapy vectors could be constructed with modifications to these highly immunogenic sites on the viral particle. Given the non-cytopathic nature of AAV, the virus does not lend itself well to traditional plaque assays. Without such assays in hand, the ability to isolate and recover viable viral clones is severely limited. We set out to map the viral epitope to monoclonal antibody A20 by generating monoclonal antibody neutralization escape mutants in cell culture. The lack of plaque assays had prompted us to focus on the development of methods that would facilitate these selection experiments. Having successfully developed a robust and reliable method for plaquing AAV we were capable of applying it to the preliminary selection experiments. The initial application of these methods did not yield the desired mutant. Presented here are the methods developed, their preliminary applications, analysis of the results and future prospective for escape mutant production.
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Date Issued: 2008
Identifier: FSU_migr_etd-2382
Format: Thesis

Department of Physics (237)	+ -
Theses and Dissertations (58)	+ -
National High Magnetic Field Laboratory (14)	+ -
Department of Chemistry and Biochemistry (4)	+ -
Department of Mathematics (3)	+ -

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Department of Biological Science (2)	+ -
Department of Earth, Ocean and Atmospheric Science (2)	+ -
Center for Ocean-Atmospheric Prediction Studies (1)	+ -
Department of Biomedical Sciences (1)	+ -
Undergraduate Honors Theses (1)	+ -

Biochemistry (30)	+ -
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Chemistry, Physical and theoretical (4)	+ -

Microphysics (4)	+ -
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Optics (3)	+ -
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Endoribonucleases/metabolism (2)	+ -
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Physics (2)	+ -
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