Hydrogen Exchange Mass Spectrometry For Studying Protein Ligand Interactions

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Hydrogen Exchange Mass Spectrometry for Studying Protein-ligand Interactions

Author : Modupeola A. Sowole
Publisher :
ISBN 13 :
Total Pages : 354 pages
Book Rating : 4.:/5 (16 download)

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Book Synopsis Hydrogen Exchange Mass Spectrometry for Studying Protein-ligand Interactions by : Modupeola A. Sowole

Download or read book Hydrogen Exchange Mass Spectrometry for Studying Protein-ligand Interactions written by Modupeola A. Sowole and published by . This book was released on 2015 with total page 354 pages. Available in PDF, EPUB and Kindle. Book excerpt: 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.

Hydrogen Exchange Mass Spectrometry of Proteins

Author : David D. Weis
Publisher : John Wiley & Sons
ISBN 13 : 1118703693
Total Pages : 376 pages
Book Rating : 4.1/5 (187 download)

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Book Synopsis Hydrogen Exchange Mass Spectrometry of Proteins by : David D. Weis

Download or read book Hydrogen Exchange Mass Spectrometry of Proteins written by David D. Weis and published by John Wiley & Sons. This book was released on 2016-01-11 with total page 376 pages. Available in PDF, EPUB and Kindle. Book excerpt: Hydrogen exchange mass spectrometry is widely recognized for its ability to probe the structure and dynamics of proteins. The application of this technique is becoming widespread due to its versatility for providing structural information about challenging biological macromolecules such as antibodies, flexible proteins and glycoproteins. Although the technique has been around for 25 years, this is the first definitive book devoted entirely to the topic. Hydrogen Exchange Mass Spectrometry of Proteins: Fundamentals, Methods and Applications brings into one comprehensive volume the theory, instrumentation and applications of Hydrogen Exchange Mass Spectrometry (HX-MS) - a technique relevant to bioanalytical chemistry, protein science and pharmaceuticals. The book provides a solid foundation in the basics of the technique and data interpretation to inform readers of current research in the method, and provides illustrative examples of its use in bio- and pharmaceutical chemistry and biophysics In-depth chapters on the fundamental theory of hydrogen exchange, and tutorial chapters on measurement and data analysis provide the essential background for those ready to adopt HX-MS. Expert users may advance their current understanding through chapters on methods including membrane protein analysis, alternative proteases, millisecond hydrogen exchange, top-down mass spectrometry, histidine exchange and method validation. All readers can explore the diversity of HX-MS applications in areas such as ligand binding, membrane proteins, drug discovery, therapeutic protein formulation, biocomparability, and intrinsically disordered proteins.

Mass Spectrometry of Protein Interactions

Author : Kevin Downard
Publisher : John Wiley & Sons
ISBN 13 : 047014632X
Total Pages : 153 pages
Book Rating : 4.4/5 (71 download)

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Book Synopsis Mass Spectrometry of Protein Interactions by : Kevin Downard

Download or read book Mass Spectrometry of Protein Interactions written by Kevin Downard and published by John Wiley & Sons. This book was released on 2007-08-24 with total page 153 pages. Available in PDF, EPUB and Kindle. Book excerpt: 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.

Characterizing Protein Dynamics of Protein-Ligand Interactions by Hydrogen-Deuterium Exchange Mass Spectrometry

Author : Diana Resetca
Publisher :
ISBN 13 :
Total Pages : pages
Book Rating : 4.:/5 (13 download)

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Book Synopsis Characterizing Protein Dynamics of Protein-Ligand Interactions by Hydrogen-Deuterium Exchange Mass Spectrometry by : Diana Resetca

Download or read book Characterizing Protein Dynamics of Protein-Ligand Interactions by Hydrogen-Deuterium Exchange Mass Spectrometry written by Diana Resetca and published by . This book was released on 2014 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt:

Application of Hydrogen Deuterium Exchange Mass Spectrometry in Protein-ligand and Protein-protein Interactions

Author : Siqi Guan
Publisher :
ISBN 13 :
Total Pages : 322 pages
Book Rating : 4.:/5 (131 download)

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Book Synopsis Application of Hydrogen Deuterium Exchange Mass Spectrometry in Protein-ligand and Protein-protein Interactions by : Siqi Guan

Download or read book Application of Hydrogen Deuterium Exchange Mass Spectrometry in Protein-ligand and Protein-protein Interactions written by Siqi Guan and published by . This book was released on 2016 with total page 322 pages. Available in PDF, EPUB and Kindle. Book excerpt: 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.

Probing Protein-ligand Interactions Via Solution Phase Hydrogen Exchange Mass Spectrometry

Author : Stefan Theo Esswein
Publisher :
ISBN 13 :
Total Pages : 220 pages
Book Rating : 4.:/5 (781 download)

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Book Synopsis Probing Protein-ligand Interactions Via Solution Phase Hydrogen Exchange Mass Spectrometry by : Stefan Theo Esswein

Download or read book Probing Protein-ligand Interactions Via Solution Phase Hydrogen Exchange Mass Spectrometry written by Stefan Theo Esswein and published by . This book was released on 2010 with total page 220 pages. Available in PDF, EPUB and Kindle. Book excerpt: Mass spectrometry is a versatile, sensitive and fast technique with which to probe biophysical properties in biological systems and one of the most important analytical tools in the multidisciplinary field of proteomics. The study of nativestate proteins and their complexes in the gas-phase is well established and direct infusion electrospray ionisation mass spectrometry (DI-ESI-MS) techniques are becoming increasingly popular as a tool for screening and determining quantitative information on protein-protein and protein-ligand interactions. However, complexes retained by ESI-MS are not always representative of those in solution and care must be taken in interpreting purely gas-phase results. This thesis details modification and advancement of solution phase techniques devised by Gross et al. utilising ESI-MS and Fitzgerald et al. applying matrix assisted laser desorption ionisation (MALDI)-MS termed PLIMSTEX (protein-ligand interactions by mass spectrometry, titration and hydrogen-deuterium-exchange)[1] and SUPREX (Stability of unpurified proteins from rates of H/D exchange)[2] to quantify these interactions with regards to high throughput analysis. The first part of this thesis describes the different developmental stages of the devised HPLC-front ends and their optimisation with myoglobin and insulin. The successfully developed HPLC-front end in conjunction with PLIMSTEX and SUPREX and ESI-MS then gets tested with self expressed and purified cyclophilin A(CypA)- cyclosporin A (CsA) system, followed by a test screen with potential CypA binding ligands. Dissociation constants (Kd's) within one order of magnitude to reported values are determined. In the third part of this thesis the application of the devised ESI-SUPREX methodology has been applied to anterior gradient 2 (AGr2) and the factor H complement control proteins module 19-20 (fH19-20) exhibiting binding potential to a taggedhexapeptide and a synthetic pentasaccharide, respectively, resulting in thermodynamical data for these protein-ligand interactions. For the AGr2 system another dimension of investigation has been added by temperature controlling the devised ESI-SUPREX approach, revealing a phase transition in the protein at higher temperatures. The final part of this thesis describes the application of the ESI-SUPREX methodology to probe folding properties of CypA in the presence of the self expressed and purified E. coli chaperonin groEL. Thereby the denaturing properties of groEL have been emphasised along with the stabilisation of a denatured CypA species.

Mass Spectrometry Analysis for Protein-Protein Interactions and Dynamics

Author : M. Chance
Publisher : John Wiley & Sons
ISBN 13 : 0470258861
Total Pages : 325 pages
Book Rating : 4.4/5 (72 download)

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Book Synopsis Mass Spectrometry Analysis for Protein-Protein Interactions and Dynamics by : M. Chance

Download or read book Mass Spectrometry Analysis for Protein-Protein Interactions and Dynamics written by M. Chance and published by John Wiley & Sons. This book was released on 2008-09-22 with total page 325 pages. Available in PDF, EPUB and Kindle. Book excerpt: Presents a wide variety of mass spectrometry methods used to explore structural mechanisms, protein dynamics and interactions between proteins. Preliminary chapters cover mass spectrometry methods for examining proteins and are then followed by chapters devoted to presenting very practical, how-to methods in a detailed way. Includes footprinting and plistex specifically, setting this book apart from the competition.

Investigating Protein-carbohydrate Interactions with Hydrogen/deuterium Exchange Mass Spectrometry (HDX-MS)

Author : Jingjing Zhang
Publisher :
ISBN 13 :
Total Pages : 7 pages
Book Rating : 4.:/5 (9 download)

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Book Synopsis Investigating Protein-carbohydrate Interactions with Hydrogen/deuterium Exchange Mass Spectrometry (HDX-MS) by : Jingjing Zhang

Download or read book Investigating Protein-carbohydrate Interactions with Hydrogen/deuterium Exchange Mass Spectrometry (HDX-MS) written by Jingjing Zhang and published by . This book was released on 2014 with total page 7 pages. Available in PDF, EPUB and Kindle. Book excerpt: The application of hydrogen/deuterium exchange mass spectrometry (HDX-MS) to investigating protein-carbohydrate interactions is described. Proteins from three bacterial toxins, the B subunit homopentamers of Cholera toxin (CTB5) and Shiga toxin type 1 (Stx1B5) and a fragment of Clostridium difficile toxin A (TcdA-A2), and their interactions with native carbohydrate receptors, GM1 pentasaccharide (GM1-os), Pk trisaccharide and CD-grease, respectively, were first served as model systems for this study. The results suggested that HDX-MS can serve as a useful tool for localizing the ligand binding sites in carbohydrate-binding proteins. Following this, HDX-MS measurements were applied to explore the existence of distinct HMOs binding sites on toxins. Altogether, two toxins were studied, CTB5 and TcdA-A2, and their interactions with HMOs, 2'-fucosyllactose (2'-FL) and lacto-N-tetraose (LNT), respectively. For CTB5 and its interaction with 2'-FL, a novel binding site was localized for 2'-FL, different from the one for native receptor GM1-os. For TcdA-A2 and its interaction with LNT, however, the localized binding site was the same as its native carbohydrate receptor CD-grease. A HDX-MS based titration method Protein-Ligand Interactions in solution by Mass Spectrometry, Titration and hydrogen/deuterium Exchange (PLIMSTEX), was also applied to CTB5 and its interactions GM1-os, to test the reliability of using peptides as indicators to obtain the protein-carbohydrate binding affinities. The average apparent association constant measured for the addition of GM1-os to CTB at pH 7.0 and 20 °C was found to be (1.6 ± 0.2) * 106 M-1. This is in reasonable agreement with the reported value of (3.2 ± 0.2) * 106 M-1, which was measured using direct ESI-MS assay at pH 6.9 and room temperature.

Mass Spectrometry in Biophysics

Author : Igor A. Kaltashov
Publisher : John Wiley & Sons
ISBN 13 : 0471705160
Total Pages : 320 pages
Book Rating : 4.4/5 (717 download)

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Book Synopsis Mass Spectrometry in Biophysics by : Igor A. Kaltashov

Download or read book Mass Spectrometry in Biophysics written by Igor A. Kaltashov and published by John Wiley & Sons. This book was released on 2005-05-06 with total page 320 pages. Available in PDF, EPUB and Kindle. Book excerpt: The first systematic summary of biophysical mass spectrometrytechniques Recent advances in mass spectrometry (MS) have pushed the frontiersof analytical chemistry into the biophysical laboratory. As aresult, the biophysical community's acceptance of MS-based methods,used to study protein higher-order structure and dynamics, hasaccelerated the expansion of biophysical MS. Despite this growing trend, until now no single text has presentedthe full array of MS-based experimental techniques and strategiesfor biophysics. Mass Spectrometry in Biophysics expertly closesthis gap in the literature. Covering the theoretical background and technical aspects of eachmethod, this much-needed reference offers an unparalleled overviewof the current state of biophysical MS. Mass Spectrometry inBiophysics begins with a helpful discussion of general biophysicalconcepts and MS-related techniques. Subsequent chaptersaddress: * Modern spectrometric hardware * High-order structure and dynamics as probed by various MS-basedmethods * Techniques used to study structure and behavior of non-nativeprotein states that become populated under denaturingconditions * Kinetic aspects of protein folding and enzyme catalysis * MS-based methods used to extract quantitative information onprotein-ligand interactions * Relation of MS-based techniques to other experimental tools * Biomolecular properties in the gas phase Fully referenced and containing a helpful appendix on the physicsof electrospray mass spectrometry, Mass Spectrometry in Biophysicsalso offers a compelling look at the current challenges facingbiomolecular MS and the potential applications that will likelyshape its future.

Protein-ligand Interactions, Structure and Spectroscopy

Author : Stephen E. Harding
Publisher : Oxford University Press, USA
ISBN 13 : 9780199637478
Total Pages : 474 pages
Book Rating : 4.6/5 (374 download)

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Book Synopsis Protein-ligand Interactions, Structure and Spectroscopy by : Stephen E. Harding

Download or read book Protein-ligand Interactions, Structure and Spectroscopy written by Stephen E. Harding and published by Oxford University Press, USA. This book was released on 2001 with total page 474 pages. Available in PDF, EPUB and Kindle. Book excerpt: This text on protein-ligand interactions offers a selection of the most useful and easily applied methods and acts as a guide to the principal techniques used.

Hydrogen Deuterium Exchange Mass Spectrometry for Protein-protein Interaction and Structural Dynamics

Author : Harsimran Singh
Publisher :
ISBN 13 :
Total Pages : 159 pages
Book Rating : 4.:/5 (87 download)

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Book Synopsis Hydrogen Deuterium Exchange Mass Spectrometry for Protein-protein Interaction and Structural Dynamics by : Harsimran Singh

Download or read book Hydrogen Deuterium Exchange Mass Spectrometry for Protein-protein Interaction and Structural Dynamics written by Harsimran Singh and published by . This book was released on 2013 with total page 159 pages. Available in PDF, EPUB and Kindle. Book excerpt: 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.

Biological Mass Spectrometry

Author : A.L. Burlingame
Publisher : Gulf Professional Publishing
ISBN 13 : 9780121828073
Total Pages : 520 pages
Book Rating : 4.8/5 (28 download)

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Book Synopsis Biological Mass Spectrometry by : A.L. Burlingame

Download or read book Biological Mass Spectrometry written by A.L. Burlingame and published by Gulf Professional Publishing. This book was released on 2005-11-28 with total page 520 pages. Available in PDF, EPUB and Kindle. Book excerpt: Describes and integrates the techniques of many advances in both chromatographic and mass spectrometric technologies. This book also covers various biophysical applications, such as H/D exchange for study of conformations, protein-protein and protein-metal and ligand interactions. It also describes atto-to-zepto-mole quantitation of 14C and 3H.

Mass Spectrometry-based Strategies for Protein Characterization

Author : Ke Li (Chemist)
Publisher :
ISBN 13 :
Total Pages : 189 pages
Book Rating : 4.:/5 (19 download)

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Book Synopsis Mass Spectrometry-based Strategies for Protein Characterization by : Ke Li (Chemist)

Download or read book Mass Spectrometry-based Strategies for Protein Characterization written by Ke Li (Chemist) and published by . This book was released on 2018 with total page 189 pages. Available in PDF, EPUB and Kindle. Book excerpt: Mass spectrometry (MS)-based protein footprinting characterizes protein structure and protein-ligand interactions by interrogating protein solvent-accessible surfaces by using chemical reagents as probes. The method is highly applicable to protein or protein-ligand complexes that are difficult to study by conventional means such as X-ray crystallography and nuclear magnetic resonance. In this dissertation, we describe the development and application of MS-based protein footprinting from three perspectives, including I) protein aggregation and amyloid formation (Chapter 2-3), II) protein-ligand interactions (Chapter 4-5), and III) in-cellulo structures and dynamic motion of membrane proteins (Chapter 6). Fast Photochemical Oxidation of Proteins (FPOP) is the main methodology implemented in the work presented in this dissertation. Chapter 1 provides an overview of FPOP and discusses its fundamentals as well as its important applications in both academic research and biotechnology drug development. In Part I, Chapter 2 covers the early method development of FPOP for monitoring amyloid beta (A[beta]) aggregation. In this work, we demonstrated the high sensitivity and spatial resolution of the method in probing the solvent accessibility of A[beta] at global, sub-regional, and some amino-acid residue levels as a function of its aggregation, and revealed A[beta] species at various oligomeric states identified by their characteristic modification levels. In Chapter 3, we extended the application of the platform to assess the effect of a putative polyphenol inhibitor on amyloid formation and to provide insights into the mechanism of action of the inhibitor in remodeling A[beta] aggregation pathways. In Part II, we evaluated different protein footprinting techniques, including FPOP, hydrogen-deuterium exchange (HDX), and carboxyl group footprinting, for probing protein-ligand (drug candidates) interaction in the context of a therapeutic development. Chapter 4 focused on protein-protein interaction by investigating the epitope of IL-6 receptor for two adnectins that have similar apparent biophysical properties. In Chapter 5, we probed the hydrophobic binding cavity of bromodomain protein for a small molecule inhibitor. This study serves as an example of interrogating protein-small molecule interactions. The two studies in Part II demonstrate the unique capabilities and limitations of protein footprinting methods in protein structural characterization. In Part III, we pushed the boundary of MS-based protein footprinting by applying the method to footprint live cells and investigate the dynamic structures/motion of membrane-transport proteins in their native cellular environment. We employed protein engineering, suspension cell expression and isotopic-encoded carboxyl group footprinting to identify salt bridges in two proteins, GLUT1 and GLUT5, that control their alternating access motions for substrate translocation. With functional analysis and mutagenesis, live-cell footprinting provides new insights into the transport mechanism of proteins in the major facilitator superfamily. The five studies in the dissertation demonstrate the powerful capability of MS-based protein footprinting in protein structural biology and biophysics research. The method also holds great potential in studying more complicated biological systems and solving demanding problems related to protein structure and properties.

Mass Spectrometry-based Strategies for Protein Footprinting

Author : Jing Li
Publisher :
ISBN 13 :
Total Pages : 198 pages
Book Rating : 4.:/5 (958 download)

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Book Synopsis Mass Spectrometry-based Strategies for Protein Footprinting by : Jing Li

Download or read book Mass Spectrometry-based Strategies for Protein Footprinting written by Jing Li and published by . This book was released on 2016 with total page 198 pages. Available in PDF, EPUB and Kindle. Book excerpt: Mass spectrometry (MS) has emerged as a powerful tool for epitope mapping, protein-ligand interaction, protein-protein interaction, aggregation, and effect of solution environment on protein conformation because they provide high-throughput data with relatively high structural resolution. Two popular MS-based approaches are hydrogen deuterium exchange-mass spectrometry (HDX-MS) and fast photochemical oxidation of proteins (FPOP), which complement classical biophysical and biochemical techniques in achieving higher structural resolution. The research presented in this dissertation is focused on the application of mass spectrometry-based footprinting techniques in characterizing the biophysical properties of Part I: pH-dependent conformation change of diphtheria toxin T domain (Chapters 2-4)); Part II: Ca2+ binding proteins and the role of Ca2+ regulation (Chapters 5-6); and Part III: protein-protein interaction including epitope mapping of IL-23 (Chapter 7) and Marburg virus protein VP24 (Chapter 8). Chapter 1 serves as an introduction to mass spectrometry instrumentation and standard LC-MS workflow. Two mass spectrometry based-footprinting techniques are introduced: (1) hydrogen deuterium exchange (HDX), and (2) fast photochemical oxidation of proteins (FPOP). Part I focuses on the development of pH-dependent HDX-MS for the conformation study of diphtheria toxin T domain. In Chapter 2, we describe the use pH-dependent HDX to study the pH-dependent conformation change of wild-type diphtheria toxin T domain monomer along its translocation pathway. In Chapter 3, we study the pH-dependent dissociation and reformation of T domain dimer. In Chapter 4, we apply the same method to a T domain mutant H223Q to further investigate the role of key histidine residues in triggering the conformation change. Part II focuses on the application of HDX mass spectrometry for the study of calcium binding proteins. Chapter 5 describes the Ca2+-binding property of ACaM and its Ca2+-regulated interaction with myosin VI. In chapter 6, HDX is also applied to an EF-hand Ca2+ binding protein, DREAM, for the study of its Ca2+ binding sites and stoichiometry. Part III of the dissertation focuses on the development and application of MS-based footprinting methods to investigate protein-protein interaction. Chapter 7 describes the methodology of fast photochemical oxidation of proteins (FPOP) for epitope mapping of IL-23 interacting a therapeutic antibody from Bristol-Myers Squibb. Chapter 8 discusses the use of HDX, FPOP, and NEM chemical labeling for the study of Marburg virus protein VP24 and its interaction with the host protein Keap1 Kelch domain. These seven studies on characterization of protein conformation dynamics, Ca2+ binding protein, and protein-protein interaction show the successful application of mass spectrometry in the structural study of large biomolecules.

Protein Complex Assembly

Author : Joseph A. Marsh
Publisher : Humana
ISBN 13 : 9781493992775
Total Pages : 506 pages
Book Rating : 4.9/5 (927 download)

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Book Synopsis Protein Complex Assembly by : Joseph A. Marsh

Download or read book Protein Complex Assembly written by Joseph A. Marsh and published by Humana. This book was released on 2019-04-15 with total page 506 pages. Available in PDF, EPUB and Kindle. Book excerpt: This volume details the importance of multiple experimental techniques and computational methods needed to obtain the comprehensive picture of protein complex structure, dynamics and assembly afforded by the emerging field of integrative structural biology. Chapters guide readers through the broad spectrum of approaches required for a complete representation of protein complexes, including expression and purification, experimental characterization of structure and assembly, and computational methods for identifying protein complexes and modelling their assembly. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, Protein Complex Assembly: Methods and Protocols aims to ensure successful results in the further study of this vital field.

Protein Mass Spectrometry

Author : Julian Whitelegge
Publisher : Elsevier
ISBN 13 : 0080932037
Total Pages : 563 pages
Book Rating : 4.0/5 (89 download)

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Book Synopsis Protein Mass Spectrometry by : Julian Whitelegge

Download or read book Protein Mass Spectrometry written by Julian Whitelegge and published by Elsevier. This book was released on 2008-10-09 with total page 563 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book is designed to be a central text for young graduate students interested in mass spectrometry as it relates to the study of protein structure and function as well as proteomics. It is a definite must-have work for:- libraries at academic institutions with Master and Graduate programs in biochemistry, molecular biology, structural biology and proteomics- individual laboratories with interests covering these areas - libraries and individual laboratories in the pharmaceutical and biotechnology industries. *Serves as an essential reference to those working in the field*Incorporates the contributions of prominent experts *Features comprehensive coverage and a logical structure

Peptide-level Biophysical Characterization of Protein-ligand Interactions by H/D Exchange Mass Spectrometry

Author : Justin Baxter Sperry
Publisher :
ISBN 13 :
Total Pages : 372 pages
Book Rating : 4.:/5 (261 download)

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Book Synopsis Peptide-level Biophysical Characterization of Protein-ligand Interactions by H/D Exchange Mass Spectrometry by : Justin Baxter Sperry

Download or read book Peptide-level Biophysical Characterization of Protein-ligand Interactions by H/D Exchange Mass Spectrometry written by Justin Baxter Sperry and published by . This book was released on 2008 with total page 372 pages. Available in PDF, EPUB and Kindle. Book excerpt: