Membrane Protein Complexes

This edited book contains a compilation of 14 advanced academic chapters dealing with the structure and function of membrane protein complexes. This rapidly advancing important field of study closely parallels those on soluble protein complexes, and viral protein and nucleoprotein complexes. Diverse topics are included in this book, ranging from membrane–bound enzymes to ion channels, proton pumps and photosystems. Data from X-ray crystallography, cryo-electron microscopy and other biophysical and biochemical techniques are presented throughout the book. There is extensive use of colour figures of protein structures. Throughout the book structure and function are closely correlated. The two editors, Egbert Boekema and J. Robin Harris, have worked on aspects of membrane and soluble proteins throughout their scientific careers and also have much publishing experience. The Subcellular Biochemistry series has expanded considerably in recent years, including several related volumes. The theme of protein complexes will be continued within several future volumes, thereby creating encyclopaedic coverage. The chapter topics within this book are particularly relevant to those involved in the biological and biomedical sciences. It is aimed at the advanced undergraduates, postgraduates and established researchers within this broad field. It is hoped that the book will be of interest and use to those involved with the study of cellular membranes and their associated proteins.

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.

With the aim of providing a deeper insight into possible mechanisms of biological self-organization, this thesis presents new approaches to describe the process of self-assembly and the impact of spatial organization on the function of membrane proteins, from a statistical physics point of view. It focuses on three important scenarios: the assembly of membrane proteins, the collective response of mechanosensitive channels and the function of the twin arginine translocation (Tat) system. Using methods from equilibrium and non-equilibrium statistical mechanics, general conclusions were drawn that demonstrate the importance of the protein-protein interactions. Namely, in the first part a general aggregation dynamics model is formulated, and used to show that fragmentation crucially affects the efficiency of the self-assembly process of proteins. In the second part, by mapping the membrane-mediated forces into a simplified many-body system, the dynamic and equilibrium behaviour of interacting mechanosensitive channels is derived, showing that protein agglomeration strongly impacts its desired function. The final part develops a model that incorporates both the agglomeration and transport function of the Tat system, thereby providing a comprehensive description of this self-organizing process.

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

This book collects up-to-date advanced protocols and advice from leading experts in the area of membrane protein biology that can be applied to structural and functional studies of any membrane protein system. The contents explore methods for cloning and expression of membrane proteins and membrane protein complexes in prokaryotic and eukaryotic systems, approaches for protein purification, nanobody applications, as well as biophysical characterization and much more. Written for the highly successful Methods in Molecular Biology series, 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 thorough, Expression, Purification, and Structure Biology of Membrane Proteins serves to guide and encourage young researchers and newcomers to the field to tackle bold new studies on membrane proteins. Chapter 11 is available open access under a CC-BY 4.0 license via link.springer.com.

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