Chemical Crosslinking and Mass Spectrometry Studies of the Structure and Dynamics of Membrane Proteins and Receptors PDF Download
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Author: Joseph S. Schoeniger
Publisher:
ISBN:
Category :
Languages : en
Pages : 60
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Book Description
Membrane proteins make up a diverse and important subset of proteins for which structural information is limited. In this study, chemical cross-linking and mass spectrometry were used to explore the structure of the G-protein-coupled photoreceptor bovine rhodopsin in the dark-state conformation. All experiments were performed in rod outer segment membranes using amino acid 'handles' in the native protein sequence and thus minimizing perturbations to the native protein structure. Cysteine and lysine residues were covalently cross-linked using commercially available reagents with a range of linker arm lengths. Following chemical digestion of cross-linked protein, cross-linked peptides were identified by accurate mass measurement using liquid chromatography-fourier transform mass spectrometry and an automated data analysis pipeline. Assignments were confirmed and, if necessary, resolved, by tandem MS. The relative reactivity of lysine residues participating in cross-links was evaluated by labeling with NHS-esters. A distinct pattern of cross-link formation within the C-terminal domain, and between loop I and the C-terminal domain, emerged. Theoretical distances based on cross-linking were compared to inter-atomic distances determined from the energy-minimized X-ray crystal structure and Monte Carlo conformational search procedures. In general, the observed cross-links can be explained by re-positioning participating side-chains without significantly altering backbone structure. One exception, between C3 16 and K325, requires backbone motion to bring the reactive atoms into sufficient proximity for cross-linking. Evidence from other studies suggests that residues around K325 for a region of high backbone mobility. These findings show that cross-linking studies can provide insight into the structural dynamics of membrane proteins in their native environment.
Author: Joseph S. Schoeniger
Publisher:
ISBN:
Category :
Languages : en
Pages : 60
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Book Description
Membrane proteins make up a diverse and important subset of proteins for which structural information is limited. In this study, chemical cross-linking and mass spectrometry were used to explore the structure of the G-protein-coupled photoreceptor bovine rhodopsin in the dark-state conformation. All experiments were performed in rod outer segment membranes using amino acid 'handles' in the native protein sequence and thus minimizing perturbations to the native protein structure. Cysteine and lysine residues were covalently cross-linked using commercially available reagents with a range of linker arm lengths. Following chemical digestion of cross-linked protein, cross-linked peptides were identified by accurate mass measurement using liquid chromatography-fourier transform mass spectrometry and an automated data analysis pipeline. Assignments were confirmed and, if necessary, resolved, by tandem MS. The relative reactivity of lysine residues participating in cross-links was evaluated by labeling with NHS-esters. A distinct pattern of cross-link formation within the C-terminal domain, and between loop I and the C-terminal domain, emerged. Theoretical distances based on cross-linking were compared to inter-atomic distances determined from the energy-minimized X-ray crystal structure and Monte Carlo conformational search procedures. In general, the observed cross-links can be explained by re-positioning participating side-chains without significantly altering backbone structure. One exception, between C3 16 and K325, requires backbone motion to bring the reactive atoms into sufficient proximity for cross-linking. Evidence from other studies suggests that residues around K325 for a region of high backbone mobility. These findings show that cross-linking studies can provide insight into the structural dynamics of membrane proteins in their native environment.
Author: David D. Weis
Publisher: John Wiley & Sons
ISBN: 1118616499
Category : Science
Languages : en
Pages : 422
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Book Description
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.
Author: Nagarajan Vaidehi
Publisher: Humana Press
ISBN: 9781627030243
Category : Science
Languages : en
Pages : 357
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Book Description
Membrane proteins play key roles in numerous cellular processes, in particular mediating cell-to-cell communication and signaling events that lead to a multitude of biological effects. Membrane proteins have also been implicated in many critical diseases such as atherosclerosis, hypertension, diabetes and cancer. In Membrane Protein Structure Predictions Methods: Methods and Protocols, expert researcher in the field detail the advances in both experimental and computational approaches of the structure, dynamics and interactions of membrane proteins dividing the volume into two sections. The first section details the procedures used for measurements of structure and dynamics of membrane proteins. While the second section contains a survey of the computational methods that have played a critical role in membrane protein structure prediction as well as in providing atomic level insight into the mechanism of the dynamics of membrane receptors. Written in the highly successful Methods in Molecular BiologyTM series format, the chapters include the kind of detailed description and implementation advice that is crucial for getting optimal results in the laboratory. Thorough and intuitive, Membrane Protein Structure Predicitons: Methods and Protocols seeks to aid scientists in the further study of membrane protein structure and function.
Author: Norbert Latruffe
Publisher: Springer Science & Business Media
ISBN: 3642739059
Category : Science
Languages : en
Pages : 290
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Book Description
This manual on "Dynamics of Membrane Proteins and Cellular Energetics" is the result of a FEBS-CNRS Course held in Grenoble and Besanc;{on in September 1987.' It appears to be, after the first, published in 1979 the fifth of the series. After focussing on the "Biochemistry of Membranes" (1979) it was the turn of "Membrane Proteins" (1981) and of Enzymes, Receptors and Carriers of Biological Membranes (1983), fo1- wed by "Membrane Proteins Isolation and Characterization" ( 1986) . Al though the central issue has always been the its components and its functions, each biological membrane, manual has put the accent on somewhat different issues corresponding to the most innovative, interesting research in the field. After almost a decade this new manual appears, which stresses the aspect of the integration of membrane research at a cellular level. Such a novel emphasis is the consequence of the common interest of cell biology and biochemistry to understand the results of the biochemical analysis of membrane proteins in the context of the cell complexity. Consequently most of the experimental protocols are dealing with cellular models, but with clear reference to the function and structure of isolated membrane proteins.
Author: Stephen H White
Publisher: Springer
ISBN: 1461475155
Category : Science
Languages : en
Pages : 403
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Book Description
Studies of receptors, ion channels, and other membrane proteins require a solid understanding of the structural principles of these important biomolecules. Membrane protein structure is, however, a very challenging field. The structures of only three types of transmembrane proteins have been determined to moderate or high resolution during the last two decades, a period during which the amino acid sequences of hundreds, if not thousands, of membrane proteins have been reported. As a result, the creation of structural models to serve as guides for studies of receptors, channels, and other membrane proteins has become crucially important. This book has been assembled in order to share the experiences and findings of expert researchers in protein structure and structure-prediction methods as well as membrane biophysics and lipid physical chemistry, whose work establishes the basis for the development of suitable model structures. The reviews presented here emphasize fundamental ideas and provide an entry to the diverse and complex literature. The four major sections deal with the general nature of the membrane protein structure problem, biochemical and molecular biological approaches to protein topology, direct structural methods, and model and physicochemical approaches. The work will be of interest to physiologists, cellular and molecular biologists, biophysicists, and biochemists working on the function of membrane proteins such as receptors, ion channels, and transporters, as well as senior graduate students and independent investigators.
Author: Mario Díaz
Publisher: Frontiers Media SA
ISBN: 2889457192
Category :
Languages : en
Pages : 85
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Book Description
Author: Yan Pan
Publisher:
ISBN:
Category :
Languages : en
Pages : 400
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Book Description
Membrane proteins continue to represent a major challenge for most analytical techniques. Using bacteriorhodopsin (BR) as model system, this work aims to develop mass spectrometry (MS)-based approaches for exploring the structure, dynamics and folding of membrane proteins. As the first step, BR in its native lipid environment was exposed to hydroxyl radicals, which were produced by laser photolysis of hydrogen peroxide. It was found that the resulting methionine (Met) labeling pattern was consistent with the known BR structure. This finding demonstrates that laser-induced oxidative Met labeling can provide structural information on membrane proteins. In subsequent experiments, the effects of different denaturing agents (heat, acid, and SDS) on the BR conformation were investigated. It was demonstrated that each of these non-native conditions results in unique structural features that give rise to characteristic Met labeling patterns. These results highlight the ability of laser-induced oxidative labeling to detect conformational changes of membrane proteins. Obtaining better insights into the structural properties of SDS-denatured BR is particularly important because this form of protein is widely used as starting point for folding studies. Combining oxidative labeling with site-directed mutagenesis and fluorescence measurements, this work yielded a detailed structural model of SDS-denatured BR. Subsequently, pulsed oxidative labeling coupled with rapid mixing and MS was used to characterize short-lived intermediates that become populated during BR refolding. The combination of pulsed oxidative labeling and stopped-flow spectroscopy provided key structural insights into the kinetic mechanism by which the SDS-denatured protein inserts and folds into the lipid bilayer. Complementary to oxidative labeling, hydrogen/deuterium exchange (HDX) MS was employed to examine the structure and dynamics of BR under various physiochemical conditions. Structural features of different detergent/lipid-bound BR samples were characterized by their HDX kinetics. Comparative HDX experiments of BR were carried out in the dark (resting state) and under illumination where the induced retinal isomerization mediates proton transport (functioning state). Isotope exchange was found to be much faster during light exposure than in the dark. This observation reveals that structural dynamics of the protein scaffold are "accelerated" by motions of the retinal reflecting a direct coupling between protein dynamics and function.
Author: A. Pullman
Publisher: Springer Science & Business Media
ISBN: 9401127182
Category : Science
Languages : en
Pages : 500
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Book Description
The 25th Jerusalem Symposium represents a most significant highlight in the development and history of these meetings. Living within the decimal system we have celebrated with much pleasure the lath and the 20th Jerusalem Symposia. With this one we experience a feeling of particular satisfaction because 25 years is different from, is more than, two decades and a half. It is a quarter of a century. It seems thus as if we have changed the dimension of our endeavour. In no way do we loose the sense of modesty with respect to the significance of these meetings. For the organizers, however, they do represent a continuity of efforts which we feel happy to have been able to carry out. At this occasion it seems useful to say a few words about the origin of the Jerusalem Symposia and to recall the name of a colleague who played an essential role in their creation and has been a most efficient and devoted co organizer of the seven first of them. This was Professor Ernst Bergmann, one of the most distinguished founders of Israeli Science and a world famous physico-organic chemist.
Author: Anne Skaja Robinson
Publisher: John Wiley & Sons
ISBN: 3527634533
Category : Science
Languages : en
Pages : 631
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Book Description
Designed as a research-level guide to current strategies and methods of membrane protein production on the small to intermediate scale, this practice-oriented book provides detailed, step-by-step laboratory protocols as well as an explanation of the principles behind each method, together with a discussion of its relative advantages and disadvantages. Following an introductory section on current challenges in membrane protein production, the book goes on to look at expression systems, emerging methods and approaches, and protein specific considerations. Case studies illustrate how to select or sample the optimal production system for any desired membrane protein, saving both time and money on the laboratory as well as the technical production scale. Unique in its coverage of "difficult" proteins with large membrane-embedded domains, proteins from extremophiles, peripheral membrane proteins, and protein fragments.
Author:
Publisher: Elsevier
ISBN: 0443193436
Category : Science
Languages : en
Pages : 448
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Book Description
Advances in Protein Chemistry and Structural Biology, Volume 138 covers reviews of methodology and research in all aspects of protein chemistry, including purification/expression, proteomics, modeling and structural determination and design. Chapters in this release include Proteomic Applications in Identifying Protein-Protein Interactions, Understanding functions of eEF1 translation elongation factors beyond translation. A proteomic approach, Proteomics provides insights into theranostic potential of extracellular vesicles, Towards a shareable functional analysis of the structural proteome, Functional unfoldomics, In-silico Network Pharmacology Study on Glycyrrhiza glabra: Analyzing the Immune-Boosting phytochemical properties of Siddha Medicinal Plant against COVID-19, and more.Other chapters cover In silico Network Pharmacology Analysis and Molecular Docking Validation of Swasa Kudori for Screening Druggable Phytoconstituents of Asthma, Proteomics and Genomics Insights on Malignant Osteosarcoma, Application of functional proteomics in understanding RNA Virus-Mediated Infection, Biofilm proteome of Staphylococcus aureus: implications for therapeutic interventions to biofilm-associated infections, A computational pipeline elucidating functions of conserved hypothetical Trypanosoma cruzi proteins based on public proteomic data, Functional Proteomics based on Protein Microarray Technology for Biomedical Research, and an Analysis of endoglucanases production using proteomics and metatranscriptomics. - Includes new information about Protein Aggregation - Presents chapters by a wide range of leading experts - Cover new, cutting-edge information that will serve as an essential addition to any bookshelf or laboratory
