Download or read book Structural and Dynamics Characterization of Membrane Proteins Using Static Solid state Nuclear Magnetic Resonance Spectroscopy written by Conggang Li and published by . This book was released on 2007 with total page 116 pages. Available in PDF, EPUB and Kindle. Book excerpt: Solid-state nuclear magnetic resonance (NMR) provides a unique approach for structure determination and functional studies of membrane proteins in a native lipid bilayer environment. Here, polarization inversion spin exchange at magic angle (PISEMA) experiments of aligned samples were applied to study the proton channel, M2 protein transmembrane portion (M2-TMD) from influenza A virus and other intact full length membrane proteins. The challenges of membrane protein structure characterization utilizing static aligned sample, including sample preparation, sample stability induced by RF heating, PISEMA experiment set up were discussed.

Download or read book Characterization of Structure and Dynamics of Membrane Proteins from Solid state NMR written by Byungsu Kwon and published by . This book was released on 2018 with total page 144 pages. Available in PDF, EPUB and Kindle. Book excerpt: Solid-state nuclear magnetic resonance (ssNMR) spectroscopy is an essential tool for elucidating the structure, dynamics, and function of biomolecules. ssNMR is capable of studying membrane proteins in near-native lipid bilayers and is thus preferred over other biophysical techniques for characterizing the structure and dynamics of membrane proteins. This thesis primarily focuses on the study of the following membrane proteins: 1) the N-terminal ectodomain and C-terminal cytoplasmic domain of influenza A virus M2 and 2) HIV-1 glycoprotein gp4l membrane-proximal external region and transmembrane domain (MPER-TMD) in a near native membrane environment. The cytoplasmic domain of M2 is necessary for membrane scission and virus shedding. The M2(22-71) construct shows random-coil chemical shifts, large motional amplitudes, and a membrane surface-bound location with close proximity to water, indicating the post-amphipathic helix (AH) cytoplasmic domain is a dynamic random coil near the membrane surface. The influenza M2 ectodomain contains highly conserved epitopes but its structure is largely unknown. The M2(1-49) construct containing both the ectodomain and transmembrane domain exhibits an entirely unstructured ectodomain with a motional gradient in which the motion is slower for residues near the TM domain, which attributed to the formation of a tighter helical bundle in the presence of drug that should cause the more tightened C-terminal ectodomain, thereby slowing its local motions. HIV-1 virus gp4l is directly involved in virus-cell membrane fusion. However, the structural topologies of the gp4l MPER-TMD are still controversial and the biologically-relevant intrinsic conformational state of MPER has not yet been determined. In order to obtain near native structural information of gp4l, we have studied gp41 (665-704) and found a primarily a-helical conformation, membrane-anchored trimeric TMD and water-exposed membrane surface-bound MPER. Intra- and intermolecular distances measured using 19C-19F REDOR and 19F-19F CODEX revealed that MPER-TMD has a significant kink between MPER and TMD, which has aided a deeper understanding of the HIV virus entry mechanism and the design of vaccines.

Download or read book Advances in Biological Solid State NMR written by Frances Separovic and published by Royal Society of Chemistry. This book was released on 2014 with total page 632 pages. Available in PDF, EPUB and Kindle. Book excerpt: Advances in Biological NMR brings the reader up to date with chapters from international leaders of this growing field, covering the most recent developments in the methodology and applications of solid state NMR to studies of membrane interactions and molecular motions

Download or read book Structural Biology in Drug Discovery written by Jean-Paul Renaud and published by John Wiley & Sons. This book was released on 2020-01-09 with total page 1367 pages. Available in PDF, EPUB and Kindle. Book excerpt: With the most comprehensive and up-to-date overview of structure-based drug discovery covering both experimental and computational approaches, Structural Biology in Drug Discovery: Methods, Techniques, and Practices describes principles, methods, applications, and emerging paradigms of structural biology as a tool for more efficient drug development. Coverage includes successful examples, academic and industry insights, novel concepts, and advances in a rapidly evolving field. The combined chapters, by authors writing from the frontlines of structural biology and drug discovery, give readers a valuable reference and resource that: Presents the benefits, limitations, and potentiality of major techniques in the field such as X-ray crystallography, NMR, neutron crystallography, cryo-EM, mass spectrometry and other biophysical techniques, and computational structural biology Includes detailed chapters on druggability, allostery, complementary use of thermodynamic and kinetic information, and powerful approaches such as structural chemogenomics and fragment-based drug design Emphasizes the need for the in-depth biophysical characterization of protein targets as well as of therapeutic proteins, and for a thorough quality assessment of experimental structures Illustrates advances in the field of established therapeutic targets like kinases, serine proteinases, GPCRs, and epigenetic proteins, and of more challenging ones like protein-protein interactions and intrinsically disordered proteins

Download or read book Structural Characterization of Membrane Proteins by Solid state NMR Spectroscopy written by and published by . This book was released on 2008 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Structural Characterization of Membrane Proteins by Solid state NMR Spectroscopy written by Karsten Seidel and published by . This book was released on 2008 with total page 213 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Solid State NMR Spectroscopy for Biopolymers written by Hazime Saitô and published by Springer Science & Business Media. This book was released on 2006-08-05 with total page 455 pages. Available in PDF, EPUB and Kindle. Book excerpt: ‘‘Biopolymers’’ are polymeric materials of biological origin, including globular, membrane, and fibrous proteins, polypeptides, nucleic acids, po- saccharides, lipids, etc. and their assembly, although preference to respe- ive subjects may be different among readers who are more interested in their biological significance or industrial and/or medical applications. Nevert- less, characterizing or revealing their secondary structure and dynamics may be an equally very important and useful issue for both kinds of readers. Special interest in revealing the 3D structure of globular proteins, nucleic acids, and peptides was aroused in relation to the currently active Structural Biology. X-ray crystallography and multidimensional solution NMR sp- troscopy have proved to be the standard and indispensable means for this purpose. There remain, however, several limitations to this end, if one intends to expand its scope further. This is because these approaches are not always straightforward to characterize fibrous or membrane proteins owing to extreme difficulty in crystallization in the former, and insufficient spectral resolution due to sparing solubility or increased effective molecular mass in the presence of surrounding lipid bilayers in the latter.

Download or read book Biological NMR Spectroscopy written by John L. Markley and published by Oxford University Press. This book was released on 1997-01-30 with total page 375 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book presents a critical assessment of progress on the use of nuclear magnetic resonance spectroscopy to determine the structure of proteins, including brief reviews of the history of the field along with coverage of current clinical and in vivo applications. The book, in honor of Oleg Jardetsky, one of the pioneers of the field, is edited by two of the most highly respected investigators using NMR, and features contributions by most of the leading workers in the field. It will be valued as a landmark publication that presents the state-of-the-art perspectives regarding one of today's most important technologies.

Download or read book Solid State NMR Spectroscopy written by Melinda J. Duer and published by John Wiley & Sons. This book was released on 2008-04-15 with total page 592 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book is for those familiar with solution-state NMR who are encountering solid-state NMR for the first time. It presents the current understanding and applications of solid-state NMR with a rigorous but readable approach, making it easy for someone who merely wishes to gain an overall impression of the subject without details. This dual requirement is met through careful construction of the material within each chapter. The book is divided into two parts: "Fundamentals" and "Further Applications." The section on Fundamentals contains relatively long chapters that deal with the basic theory and practice of solid-state NMR. The essential differences and extra scope of solid-state NMR over solution-state is dealt with in an introductory chapter. The basic techniques that all chapters rely on are collected into a second chapter to avoid unnecessary repetition later. Remaining chapters in the "Fundamentals" part deal with the major areas of solid-state NMR which all solid-state NMR spectroscopists should know about. Each begins with an overview of the topic that puts the chapter in context. The basic principles upon which the techniques in the chapter rely are explained in a separate section. Each of these chapters exemplifies the principles and techniques with the applications most commonly found in current practice. The "Further Applications" section contains a series of shorter chapters which describe the NMR techniques used in other, more specific areas. The basic principles upon which these techniques rely will be expounded only if not already in the Fundamentals part.

Download or read book Development of Solid state NMR Spectroscopy for Membrane Proteins written by Jiaozhi George Lu and published by . This book was released on 2014 with total page 220 pages. Available in PDF, EPUB and Kindle. Book excerpt: Atomic-resolution membrane protein structures can be determined by solid-state Nuclear Magnetic Resonance (NMR) spectroscopy, and the unique advantage of the approach is that membrane proteins reside in near-native lipid bilayer environment at physiological pH and temperature, which minimizes the potential distortions of the protein structure caused by the environment. Here, the full-length mercury transporter protein, MerF, is the focus of the structural studies, and the protein is an essential part of the bacterial mercury detoxification system that has been exploited as a potential engineering target for mercury bioremediation strategies. The backbone structures of the full-length MerF are determined in two environments, (i) magnetically aligned bicelles by oriented-sample (OS) solid-state NMR and (ii) proteoliposome by rotationally aligned (RA) solid-state NMR; and notably, both environments provide the planar lipid bilayer environment for the protein. The structural study of MerF in aligned bicelle has initially been challenging for the OS solid-state NMR, and consequently, methods have been developed to tackle the two major obstacles, the spectral resolution and resonance assignments. New pulse sequence, MSHOT-Pi4/Pi, has demonstrated a reduction of the 1H resonance line width by more than a factor of two, a significant improvement in spectral resolution. New resonance assignment method, Dipolar Coupling Correlated Isotropic Chemical Shift (DCCICS) Analysis, has been developed that is able to transfer resonance assignment from isotropic NMR methods to OS solid-state NMR spectra. The combined usage of several resonance assignment strategies and special tactics, such as applying DCCICS to the new high-resolution proton-evolved local field experiments for terminal and loop residues, has resulted in the complete assignment of all backbone immobile residues of the full-length MerF protein in magnetically aligned bicelle. Meanwhile, RA solid-state NMR is developed in the lab as a new method that combines the strength of magic-angle-spinning (MAS) solid-state NMR in obtaining resonance assignment and the concept of molecular alignment from OS solid-state NMR in obtaining angular restraints. In applying to the structural study of MerF, the method is further incorporated with multi-contact cross polarization and sequential backbone "walk" with three three-dimensional experiments, and the first structure of full-length MerF is determined with the method. In comparison to the previously determined structure of the truncated MerF (MerFt), the full-length structure reveals that the protein truncation has caused large conformational rearrangement at a place more than ten residues away from the truncation site, which serves as an example to demonstrate the importance of studying the full-length unmodified proteins by structural biologists. Additionally, the structure reveals that both mercury-binding sites are located at the intracellular side of the membrane, hinting at the observation of a conformation that allows intramolecular transfer of mercury ions. Subsequently after the complete assignment of MerF in OS solid-state NMR, the MerF structure determined by RA solid-state NMR is further improved by incorporating additional angular restraints from OS solid-state NMR and by the new treatment of dihedral restraints derived from the experimental study of C-terminal dynamics. Lastly, as a side project, the theoretical foundation of MSHOT-Pi4 pulse sequence is further explored. The observation that the pulse sequence selectively improves the resolution of membrane protein samples but not of standard single crystal sample has been analytically generalized as the principle of "motion-adapted" pulse sequence, where it is found that the interference between sample's spatial rotational motion and the radio-frequency pulse rotation in the quantum spin space is the cause of the selectivity. As a related endeavor, the mechanisms of dilute spin exchange and the magic-angle 1H spin-lock pulses have been analyzed theoretically and demonstrated in standard and biological samples. Mixed-order proton-relay mechanism is proposed to be the main contributor to dilute spin exchange in stationary aligned sample, and once more, the difference of pulse performance between standard and biological samples is observed that may be a consequence of several causes including sample motion. In conclusion, the development of various methods in OS and RA solid-state NMR are likely to find their usage in future structural studies of membrane proteins; the theoretical principle of motion-adapted property opens up new avenue to develop pulse sequences for membrane protein samples; and the atomic-resolution backbone structures of MerF contribute information for structural biologist and for the mechanistic study of mercury transportation.

Download or read book Structure and Dynamics of Membrane Proteins from Solid state NMR written by Myungwoon Lee and published by . This book was released on 2018 with total page 241 pages. Available in PDF, EPUB and Kindle. Book excerpt: Solid-state nuclear magnetic resonance (SSNMR) spectroscopy is an essential tool to elucidate the structure, dynamics, and function of biomolecules. This thesis mainly focuses on the structure determination of the hydrophobic domains for fusion proteins which are involved in membrane fusion between the cell membrane and viral envelope. Although extensive structural studies have been conducted on the soluble ectodomain by crystallography, the structural topologies of the hydrophobic TMD of the fusion proteins have been poorly understood. Here, we introduced SSNMR to investigate the secondary structure and oligomeric states of the TMD of two fusion proteins, PIV5 F and HIV gp41. For the PIV5 TMD, the membrane dependent secondary structure was determined by measuring the chemical shifts: predominant a-helical conformation in the POPC/cholesterol membrane shifts to the [Beta]-strand in the POPE membrane. Using 19F spin diffusion experiments on the fluorinated TMD, we have determined that the TMD forms a trimeric helical bundle. For the HIV gp4l MPER-TMD, we found the presence of a turn between the MPER helix and the TMD helix by measuring intramolecular distances and probing the lipid-peptide and water-peptide interactions. Intermolecular 19F- 19F distances of the fluorinated peptides indicate that the MPER-TMD is a trimeric. In addition to membrane fusion proteins, we have studied the oligomeric structure and the zinc-bound coordination geometry of a de novo designed amyloid fibril that catalyzes ester hydrolysis. By measuring the intermolecular contacts, we determined that peptides form parallel-in- register P-sheets and further assemble into stacked bilayers in an antiparallel orientation. The zinc binding sites were confirmed by the chemical shifts perturbation of histidines with zinc and the specific zinc-bound geometry was identified by measuring intra-residue distances of histidines. We also investigated the effects of cryoprotectants on the spectral resolution of lipid membranes and membrane peptides at low temperature. 13C and 1H MAS spectra of various cryoprotected membranes showed that DMSO provides the best resolution enhancement with the best ice formation retardation at low temperature and DLPE lipid exhibits the excellent resolution.

Download or read book Improved Methods for Characterization of Protein Dynamics by NMR spectroscopy and Studies of the EphB2 Kinase Domain written by Alexandra Ahlner and published by Linköping University Electronic Press. This book was released on 2015-04-15 with total page 79 pages. Available in PDF, EPUB and Kindle. Book excerpt: Proteins are essential for all known forms of life and in many lethal diseases protein failure is the cause of the disease. To understand proteins and the processes they are involved in, it is valuable to know their structures as well as their dynamics and interactions. The structures may not be directly inspected because proteins are too small to be visible in a light microscope, which is why indirect methods such as nuclear magnetic resonance (NMR) spectroscopy have to be utilized. This method provides atomic information about the protein and, in contrast to other methods with similar resolution, the measurements are performed in solution resulting in more physiological conditions, enabling analysis of dynamics. Important dynamical processes are the ones on the millisecond timeframe, which may contribute to interactions of proteins and their catalysis of chemical reactions, both of significant value for the function of the proteins. To better understand proteins, not only do we need to study them, but also develop the methods we are using. This thesis presents four papers about improved NMR techniques as well as a fifth where the kinase domain of ephrinB receptor 2 (EphB2) has been studied regarding the importance of millisecond dynamics and interactions for the activation process. The first paper presents the software COMPASS, which combines statistics and the calculation power of a computer with the flexibility and experience of the user to facilitate and speed up the process of assigning NMR signals to the atoms in the protein. The computer program PINT has been developed for easier and faster evaluation of NMR experiments, such as those that evaluate protein dynamics. It is especially helpful for NMR signals that are difficult to distinguish, so called overlapped peaks, and the soft- ware also converts the detected signals to the indirectly measured physical quantities, such as relaxation rate constants, principal for dynamics. Next are two new versions of the Carr-Purcell-Maiboom-Gill (CPMG) dispersion pulse sequences, designed to measure millisecond dynamics in a way so that the signals are more separated than in standard experiments, to reduce problems with overlaps. To speed up the collection time of the data set, a subset is collected and the entire data set is then reconstructed, by multi-dimensional decomposition co-processing. Described in the thesis is also a way to produce suitably labeled proteins, to detect millisecond dynamics at C? positions in proteins, using the CPMG dispersion relaxation experiment at lower protein concentrations. Lastly, the kinase domain of EphB2 is shown to be more dynamic on the millisecond time scale as well as more prone to interact with itself in the active form than in the inactive one. This is important for the receptor function of the protein, when and how it mediates signals. To conclude, this work has extended the possibilities to study protein dynamics by NMR spectroscopy and contributed to increased understanding of the activation process of EphB2 and its signaling mechanism.

Download or read book Membrane Protein Structure Determination written by Jean-Jacques Lacapère and published by Methods in Molecular Biology. This book was released on 2010-08-06 with total page 482 pages. Available in PDF, EPUB and Kindle. Book excerpt: Membrane proteins, representing nearly 40% of all proteins, are key components of cells involved in many cellular processes, yet only a small number of their structures have been determined. Membrane Protein Structure Determination: Methods and Protocols presents many detailed techniques for membrane protein structure determination used today by bringing together contributions from top experts in the field. Divided into five convenient sections, the book covers various strategies to purify membrane proteins, approaches to get three dimensional crystals and solve the structure by x-ray diffraction, possibilities to gain structural information for a membrane protein using electron microscopy observations, recent advances in nuclear magnetic resonance (NMR), and molecular modelling strategies that can be used either to get membrane protein structures or to move from atomic structure to a dynamic understanding of a molecular functioning mechanism. Written in the highly successful Methods in Molecular BiologyTM 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. Comprehensive and easy to use, Membrane Protein Structure Determination: Methods and Protocols serves as an ideal reference for scientists seeking to further our knowledge of these vital and versatile proteins as well as our overall understanding of the complicated world of cell biology.

Download or read book High resolution Structural Studies of Paramagnetic Proteins by Multidimensional Solid state Nuclear Magnetic Resonance Spectroscopy written by Philippe S. Nadaud and published by . This book was released on 2010 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Abstract: Nuclear magnetic resonance (NMR) is one of the major spectroscopic techniques available for the characterization of molecular structure and conformational dynamics with atomic level detail. NMR relies on the intrinsic magnetic properties of certain nuclear isotopes, such as 1H, 13C, 15N, and 31P, which provide convenient, site-specific structural probes when placed inside a large external magnetic field. Recent developments in solid-state NMR (SSNMR) spectroscopy promise to enable detailed structural studies to be performed for important biological macromolecules, which are inherently insoluble but at the same time cannot be readily crystallized for analysis by X-ray diffraction. Prominent examples of systems of this type include large macromolecular complexes, membrane-bound peptides and proteins important in cell signaling, and fibrillar protein aggregates associated with the development of systemic and neurodegenerative human disorders, including Alzheimer's and Parkinson's diseases and type II diabetes.

Download or read book Investigations of Membrane Protein Dynamics Using Solid State NMR written by Daryl Good and published by . This book was released on 2020 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Proteins populate ensembles of conformations at ambient temperature. Protein dynamics result from transitions between conformational states and occur over a broad range of time scales. Although dynamics play important roles in protein function, understanding how the proteins' interactions with the surrounding environment, e.g., with solvent, lipids and other proteins, affect the energetics of internal motions is a major challenge. In this Thesis, we focus on the analysis of motions in membrane proteins. They reside in a highly anisotropic environment of a lipid bilayer, and are not easily studied by commonly used techniques, such as x-ray crystallography, electron microscopy, or solution NMR. We use a novel technique of solid-state NMR (SSNMR) spectroscopy to investigate internal motions in two alpha-helical transmembrane microbial rhodopsins. First, we combine multidimensional SSNMR relaxation and order parameter measurements to characterize conformational dynamics of Anabaena Sensory Rhodopsin (ASR), a light sensing protein. We demonstrate that both fast picosecond and slow nanosecond motions occur in ASR; the amplitudes of the slower motions are small in the well-structured TM regions and increase towards the cytoplasmic ends of helices and the interhelical loop regions. Larger amplitudes of nanosecond motions on the cytoplasmic side of the TM helices correlates with the location of the binding sites where ASR is likely to undergo large conformational changes during the binding/unbinding of a soluble transducer. Second, we measure SSNMR bulk relaxation rates over the temperature range from 104 K - 289 K in the light-driven proton pump green proteorhodopsin (GPR). Using model-free analysis, we directly determine the activation energies of the local backbone and sidechain fluctuations as well as of the sidechain rotations and collective backbone and side chain motions. We relate these motions to the dynamics of the solvent and the lipids, thus highlighting the influence of the surrounding environment on membrane protein dynamics. From a structural perspective, ASR and GPR share their alpha-helical bundle architecture with other membrane proteins and in particular, with G-protein coupled receptors. Understanding the nature of motions in ASR and GPR provides a general framework to understanding the factors determining stability and motions in other membrane proteins.

Download or read book Structure Computation and Dynamics in Protein NMR written by N. Rama Krishna and published by Springer. This book was released on 2013-03-29 with total page 554 pages. Available in PDF, EPUB and Kindle. Book excerpt: Volume 17 is the second in a special topic series devoted to modern techniques in protein NMR, under the Biological Magnetic Resonance series. Volume 16, with the subtitle Modern Techniques in Protein NMR , is the first in this series. These two volumes present some of the recent, significant advances in the biomolecular NMR field with emphasis on developments during the last five years. We are honored to have brought together in these volume some of the world s foremost experts who have provided broad leadership in advancing this field. Volume 16 contains - vances in two broad categories: I. Large Proteins, Complexes, and Membrane Proteins and II. Pulse Methods. Volume 17 contains major advances in: I. Com- tational Methods and II. Structure and Dynamics. The opening chapter of volume 17 starts with a consideration of some important aspects of modeling from spectroscopic and diffraction data by Wilfred van Gunsteren and his colleagues. The next two chapters deal with combined automated assignments and protein structure determination, an area of intense research in many laboratories since the traditional manual methods are often inadequate or laborious in handling large volumes of NMR data on large proteins. First, Werner Braun and his associates describe their experience with the NOAH/DIAMOD protocol developed in their laboratory.

Download or read book Dynamic and Structural Studies of Membrane Proteins by Oriented Sample Solid State NMR written by Emmanuel Oluwaseyi Awosanya and published by . This book was released on 2020 with total page 97 pages. Available in PDF, EPUB and Kindle. Book excerpt: