Hydrogen deuterium exchange mass spectrometry has emerged as an important technique to probe protein structure and conformational dynamics. The rate of exchange of hydrogen with deuterium by the peptide backbone is dependent on the solvent accessibility, extent of hydrogen bonding in secondary structural elements and protein dynamics. The extent and the rate of deuterium incorporation are affected by changes in protein structure, interaction with ligand, protein-protein interaction and environmental factors such as pH and temperature. These conformational changes can be global and/or local. The increase in the mass is used to localize the deuterium incorporation after pepsin digestion of the protein and analysis by electrospray ionization mass spectrometry. In this dissertation traditional HDX-MS and a new deuterium trapping assay were used to probe the interaction sites between E. coli cysteine desulfurase SufS and acceptor protein SufE. SufS and SufE form an important part of the SUF pathway, essential for the biosynthesis of Fe-S clusters under oxidative stress and iron depletion conditions. In addition, SufE is known to stimulate SufS cysteine desulfurase activity, but the mechanism is unknown. The HDX-MS results show that the regions affected by the SufS-SufE interaction are dependent on the catalytic intermediate states of the two proteins. HDX-MS was also used to probe the conformational changes resulting upon persulfuration of SufS of Cys364 in the active site. The persulfuration of SufS not only affected regions in the active site cavity, but also had other conformational changes in more distal regions. Based on our findings a model for the interaction SufS and SufE was proposed. A mechanism for the enhancement of SufS cysteine desulfurase activity upon interaction with SufE was also postulated. In all this work demonstrates that hydrogen deuterium exchange mass spectrometry and the deuterium trapping methodology optimized for this system can be easily and effectively used to study the protein-protein interactions and the accompanying changes in structural dynamics for other proteins. Deuterium trapping was demonstrated to be fast, sensitive and reliable method to deduce the changes in solvent accessibility between two or more states of a protein. Both techniques can easily be applied to large number of protein complexes to determine the regions of interaction as well as gain mechanistic information not available through traditional methods such as X-ray crystallography and NMR.

The authoritative guide to analyzing protein interactions by mass spectrometry Mass spectrometry (MS) is playing an increasingly important role in the study of protein interactions. Mass Spectrometry of Protein Interactionspresents timely and definitive discussions of the diverse range of approaches for studying protein interactions by mass spectrometry with an extensive set of references to the primary literature. Each chapter is written by authors or teams of authors who are international authorities in their fields. This leading reference text: * Discusses the direct detection of protein interactions through electrospray ionization (ESI-MS); ion mobility analysis; and matrix-assisted laser desorption/ionization (MALDI-MS) * Covers the indirect analysis of protein interactions through hydrogen-deuterium exchange (HX-MS); limited proteolysis; cross-linking; and radial probe (RP-MS) * Guides researchers in the use of mass spectrometry in structural biology, biochemistry, and protein science to map and define the huge number and diversity of protein interactions * Reviews the latest discoveries and applications and addresses new and ongoing challenges This is a comprehensive reference for researchers in academia and industry engaged in studies of protein interactions and an excellent text for graduate and postgraduate students.

Hydrogen deuterium exchange (HDX) coupled with mass spectrometry is widely used for probing protein structure and dynamics. Protein-ligand interactions usually induce a reduction in the measured HDX rates an effect that may be ascribed to stabilization of the protein structure. This work aims to improve the general understanding of the changes in HDX patterns associated with ligand binding. We initially applied HDX for studying differences between oxy -hemoglobin (Oxy- Hb) and aquomet-hemoglobin (Chapter 2). The results show that the and subunits respond differently to the oxy to aquomet transition with the heme binding pocket being destabilized in both cases. The results suggest that enhanced structural dynamics in the heme binding pocket may have adverse effects on heme-protein interactions. Chapter 3 focuses on the different scenarios that can be encountered in an HDX experiment upon ligand binding. Myoglobin and hemoglobin were used as model systems, focusing on the oxy and deoxy states of both proteins. Our results demons trate that ligand binding can be stabilizing or destabilizing, leading to decreased or increased HDX rates respectively. In Chapters 4 HDX was used to probe the changes in structural dynamics of caseinolytic protease P (ClpP), an antibiotic drug target, after binding ADEP antibiotics. The mechanism of ADEP binding and the N-terminal structure of ClpP is not well understood with conflicting x-ray structures reported in literature. Our findings demonstrate that the N- terminus of ClpP remains quite unstructured after ADEP binding, while belt region undergoes tightening. Pin 1, a peptidyl prolyl isomerase, binding to a cyclic peptide inhibitor was studied in Chapter 5. Characterization of Pin1-CRYPEVEIC interactions by ot her techniques has been difficult. This study demonstrates that binding of the inhibitor triggers an overall stabilization of Pin 1. We identify a loop that interacts with basic sites of the ligand and that becomes destabilized upon ligand binding. This destabilization is ascribed to steric clashes between the peptide inhibitor and the protein.

Proteins are not static objects. They have a great variety of internal motions with different amplitudes and different timescales. These internal motions play an important role in catalytic processes. Therefore, the existence of an intimate relationship between protein dynamics and protein function is widely accepted. Due to the significance of protein dynamics, techniques have been developed to study protein dynamics including nuclear magnetic resonance (NMR) spectroscopy, electron paramagnetic resonance (EPR) spectroscopy, and mass spectrometry (MS). Compared with NMR and EPR spectroscopy, MS has less stringent sample requirements, including protein concentration and protein size. Moreover, the mass accuracy, sensitivity, and faster data analysis also have contributed to the rapid growth of MS based techniques. Hydrogen-deuterium exchange mass spectrometry (HDX-MS), a combination of HPLC and MS, has become a common and sensitive tool to probe protein structural flexibility and solution dynamics. In this dissertation, HDX-MS was applied to study dynamic changes of proteins due to substrate binding and protein-protein interactions. The GT-A glycosyltransferase glucosyl-3-phosphoglycerate synthase from Mycobacterium tuberculosis (MtGpgS) catalyzes the first step of biosynthesis of 6-O-methylglucose lipopolysaccharides (MGLPs), which are essential to growth and existence of mycobacterium. The HDX-MS data revealed that the two substrates UDP-glucose (UDPG) and 3-phosphoglycerate (3PGA) can bind to MtGpgS independently, disagreeing with the previous proposal that 3PGA can only bind to MtGpgS after UDPG. Moreover, 3PGA was found to bind to or allosterically affect the UDPG binding site. Furthermore, the HDX-MS data revealed that MtGpgS may provide a necessary conformation for UDPG binding, while it goes through a large conformational change on 3PGA binding. The GT-B glycosyltransferase MshA from Corynebacterium glutamicum (CgMshA) catalyzes the initial step of mycothiol biosynthesis. A large conformational change was observed in CgMshA on nucleotide binding by superimposing APO structure of CgMshA and complex structure with UDP. HDX-MS was utilized to study conformational changes of CgMshA on substrate binding from the aspect of dynamics, providing a complementary to static structures. The HDX-MS data showed that both substrates uridine diphosphate glucose-N-acetylglucosamine (UDP-GlcNAc) and 1-L-myo-inositol-1-phosphate (I1P) can bind to CgMshA independently, but the I1P binding is not productive since it binds to an uncorrect site. Moreover, the I1P binding can lead to dynamic changes of CgMshA, while only UDP-GlcNAc can induce the major conformational change of CgMshA. Furthermore, the 3PGA binding cannot induce further dynamic changes of CgMshA in the presence of UDP. HDX-MS was also employed to study dynamic changes of protein complex SufBC2D from Escherichia coli on ADP/Mg2+ binding. This complex is responsible for Fe-S cluster assembly under oxidative stress. The crystal structure of SufBC2D complex has been determined, while little dynamic information is known. So HDX-MS was applied to study dynamic changes of the SufBC2D complex. The HDX-MS data revealed that SufC has a significant conformational change, which may be required by ATP binding and hydrolysis. Moreover, SufB and SufD are detected to have dynamic changes due to SufC conformational changes. These dynamic changes suggest that SufB-SufD protomer may have a conformational change in order to provide a suitable conformation for Fe-S cluster assembly. This work demonstrates that HDX-MS can be effectively used to study protein-ligand and protein-protein interactions, as well as the accompanying changes in structural dynamics. HDX-MS data detects substrate binding mechanism and conformational changes that are not available through x-ray crystallography. With these advantages, HDX-MS has been applied in study of protein structure and dynamics, studying protein-ligand and protein-protein interactions, protein folding, as well as protein therapeutics discovery and development.