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Probing Protein Conformation and Protein-Protein Interaction by Hydrogen Deuterium Exchange Coupled with FT-ICR MS

Title: Probing Protein Conformation and Protein-Protein Interaction by Hydrogen Deuterium Exchange Coupled with FT-ICR MS.
Name(s): Tao, Yeqing, author
Marshall, Alan G. (Alan George), 1944-, professor directing dissertation
Blaber, Michael, university representative
Dorsey, John G., committee member
Li, Hong, committee member
Florida State University, degree granting institution
College of Arts and Sciences, degree granting college
Department of Chemistry and Biochemistry, degree granting department
Type of Resource: text
Genre: Text
Issuance: monographic
Date Issued: 2016
Publisher: Florida State University
Place of Publication: Tallahassee, Florida
Physical Form: computer
online resource
Extent: 1 online resource (144 pages)
Language(s): English
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 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.
Identifier: FSU_FA2016_Tao_fsu_0071E_13544 (IID)
Submitted Note: A Dissertation submitted to the Department of Chemistry and Biochemistry in partial fulfillment of the Doctor of Philosophy.
Degree Awarded: Fall Semester 2016.
Date of Defense: October 31, 2016.
Bibliography Note: Includes bibliographical references.
Advisory Committee: Alan G. Marshall, Professor Directing Dissertation; Michael Blaber, University Representative; John G. Dorsey, Committee Member; Hong Li, Committee Member.
Subject(s): Chemistry
Persistent Link to This Record:
Owner Institution: FSU

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Tao, Y. (2016). Probing Protein Conformation and Protein-Protein Interaction by Hydrogen Deuterium Exchange Coupled with FT-ICR MS. Retrieved from