Membrane Protein Purification And Crystallization

This second edition of Membrane Protein Purification and Crystallization, A Practical Guide is written for bench scientists working in the fields of biochemistry, biology, and proteomic research. This guide presents isolation and crystallization techniques in a concise form, emphasizing the critical aspects unique to membrane proteins. It explains the principles of the methods and provides protocols of general use, permitting researchers and students new to this area to adapt these techniques to their particular needs. This edition is not only an update but is comprised mainly of new contributions. It is the first monograph compiling the essential approaches for membrane protein crystallization, and emphasizes recent progress in production and purification of recombinant membrane proteins. Provides general guidelines and strategies for isolation and crystallization of membrane proteins Gives detailed protocols that have wide application, and low specialized equipment needs Emphasizes recent progress in production and purification of recombinant membrane proteins, especially of histidine-tagged and other affinity-epitope-tagged proteins Summarizes recent developments of Blue-Native PAGE, a high resolution separation technique, which is independent of the use of recombinant techniques, and is especially suited for proteomic analyses of membrane protein complexes Gives detailed protocols for membrane protein crystallization, and describes the production and use of antibody fragments for high resolution crystallization Presents a comprehensive guide to 2D-crystallization of membrane proteins

This volume of Current Topics in Membranes focuses on Membrane Protein Crystallization, beginning with a review of past successes and general trends, then further discussing challenges of mebranes protein crystallization, cell free production of membrane proteins and novel lipids for membrane protein crystallization. This publication also includes tools to enchance membrane protein crystallization, technique advancements, and crystallization strategies used for photosystem I and its complexes, establishing Membrane Protein Crystallization as a needed, practical reference for researchers.

The precise knowledge of the structure of biological macromolecules forms the basis of understanding their function and their mechanism of action. It also lays the foundation for rational protein and drug design. The only method to obtain this knowledge is still crystallography. At present, the structures of about 400 proteins are known at or nearly at atomic proteins. However, only two of them are membrane proteins or complexes of the membrane proteins. The reasons for the difference is not the crystals of membrane proteins resists forming special problems when being analysed. The reason is that the membrane proteins resist into forming into well-ordered crystals. The intention of this book is to help to produce well-ordered crystals proteins and to provide guidelines, it is aimed at both biochemists and protein crystallographer‘s.

A Practical Guide to Membrane Protein Purification is written especially for researchers who have some familarity with separation of water-soluble proteins, but who may not be aware of the pitfalls they face with membrane proteins. This guide presents techniques in a concise form, emphasizing the aspects unique to membrane proteins. The book explains the principles of the methods, permitting researchers and students new to this area to adapt these techniques to their particular needs. The second volume in the series, this book is an essential manual for investigations of structure and function of native membrane proteins, as well as for purification of these proteins for immunization and protein sequencing. Separation, Detection, and Characterization of Biological Macromolecules is a new series of laboratory guides. Each volume focuses on a topic of central interest to scientists and students in biomedical and biological research. Introductory chapters are followed by clear, step-by-step protocols that present principles and practice. These concise manuals are designed for optimal understanding of methods as well as for practical benchtop use. Provides general guidelines and strategies for isolation of membrane proteins Describes detailed practical procedures that have been the widest applications, and lowest specialized equipment needs Gives special emphasis to new native and denaturing electrophoresis techniques Explains modifications of techniques used for water-soluble proteins

Membrane Proteins – Engineering, Purification and Crystallization, a volume of Methods In Enzymology, encompasses chapters from the leading experts in the area of membrane protein biology. The chapters provide a brief overview of the topics covered and also outline step-by-step protocol for the interested audience. Illustrations and case example images are included wherever appropriate to help the readers understand the schematics and general experimental outlines. Volume of Methods In Enzymology Contains a collection of a diverse array of topics in the area of membrane protein biology ranging from recombinant expression, isolation, functional characterization, biophysical studies and crystallization

Knowledge of the three-dimensional structure of a protein is absolutely required for the complete understanding of its function. The spatial orientation of amino acids in the active site of an enzyme demonstrates how substrate specificity is defined, and assists the medicinal chemist in the design of s- cific, tight-binding inhibitors. The shape and contour of a protein surface hints at its interaction with other proteins and with its environment. Structural ana- sis of multiprotein complexes helps to define the role and interaction of each individual component, and can predict the consequences of protein mutation or conditions that promote dissociation and rearrangement of the complex. Determining the three-dimensional structure of a protein requires milligram quantities of pure material. Such quantities are required to refine crystallization conditions for X-ray analysis, or to overcome the sensitivity limitations of NMR spectroscopy. Historically, structural determination of proteins was limited to those expressed naturally in large amounts, or derived from a tissue or cell source inexpensive enough to warrant the use of large quantities of cells. H- ever, with the advent of the techniques of modern gene expression, many p- teins that are constitutively expressed in minute amounts can become accessible to large-scale purification and structural analysis.

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.