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- Title
- Ab initio Folding of a Trefoil-fold Motif Reveals Structural similarity with a β-propeller Blade Motif.
- Creator
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Tenorio, Connie, Longo, Liam, Parker, Joseph, Lee, Jihun, Blaber, Michael
- Abstract/Description
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Many protein architectures exhibit evidence of internal rotational symmetry postulated to be the result of gene duplication/fusion events involving a primordial polypeptide motif. A common feature of such structures is a domain-swapped arrangement at the interface of the N- and C-termini motifs and postulated to provide cooperative interactions that promote folding and stability. De novo designed symmetric protein architectures have demonstrated an ability to accommodate circular permutation...
Show moreMany protein architectures exhibit evidence of internal rotational symmetry postulated to be the result of gene duplication/fusion events involving a primordial polypeptide motif. A common feature of such structures is a domain-swapped arrangement at the interface of the N- and C-termini motifs and postulated to provide cooperative interactions that promote folding and stability. De novo designed symmetric protein architectures have demonstrated an ability to accommodate circular permutation of the N- and C-termini in the overall architecture; however, the folding requirement of the primordial motif are poorly understood, and tolerance to circular permutation is essentially unknown. The β-trefoil protein fold is a threefold symmetric architecture where the repeating ~42-mer “trefoil-fold” motif assembles via a domain-swapped arrangement. The trefoil-fold structure in isolation exposes considerable hydrophobic area that is otherwise buried in the intact β-trefoil trimeric assembly. The trefoil-fold sequence is not predicted to adopt the trefoil-fold architecture in ab initio folding studies; rather, the predicted fold is closely related to a compact “blade” motif from the β-propeller architecture. Expression of a trefoil-fold sequence and circular permutants shows that only the wild-type N-terminal motif definition yields an intact β-trefoil trimeric assembly, while permutants yield monomers. The results elucidate the folding requirements of the primordial trefoil-fold motif, and also suggest that this motif may sample a compact conformation that limits hydrophobic residue exposure, contains key trefoil-fold structural features, but is more structurally homologous to a β-propeller blade motif.
Show less - Date Issued
- 2020-03-03
- Identifier
- FSU_libsubv1_scholarship_submission_1583283654_54e07068, 10.1002/pro.3850
- Format
- Citation
- Title
- Investigating the Dynamics and Polyanion Binding Sites of Fibroblast Growth Factor-1 Using Hydrogen-Deuterium Exchange Mass Spectrometry.
- Creator
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Angalakurthi, Siva K, Tenorio, Connie A, Blaber, Michael, Middaugh, Russell
- Abstract/Description
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In this study, we examined the local dynamics of acidic fibroblast growth factor (FGF-1) as well as the binding sites of various polyanions including poly-sulfates (heparin and low MW heparin) and poly-phosphates (phytic acid and ATP) using hydrogen-deuterium exchange mass spectrometry (HX-MS). For local dynamics, results are analyzed at the peptide level as well as in terms of buried amides employing crystallographic B-factors and compared with a residue level heat map generated from HX-MS...
Show moreIn this study, we examined the local dynamics of acidic fibroblast growth factor (FGF-1) as well as the binding sites of various polyanions including poly-sulfates (heparin and low MW heparin) and poly-phosphates (phytic acid and ATP) using hydrogen-deuterium exchange mass spectrometry (HX-MS). For local dynamics, results are analyzed at the peptide level as well as in terms of buried amides employing crystallographic B-factors and compared with a residue level heat map generated from HX-MS results. Results show that strand 4 and 5 and the turn between them to be the most flexible regions as was previously seen by NMR. On the other hand, the C-terminal strands 8, 9 and 10 appear to be more rigid which is also consistent with crystallographic B-factors as well as local dynamics studies conducted by NMR. Crystal structures of FGF-1 in complex with heparin have shown that heparin binds to N-terminal Asn18 and to C-terminal Lys105, Tryp107, Lys112, Lys113, Arg119, Pro121, Arg122, Gln127 and Lys128 indicating electrostatic forces as dominant interactions. Heparin binding as determined by HX-MS is consistent with crystallography data. Previous studies have also shown that other polyanions including low MW heparin, phytic acid and ATP dramatically increase the thermal stability of FGF-1. Using HX-MS, we find other poly anions tested bind in a similar manner to heparin, primarily targeting the turns in the lysine rich C-terminal region of FGF-1 along with two distinct N-terminal regions that contains lysines and arginines/ histidines. This confirms the interactions between FGF-1 and polyanions are primary directed by electrostatics.
Show less - Date Issued
- 2018-04-05
- Identifier
- FSU_libsubv1_scholarship_submission_1523040928_8f170751
- Format
- Citation
- Title
- Alternative Folding Nuclei Definitions Facilitate the Evolution of a Symmetric Protein Fold from a Smaller Peptide Motif.
- Creator
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Longo, Liam, Lee, Jihun, Tenorio, Connie, Blaber, Michael
- Abstract/Description
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Protein 3° structure symmetry is a defining feature of nearly a third of protein folds and is generally thought to result from a combination of gene duplication, fusion, and truncation events. Such events represent major replication errors, involving substantial alteration of protein 3° structure as well as causing regions of exact repeating 1° structure, both of which are generally considered deleterious to protein folding. Thus, the prevalence of symmetric protein folds is counterintuitive...
Show moreProtein 3° structure symmetry is a defining feature of nearly a third of protein folds and is generally thought to result from a combination of gene duplication, fusion, and truncation events. Such events represent major replication errors, involving substantial alteration of protein 3° structure as well as causing regions of exact repeating 1° structure, both of which are generally considered deleterious to protein folding. Thus, the prevalence of symmetric protein folds is counterintuitive and suggests a specific, yet unexplained, robustness. Using a designed β-trefoil protein, we show that purely symmetric 1° structure enables utilization of alternative definitions of the critical folding nucleus in response to gross structural rearrangement. Thus, major replication errors producing 1° structure symmetry can conserve foldability. The results provide an explanation for the prevalence of symmetric protein folds, and highlight a critical role for 1° structure symmetry in protein evolution.
Show less - Date Issued
- 2013-10-17
- Identifier
- FSU_libsubv1_scholarship_submission_1456501539, 10.1016/j.str.2013.09.003
- Format
- Citation
- Title
- The Folding Nucleus Structure Persists in Thermally-Aggregated FGF-1.
- Creator
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Longo, Liam, Gao, Yuan, Tenorio, Connie, Wang, Gan, Paravastu, Anant, Blaber, Michael
- Abstract/Description
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An efficient protein folding pathway leading to target structure, and the avoidance of aggregation, is essential to protein evolution and de novo design; however, design details to achieve efficient folding and avoid aggregation are poorly understood. We report characterization of the thermally-induced aggregate of fibroblast growth factor-1 (FGF-1), a small globular protein, by solid-state NMR. NMR spectra are consistent with residual structure in the aggregate and provide evidence of a...
Show moreAn efficient protein folding pathway leading to target structure, and the avoidance of aggregation, is essential to protein evolution and de novo design; however, design details to achieve efficient folding and avoid aggregation are poorly understood. We report characterization of the thermally-induced aggregate of fibroblast growth factor-1 (FGF-1), a small globular protein, by solid-state NMR. NMR spectra are consistent with residual structure in the aggregate and provide evidence of a structured region that corresponds to the region of the folding nucleus. NMR data on aggregated FGF-1 also indicate the presence of unstructured regions that exhibit hydration-dependent dynamics and suggest that unstructured regions of aggregated FGF-1 lie outside the folding nucleus. Since it is known that regions outside the folding nucleus fold late in the folding pathway, we postulate that these regions unfold early in the unfolding pathway and that the partially folded state is more prone to intermolecular aggregation. This interpretation is further supported by comparison with a designed protein that shares the same FGF-1 folding nucleus sequence, but has different 1° structure outside the folding nucleus, and does not thermally aggregate. The results suggest that design of an efficient folding nucleus, and the avoidance of aggregation in the folding pathway, are potentially separable design criteria – the latter of which could principally focus upon the physicochemical properties of 1° structure outside the folding nucleus.
Show less - Date Issued
- 2017-10-23
- Identifier
- FSU_libsubv1_scholarship_submission_1509376995_85b5a7ca, 10.1002/pro.3332
- Format
- Citation
- Title
- Fine-sampled Photographic Quantitation of Dermal Wound Healing Senescence in Aged BALB/cByJ Mice and Therapeutic Intervention with FGF-1: Novel photographic quantitation of dermal healing.
- Creator
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Mellers, Alana, Tenorio, Connie, Lacatusu, Diana, Powell, Brett, Patel, Bhavi, Harper, Kathleen, Blaber, Michael
- Abstract/Description
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Objective: Determine quantitative parameters of dermal wound healing senescence in aged BALB/cByJ mice (an important animal model of aging) and evaluate the potential for therapeutic intervention by fibroblast growth factor-1 (FGF-1). Approach: Utilize a novel, non-invasive, fine-sampled photographic methodology to quantify wound healing parameters for healing phases from wounding through to wound closure. Results: Parameters associated with key healing phases were quantified and compared for...
Show moreObjective: Determine quantitative parameters of dermal wound healing senescence in aged BALB/cByJ mice (an important animal model of aging) and evaluate the potential for therapeutic intervention by fibroblast growth factor-1 (FGF-1). Approach: Utilize a novel, non-invasive, fine-sampled photographic methodology to quantify wound healing parameters for healing phases from wounding through to wound closure. Results: Parameters associated with key healing phases were quantified and compared for non-aged and aged cohorts of both sexes. The results identify a sexual dimorphism in dermal wound healing, with non-aged females exhibiting a greater overall healing efficiency compared to males. This enhanced healing in females, however, senesces with age such that healing parameters for aged males and females are statistically indistinguishable. Topical application of FGF-1 was identified as an effective therapeutic intervention to treat dermal healing senescence in aged females. Innovation: The FGF intervention is being analyzed using a new, recently published model. This approach significantly increases the amount of pre-clinical animal data obtainable in wound healing studies, minimizes cohort number compared to (lethal) histological studies, and permits a direct statistical comparison between different healing studies. Conclusion: Quantitative parameters of dermal wound healing, obtained from non-invasive fine-sampled photographic data, identify topical FGF-1 as an effective therapeutic to treat the senescence of dermal healing present in aged female BALB/cByJ mice.
Show less - Date Issued
- 2018-06-25
- Identifier
- FSU_libsubv1_scholarship_submission_1529889363_21ee7933
- Format
- Citation
- Title
- An S116R Phosphorylation Site Mutation in Human Fibroblast Growth Factor-1 Differentially Affects Mitogenic and Glucose-Lowering Activities.
- Creator
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Xia, Xue, Kumru, Ozan S, Blaber, Sachiko I, Middaugh, C Russell, Li, Ling, Ornitz, David M, Suh, Jae Myoung, Atkins, Annette R, Downes, Michael, Evans, Ronald M, Tenorio, Connie...
Show moreXia, Xue, Kumru, Ozan S, Blaber, Sachiko I, Middaugh, C Russell, Li, Ling, Ornitz, David M, Suh, Jae Myoung, Atkins, Annette R, Downes, Michael, Evans, Ronald M, Tenorio, Connie A, Bienkiewicz, Ewa, Blaber, Michael
Show less - Abstract/Description
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Fibroblast growth factor-1 (FGF-1), a potent human mitogen and insulin sensitizer, signals through both tyrosine kinase receptor-mediated autocrine/paracrine pathways as well as a nuclear intracrine pathway. Phosphorylation of FGF-1 at serine 116 (S116) has been proposed to regulate intracrine signaling. Position S116 is located within a ∼17 amino acid C-terminal loop that contains a rich set of functional determinants including heparin∖heparan sulfate affinity, thiol reactivity, nuclear...
Show moreFibroblast growth factor-1 (FGF-1), a potent human mitogen and insulin sensitizer, signals through both tyrosine kinase receptor-mediated autocrine/paracrine pathways as well as a nuclear intracrine pathway. Phosphorylation of FGF-1 at serine 116 (S116) has been proposed to regulate intracrine signaling. Position S116 is located within a ∼17 amino acid C-terminal loop that contains a rich set of functional determinants including heparin∖heparan sulfate affinity, thiol reactivity, nuclear localization, pharmacokinetics, functional half-life, nuclear ligand affinity, stability, and structural dynamics. Mutational targeting of specific functionality in this region without perturbing other functional determinants is a design challenge. S116R is a non-phosphorylatable variant present in bovine FGF-1 and other members of the human FGF family. We show that the S116R mutation in human FGF-1 is accommodated with no perturbation of biophysical or structural properties, and is therefore an attractive mutation with which to elucidate the functional role of phosphorylation. Characterization of S116R shows reduction in NIH 3T3 fibroblast mitogenic stimulation, increase in fibroblast growth factor receptor-1c activation, and prolonged duration of glucose lowering in ob/ob hyperglycemic mice. A novel FGF-1/fibroblast growth factor receptor-1c dimerization interaction combined with non-phosphorylatable intracrine signaling is hypothesized to be responsible for these observed functional effects.
Show less - Date Issued
- 2016-12-01
- Identifier
- FSU_pmch_27773526, 10.1016/j.xphs.2016.09.005, PMC5310217, 27773526, 27773526, S0022-3549(16)41698-9
- Format
- Citation
- Title
- Engineering a Cysteine-Free Form of Human Fibroblast Growth Factor-1 for "Second Generation" Therapeutic Application.
- Creator
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Xia, Xue, Kumru, Ozan S, Blaber, Sachiko I, Middaugh, C Russell, Li, Ling, Ornitz, David M, Sutherland, Mason A, Tenorio, Connie A, Blaber, Michael
- Abstract/Description
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Human fibroblast growth factor-1 (FGF-1) has broad therapeutic potential in regenerative medicine but has undesirable biophysical properties of low thermostability and 3 buried cysteine (Cys) residues (at positions 16, 83, and 117) that interact to promote irreversible protein unfolding under oxidizing conditions. Mutational substitution of such Cys residues eliminates reactive buried thiols but cannot be accomplished simultaneously at all 3 positions without also introducing further...
Show moreHuman fibroblast growth factor-1 (FGF-1) has broad therapeutic potential in regenerative medicine but has undesirable biophysical properties of low thermostability and 3 buried cysteine (Cys) residues (at positions 16, 83, and 117) that interact to promote irreversible protein unfolding under oxidizing conditions. Mutational substitution of such Cys residues eliminates reactive buried thiols but cannot be accomplished simultaneously at all 3 positions without also introducing further substantial instability. The mutational introduction of a novel Cys residue (Ala66Cys) that forms a stabilizing disulfide bond (i.e., cystine) with one of the extant Cys residues (Cys83) effectively eliminates one Cys while increasing overall stability. This increase in stability offsets the associated instability of remaining Cys substitution mutations and permits production of a Cys-free form of FGF-1 (Cys16Ser/Ala66Cys/Cys117Ala) with only minor overall instability. The addition of a further stabilizing mutation (Pro134Ala) creates a Cys-free FGF-1 mutant with essentially wild-type biophysical properties. The elimination of buried free thiols in FGF-1 can substantially increase the protein half-life in cell culture. Here, we show that the effective cell survival/mitogenic functional activity of a fully Cys-free form is also substantially increased and is equivalent to wild-type FGF-1 formulated in the presence of heparin sulfate as a stabilizing agent. The results identify this Cys-free FGF-1 mutant as an advantageous "second generation" form of FGF-1 for therapeutic application.
Show less - Date Issued
- 2016-04-01
- Identifier
- FSU_pmch_27019961, 10.1016/j.xphs.2016.02.010, PMC5318998, 27019961, 27019961, S0022-3549(16)00366-X
- Format
- Citation
- Title
- Engineering a Cysteine-Free Form of Human Fibroblast Growth Factor-1 for "2nd Generation" Therapeutic Application.
- Creator
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Xue, Xia, Kumru, Ozan, Blaber, Sachiko, Middaugh, Russell, Li, Ling, Ornitz, David, Sutherland, Mason, Tenorio, Connie, Blaber, Michael
- Abstract/Description
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Human fibroblast growth factor-1 (FGF-1) has broad therapeutic potential in regenerative medicine but has undesirable biophysical properties of low thermostability and three buried Cys residues (at positions 16, 83 and117) that interact to promote irreversible protein unfolding under oxidizing conditions. Mutational substitution of such Cys residues eliminates reactive buried thiols but cannot be accomplished simultaneously at all three positions without also introducing further substantial...
Show moreHuman fibroblast growth factor-1 (FGF-1) has broad therapeutic potential in regenerative medicine but has undesirable biophysical properties of low thermostability and three buried Cys residues (at positions 16, 83 and117) that interact to promote irreversible protein unfolding under oxidizing conditions. Mutational substitution of such Cys residues eliminates reactive buried thiols but cannot be accomplished simultaneously at all three positions without also introducing further substantial instability. The mutational introduction of a novel Cys residue (Ala66Cys) that forms a stabilizing disulfide bond (i.e., cystine) with one of the extant Cys residues (Cys83) effectively eliminates one Cys while increasing overall stability. This increase in stability offsets the associated instability of remaining Cys substitution mutations and permits production of a Cys-free form of FGF-1 (Cys16Ser/Ala66Cys/Cys117Ala) with only minor overall instability. The addition of a further stabilizing mutation (Pro134Ala) creates a Cys free FGF-1 mutant with essentially wild-type biophysical properties. The elimination of buried free thiols in FGF-1 can substantially increase the protein half-life in cell culture. Here we show that the effective cell survival/mitogenic functional activity of a fully Cys-free form is also substantially increased; and is equivalent to WT FGF-1 formulated in the presence of heparin sulfate as a stabilizing agent. The results identify this Cys free FGF-1 mutant as an advantageous "2nd generation" form of FGF-1 for therapeutic application.
Show less - Date Issued
- 2016-02-11
- Identifier
- FSU_libsubv1_scholarship_submission_1464366396, 10.1016/j.xphs.2016.02.010
- Format
- Citation
- Title
- An S116R Phosphorylation Site Mutation in Human FGF-1 Differentially Affects Mitogenic and Glucose Lowering Activities.
- Creator
-
Xia, Xue, Kumru, Ozan, Blaber, Sachiko, Middaugh, Russell, Li, Ling, Ornitz, David, Suh, Jae Myoung, Atkins, Annette, Downes, Michael, Evans, Ronald, Tenorio, Connie,...
Show moreXia, Xue, Kumru, Ozan, Blaber, Sachiko, Middaugh, Russell, Li, Ling, Ornitz, David, Suh, Jae Myoung, Atkins, Annette, Downes, Michael, Evans, Ronald, Tenorio, Connie, Bienkiewicz, Ewa, Blaber, Michael
Show less - Abstract/Description
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Fibroblast growth factor-1 (FGF-1), a potent human mitogen and insulin sensitizer, signals through both tyrosine kinase receptor mediated autocrine/paracrine pathways as well as a nuclear intracrine pathway. Phosphorylation of FGF-1 at serine 116 (S116) has been proposed to regulate intracrine signalling. Position S116 is located within a ~17 amino acid C-terminal loop that contains a rich set of functional determinants including heparin\heparan sulfate (HS) affinity, thiol reactivity,...
Show moreFibroblast growth factor-1 (FGF-1), a potent human mitogen and insulin sensitizer, signals through both tyrosine kinase receptor mediated autocrine/paracrine pathways as well as a nuclear intracrine pathway. Phosphorylation of FGF-1 at serine 116 (S116) has been proposed to regulate intracrine signalling. Position S116 is located within a ~17 amino acid C-terminal loop that contains a rich set of functional determinants including heparin\heparan sulfate (HS) affinity, thiol reactivity, nuclear localization, pharmacokinetics, functional half-life, nuclear ligand affinity, stability, and structural dynamics. Mutational targeting of specific functionality in this region without perturbing other functional determinants is a design challenge. S116R is a non-phosphorylatable variant present in bovine FGF-1 and other members of the human FGF family. We show that the S116R mutation in human FGF-1 is accommodated with no perturbation of biophysical or structural properties, and is therefore an attractive mutation with which to elucidate the functional role of phosphorylation. Characterization of S116R shows reduction of NIH 3T3 fibroblast mitogenic stimulation, increase in FGFR-1c activation, and prolonged duration of glucose lowering in ob/ob hyperglycemic mice. A novel FGF-1/FGFR-1c dimerization interaction combined with non-phosphorylatable intracrine signaling is hypothesized to be responsible for these observed functional effects.
Show less - Date Issued
- 2016-09-07
- Identifier
- FSU_libsubv1_scholarship_submission_1473276315
- Format
- Citation
- Title
- A Bell-shaped Dose-response of Topical FGF-1 in Accelerating Dermal Wound Healing in Aged Female BALB/cByJ Mice.
- Creator
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Hagerott, Brooke, Blumstein, Alli, McGarry, Lauren, Cohen, Hannah, Tenorio, Connie, Powell, Brett, Nagy, Tamas, Blaber, Michael
- Abstract/Description
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The objective of this study was to characterize the therapeutic dose-response characteristics for topical FGF-1 in the full-thickness dermal healing of aged female BALB/cByJ mice. The approach utilized a splinted excisional model of dermal healing, and a novel fine-sampled photographic methodology, to quantify key wound healing parameters for different doses of topical FGF-1. The histology of healed wounds, representative of each dose cohort, was also evaluated by section and staining. The...
Show moreThe objective of this study was to characterize the therapeutic dose-response characteristics for topical FGF-1 in the full-thickness dermal healing of aged female BALB/cByJ mice. The approach utilized a splinted excisional model of dermal healing, and a novel fine-sampled photographic methodology, to quantify key wound healing parameters for different doses of topical FGF-1. The histology of healed wounds, representative of each dose cohort, was also evaluated by section and staining. The results show that topical FGF-1 pharmacotherapy for accelerating dermal healing in aged BALB/cByJ female mice yields a narrow dose-response curve, with diminished therapeutic effect at high concentration (i.e. “bell-shaped” dose-response). The physiological response of FGF-1 in wound healing involves a combination of cell types (including vascular endothelial cells, epidermal keratinocytes and dermal fibroblasts). These individual cells types in culture can have different FGF-1 dose-response curves; however, only the response of fibroblasts is bell-shaped. The bell-shaped dose-response in dermal healing therefore principally reflects the effect upon fibroblasts. A narrow bell-shaped dose-response requires precise dosing of FGF-1 for therapeutic benefit. The results identify the practical dose range to elicit such a benefit.
Show less - Date Issued
- 2020-06-20
- Identifier
- FSU_libsubv1_scholarship_submission_1591883645_68fa5c0c, 10.1007/s42485-020-00040-z
- Format
- Citation
