[PDF] Advanced Computational Techniques For Structure Determination Of Membrane Proteins By Solid State Nmr eBook

Author : Kala Bharath Pilla
Publisher :
Page : 0 pages
File Size : 26,20 MB
Release : 2015
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Computational modelling of proteins that rely on either de novo or evolutionary based approaches often produce poor quality structures, primarily due to the limitations in their algorithms or forcefields. Traditional experimental techniques such as X-ray crystallography depend on narrow set of crystallographic conditions while solution/solid state nuclear magnetic resonance (NMR) spectroscopy relies on cumbersome spectral analysis and complete resonance assignments. These traditional approaches are slow and costly endeavours. Computational/experimental hybrid approaches on the other hand provide a new avenue for reliable, rapid and cost-effective structure determination. Paramagnetic NMR offers easy generation of useful and sparse structural information which can be implemented as restraints in structure prediction algorithms. Pseudocontact shifts (PCS) are the most powerful of structural restraints generated by paramagnetic NMR which are long range in nature and can be easily obtained by simple 2D NMR experiments. This thesis demonstrates different approaches involved in protein structure calculations using PCS restraints in Rosetta. Chapter 2 demonstrates structure determination using PCS restraints exclusively obtained from protein samples in microcrystalline state by magic angle spinning (MAS) NMR spectroscopy. Chapter 3 discusses the implementation of using PCS data from multiple metal centres to precisely determine the location of spins in space in a manner analogues to GPS-satellites. Chapter 4 extends the usage of PCS data from multiple metal centres to capture distinct conformational states in proteins. Chapter 5 demonstrates new techniques especially developed for structure determination of large proteins involving super secondary structure motifs (Smotifs) and data driven iterative resampling. These different computational techniques serve the goal of determining accurate 3D models using minimal experimental data, which are applicable to proteins systems that are currently beyond the realm of traditional experimental approaches.

Author : Jean-Jacques Lacapère
Publisher : Methods in Molecular Biology
Page : 482 pages
File Size : 21,37 MB
Release : 2010-08-06
Category : Science
ISBN :

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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.

Author : Isabel Moraes
Publisher : Springer
Page : 188 pages
File Size : 15,15 MB
Release : 2016-08-23
Category : Science
ISBN : 3319350722

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This book reviews current techniques used in membrane protein structural biology, with a strong focus on practical issues. The study of membrane protein structures not only provides a basic understanding of life at the molecular level but also helps in the rational and targeted design of new drugs with reduced side effects. Today, about 60% of the commercially available drugs target membrane proteins and it is estimated that nearly 30% of proteins encoded in the human genome are membrane proteins. In recent years much effort has been put towards innovative developments to overcome the numerous obstacles associated with the structure determination of membrane proteins. This book reviews a variety of recent techniques that are essential to any modern researcher in the field of membrane protein structural biology. The topics that are discussed are not commonly found in textbooks. The scope of this book includes: Expression screening using fluorescent proteins The use of detergents in membrane protein research The use of NMR Synchrotron developments in membrane protein structural biology Visualisation and X-ray data collection of microcrystals X-ray diffraction data analysis from multiple crystals Serial millisecond crystallography Serial femtosecond crystallography Membrane protein structures in drug discovery The information provided in this book should be of interest to anyone working in the area of structural biology. Students will find carefully prepared overviews of basic ideas and advanced protein scientists will find the level of detail required to apply the material directly to their day to day work. Chapters 4, 5, 6, 8 and 9 of this book are published open access under a CC BY 4.0 license at link.springer.com.

Author : Lindsay J. Sperling
Publisher :
Page : pages
File Size : 22,49 MB
Release : 2011
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Membrane proteins help control nearly every process in the cell, which is why approximately 50% of pharmaceuticals currently on the market target membrane proteins. Knowledge of structure-function relationships of these proteins could be leveraged to produce more efficient drugs. However, the traits that make membrane proteins so interesting also make them difficult targets for traditional structure elucidation techniques. X-ray crystallography relies on the use of single crystals, production of which are elusive for membrane proteins due to their inherent dynamic loops and stretches of hydrophobic residues, which contribute to aggregation and/or loss of function without the presence of a lipid environment. Solution NMR experiences difficulty dealing with slow molecular tumbling due to the large sizes of membrane proteins. Conversely, solid-state NMR (SSNMR) has no inherent size limitation and does not require the use of crystals, which presents SSNMR with the unique capability to study membrane proteins in native environments at atomic-resolution. However, this technique is still a relatively new tool for solving structures of biomolecules. Here, we begin to develop strategies for solid-state NMR de novo structure determination. We provide a 0́−divide-and-conquer0́+ investigation of an E. coli 41 kDa membrane protein complex, DsbA/DsbB. We begin by completing chemical shift assignments, the first step in structure determination in NMR studies, of the 21 kDa protein DsbA to optimize sensitivity and resolution of data collection and analysis of large systems. We then use this study to drive forward structural examination of the disulfide bond forming system DsbA/DsbB. Finally, SSNMR techniques are used to study a 144 kDa cytochrome bo3 ubiquinol oxidase demonstrating the power of this technique to investigate large membrane complexes in native environments.

Author :
Publisher :
Page : 0 pages
File Size : 43,99 MB
Release : 2013
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Three-dimensional structures of proteins determined in solution by NMR spectroscopy have the unique advantage of revealing details of molecular structure and dynamics in a physiologically relevant state; however, the many tedious steps needed to solve and validate a structure make this method challenging. The barriers to NMR structure determination become higher for larger proteins whose spectra are harder to resolve. It is clear that advances need to be made in automating protein structure determination by NMR spectroscopy. The goal of my research has been to use computational methods to advance the development of high-throughput NMR spectroscopy. Accelerating and streamlining the structure determination process will enable investigators to spend less time solving structures and more time investigating challenging biomolecular systems. My goals have been to develop an automation protocol that integrates multiple steps, ensures the robustness of each step, incorporates iterative corrections, and includes visualization tools to validate and extend the results. I developed PINE-SPARKY as a graphical interface for checking and extending automated assignments made by the PINE-NMR server. ADAPT-NMR directs fast data collection by reduced dimensionality on the basis of ongoing NMR assignments. I helped develop a version of ADAPT-NMR (originally only for Varian spectrometers) for Bruker spectrometers, and I created ADAPT-NMR Enhancer as a visualization tool for validating and extending assignments made by ADAPT-NMR on either spectrometer system. I developed the PONDEROSA package to automate the next steps. PONDEROSA carries out automatic picking of 3D-NOESY peaks and iterative structure determinations with the protein sequence and the assignments as inputs. These automation and visualization tools cover almost all of the steps involved in protein structure determination by NMR spectroscopy. As a practical test of this technology, I solved the structure of the 2A proteinase from the human rhinovirus. As a side project, I built a relational database (PACSY DB) that combines information from the Protein Data Bank (PDB) and the Biological Magnetic Resonance data Bank (BMRB) and incorporates tools for structure analysis. PACSY DB can carry out complex queries that combine atomic coordinates, NMR parameters, and structural features of proteins.

Author : Xiaolan Yao
Publisher :
Page : 400 pages
File Size : 25,85 MB
Release : 2004
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Solid-state NMR is a powerful tool in the structure and dynamic studies of biological systems. In this thesis, novel techniques are developed for this purpose and various advanced solid-state NMR techniques are used to investigate the structure-function relationships of fibrous and membrane proteins. The chemical shift tensor serves as a sensitive probe of protein conformation. Techniques that combine dipolar modulation with CSA recoupling to determine the CSA tensor orientation under magic-angle spinning are developed. The C[alpha] CAS tensor of model helical and sheet peptides with known crystal structures are determined. The experimental results agree well with ab initio calculations. We use solid-state NMR to understand the structural basis of elastin function. Our hydration dynamics studies on an 81 kDa elastin-mimetic protein show that this protein is much more mobile at 30% hydration level compared to most other globular proteins. This high mobility contributes to the entropy of the system and facilitates the entropy-driven elasticity of elastin. Our high-resolution structural characterization of an elastin-mimetic peptide shows that the protein adopts two types of structures: about 35% of molecules exhibit compact turn structures and approximately 65% of molecules extended [beta]-strand structures. Conformational equilibrium between the two states may facilitate elastin extension and relaxation. Colicin Ia is a toxin that forms ion channels in the inner membrane of the target cell. To understand colicin channel function, we have carried out resonance assignment and dynamic studies on the 21 kDa channel domain of colicin Ia. A spectral editing technique that selects the more structurally informative non-bonded 1H-13C correlations in the 2D H-C HETCOR spectrum is developed and applied to colicin to obtain the 1H chemical shift assignment. Residue type assignments of 13C and 15N are obtained by various 2D correlation experiments under MAS. The topology and dynamics of membrane bound colicin under different ionic strengths and membrane surface charge densities are investigated by 1H spin diffusion and 2D isotropic-anisotropic correlation experiments. Our results show that the optimal channel activity of colicin requires a certain degree of segmental rigidity of the protein.

Author : Daniel Chasman
Publisher : CRC Press
Page : 534 pages
File Size : 27,30 MB
Release : 2003-03-18
Category : Medical
ISBN : 0824748166

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This text offers in-depth perspectives on every aspect of protein structure identification, assessment, characterization, and utilization, for a clear understanding of the diversity of protein shapes, variations in protein function, and structure-based drug design. The authors cover numerous high-throughput technologies as well as computational methods to study protein structures and residues. A valuable reference, this book reflects current trends in the effort to solve new structures arising from genome initiatives, details methods to detect and identify errors in the prediction of protein structural models, and outlines challenges in the conversion of routine processes into high-throughput platforms.