Membrane proteins represent about one third of the proteins encoded in a cell's genome, and, because of their key physiological roles, more than half of drug targets. Detergents are traditionally used to extract proteins from membranes in order to make them amenable to the tools of biochemistry and biophysics. However, detergent-solubilized proteins are generally unstable. This has led to the development of alternative, non-conventional surfactants, such as bicelles, nanodiscs, amphipathic peptides, fluorinated surfactants, and specially designed amphipathic polymers called 'amphipols'. These novel tools, mainly developed over the past 20 years, are revolutionizing handling membrane proteins in vitro for basic and applied research, as well as for such biomedical applications as drug screening or vaccination. This book, written by a specialist of membrane proteins and one of the creators of amphipols, describes the properties and usesof these novel molecules. It opens with general introductions on membrane proteins and their natural environment, detergents, the current status of membrane protein in vitro studies, a broad panorama of non-conventional surfactants and a discussion of their respective advantages and limitations, and the preparation and properties of amphipols and membrane protein/amphipol complexes. Topical chapters cover in vitro folding, cell-free synthesis and stabilization of membrane proteins, and such biophysical and biochemical applications as electron microscopy, Xray diffraction, NMR, optical spectroscopy, mass spectrometry, the whole range of solutions studies, proteomics, and such practical applications as membrane protein immobilization and drug screening and the use of amphipols in vivo for vaccination and drug delivery. Each topical chapter is introduced with a concise, up-to-date overview of how membrane proteins are currently studied using each individual technique, before offering an exhaustive coverage and in-depth discussion of the contribution of amphipols, and concluding with hands-on protocols written by everyday practitioners of each application. In addition to a comprehensive coverage of the properties and uses of non-conventional surfactants, this book therefore also offers a concise, accessible introduction to membrane protein biochemistry and biophysics. It is meant to be used both in basic and applied research laboratories and as a teaching help.

Autorentext

Jean-Luc Popot, born 1948, studied biology, chemistry, and biophysics in Orléans and Orsay. In 1971, he took a permanent position with the Collège de France. In J.P. Changeux's laboratory, at the Pasteur Institute, he applied electrophysiology and biophysical and biochemical approaches to functional and structural studies of the nicotinic acetyl­cho­li­ne receptor. In 1982, he joined D.M. Engelman at Yale University as a visiting scientist. His work at Yale, which bore princi­pal­ly on refolding bacterio­rhod­opsin from dena­tu­red fragments and studying the refolded structure crystallographically, led the two of them to propose, in 1990, an influential model for the folding of helical membrane proteins. In 1985, he joined the laboratory of P. Joliot at the Institut de Biologie Physico-Chimique (Paris). His group car­ried out neutron diffraction and model building work on bacterio­rhod­opsin and bio­che­mical studies on Photo­sys­tem II and cytochrome b6 f. In 1996, he became Research Director at the Centre National de la Recher­­che Scienti­fi­que and created his own laboratory, where the Xray struc­tu­re of the b6 f was solved in 2003. In parallel, he pursued the development of sequence analysis approa­ches and, in collabo­ra­tion with chemists and physical chemists, designed and validated non-conventional surfact­ants aimed at facilitating membrane protein solu­tion studies, most notably amphi­­pathic polymers ('amphipols') and fluorinated surfactants. He retired in 2013 and, along with his wife, splits his time between restoring an old house in Languedoc, reading, writing, hiking, photographing, cooking, and enjoying the company of kith and kin.

Klappentext

This book is the first to be entirely devoted to the challenging art of handling membrane proteins out of their natural environment, a key process in biological and pharmaceutical research, but one plagued with difficulties and pitfalls. Written by one of the foremost experts in the field, Membrane Proteins in Aqueous Solutions is accessible to any member of a membrane biology laboratory. After presenting the structure, functions, dynamics, synthesis, natural environment and lipid interactions of membrane proteins, the author discusses the principles of extracting them with detergents, the mechanisms of detergent-induced destabilization, countermeasures, and recent progress in developing detergents with weaker denaturing properties. Non-conventional alternatives to detergents, including bicelles, nanodiscs, amphipathic peptides, fluorinated surfactants and amphipols, are described, and their relative advantages and drawbacks are compared. The synthesis and solution properties of the various types of amphipols are presented, as well as the formation and properties of membrane protein/amphipol complexes and the transfer of amphipol-trapped proteins to detergents, nanodiscs, lipidic mesophases, or living cells. The final chapters of the book deal with applications: membrane protein in vitro folding and cell-free expression, solution studies, NMR, crystallography, electron microscopy, mass spectrometry, amphipol-mediated immobilization of membrane proteins, and biomedical applications.

Important features of the book include introductory sections describing foundations as well as the state-of-the-art for each of the biophysical techniques discussed, and topical tables which organize a widely dispersed literature. Boxes and annexes throughout the book explain technical aspects, and twelve detailed experimental protocols, ranging from in vitro folding of membrane proteins to single-particle electron cryomicroscopy, have been contributed by and commented on by experienced users.

Membrane Proteins in Aqueous Solutions offers a concise, accessible introduction to membrane protein biochemistry and biophysics, as well as comprehensive coverage of the properties and uses of conventional and non-conventional surfactants. It will be useful both in basic and applied research laboratories and as a teaching aid for students, instructors, researchers, and professionals within the field.

Inhalt

Membrane proteins: functions, structures, environments.- Taking membrane proteins out of their natural environment.- Alternatives to detergents for handling membrane proteins in aqueous solutions.- Chemical structure and physical-chemical properties of amphipols.- Formation and properties of membrane protein/amphipol complexes.- Amphipol-assisted folding of membrane proteins to their native state.- Amphipol-assisted cell-free expression of membrane proteins.- Amphipols and NMR.- Amphipols and crystallography.- Amphipols and electron microscopy.- Amphipols and radiation scattering.- Amphipol-assisted immobilization of membrane proteins onto solid supports for ligand-binding studies.- Amphipols and proteomics.- Amphipols as vectors for the delivery of membrane proteins and transmembrane peptides.- Perspectives.- Annex: protocols.- Trapping membrane proteins with amphipols.- Amphipol-assisted folding of membrane proteins.- Amphipol-assisted cell-free expression of membrane proteins.- Preparing membrane protein/amphipol complexes for solution NMR.- Preparing membrane protein/amphipol complexes for electron microscopy.- Amphipol-assisted transfer of membrane proteins to lipid phases for crystallization.- Preparing membrane protein/amphipol complexes for radiation scattering measurements.- Using tagged amphipols to immobilize membrane proteins onto solid …