Membrane Protein Crystallization at the Advanced Light Source (original) (raw)

Membrane protein structural biology-How far can the bugs take us?(Review)

2007

Membrane proteins are core components of many essential cellular processes, and high-resolution structural data is therefore highly sought after. However, owing to the many bottlenecks associated with membrane protein crystallization, progress has been slow. One major problem is our inability to obtain sufficient quantities of membrane proteins for crystallization trials. Traditionally, membrane proteins have been isolated from natural sources, or for prokaryotic proteins, expressed by recombinant techniques.

Progress in the analysis of membrane protein structure and function

FEBS Letters, 2002

Structural information on membrane proteins is sparse, yet they represent an important class of proteins that is encoded by about 30% of all genes. Progress has primarily been achieved with bacterial proteins, but e¡orts to solve the structure of eukaryotic membrane proteins are also increasing. Most of the structures currently available have been obtained by exploiting the power of X-ray crystallography. Recent results, however, have demonstrated the accuracy of electron crystallography and the imaging power of the atomic force microscope. These instruments allow membrane proteins to be studied while embedded in the bi-layer, and thus in a functional state. The low signal-to-noise ratio of cryo-electron microscopy is overcome by crystallizing membrane proteins in a two-dimensional proteinl ipid membrane, allowing its atomic structure to be determined. In contrast, the high signal-to-noise ratio of atomic force microscopy allows individual protein surfaces to be imaged at subnanometer resolution, and their conformational states to be sampled. This review summarizes the steps in membrane protein structure determination and illuminates recent progress. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

Membrane Protein Structure Determination

Methods in Molecular Biology, 2010

, except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. While the advice and information in this book are believed to be true and accurate at the date of going to press, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein. Cover illustration: Membrane protein determination starts from extraction-purification and reaches atomic structure by crystal formation and X-ray diffraction or electron microscopy analysis, or nuclear magnetic resonance studies combined or not with molecular modelling.

The fine art of integral membrane protein crystallisation

Methods, 2018

Integral membrane proteins are among the most fascinating and important biomolecules as they play a vital role in many biological functions. Knowledge of their atomic structures is fundamental to the understanding of their biochemical function and key in many drug discovery programs. However, over the years, structure determination of integral membrane proteins has proven to be far from trivial, hence they are underrepresented in the protein data bank. Low expression levels, insolubility and instability are just a few of the many hurdles one faces when studying these proteins. X-ray crystallography has been the most used method to determine atomic structures of membrane proteins. However, the production of high quality membrane protein crystals is always very challenging, often seen more as art than a rational experiment. Here we review valuable approaches, methods and techniques to successful membrane protein crystallisation.

Crystallization of membrane proteins

Trends in Biochemical Sciences, 1983

Five new membrane protein structures have been determined since 1995 using X-ray crystallography: bacterial light-harvesting complex; bacterial and mitochondrial cytochrome c oxidases; mitochondrial bc 1 complex; and α-hemolysin. These successes are partly based on advances in the crystallization procedures for integral membrane proteins. Variation of the size of the detergent micelle and/or increasing the size of the polar surface of the membrane protein is the most important route to well-ordered membrane protein crystals. The use of bicontinuous lipidic cubic phases also appears to be promising.

Structure determination of an integral membrane protein at room temperature from crystals in situ

The structure determination of an integral membrane protein using synchrotron X-ray diffraction data collected at room temperature directly in vapourdiffusion crystallization plates (in situ) is demonstrated. Exposing the crystals in situ eliminates manual sample handling and, since it is performed at room temperature, removes the complication of cryoprotection and potential structural anomalies induced by sample cryocooling. Essential to the method is the ability to limit radiation damage by recording a small amount of data per sample from many samples and subsequently assembling the resulting data sets using specialized software. The validity of this procedure is established by the structure determination of Haemophilus influenza TehA at 2.3 A˚ resolution. The method presented offers an effective protocol for the fast and efficient determination of membrane-protein structures at room temperature using thirdgeneration synchrotron beamlines.

Determining membrane protein structures: still a challenge!

Trends in Biochemical Sciences, 2007

Determination of structures and dynamics events of transmembrane proteins is important for the understanding of their function. Analysis of such events requires high-resolution 3D structures of the different conformations coupled with molecular dynamics analyses describing the conformational pathways. However, the solution of 3D structures of transmembrane proteins at atomic level remains a particular challenge for structural biochemists-the need for purified and functional transmembrane proteins causes a 'bottleneck'. There are various ways to obtain 3D structures: X-ray diffraction, electron microscopy, NMR and modelling; these methods are not used exclusively of each other, and the chosen combination depends on several criteria. Progress in this field will improve knowledge of ligand-induced activation and inhibition of membrane proteins in addition to aiding the design of membrane-protein-targeted drugs. Purification and characterization Because TMPs comprise a hydrophobic core inserted into the lipid bilayer and hydrophilic domains on either side of Review

Membrane protein structure determination — The next generation

Biochimica et Biophysica Acta (BBA) - Biomembranes, 2014

The field of Membrane Protein Structural Biology has grown significantly since its first landmark in 1985 with the first three-dimensional atomic resolution structure of a membrane protein. Nearly twenty-six years later, the crystal structure of the beta2 adrenergic receptor in complex with G protein has contributed to another landmark in the field leading to the 2012 Nobel Prize in Chemistry. At present, more than 350 unique membrane protein structures solved by X-ray crystallography (http://blanco.biomol.uci.edu/mpstruc/exp/list, Stephen White Lab at UC Irvine) are available in the Protein Data Bank. The advent of genomics and proteomics initiatives combined with high-throughput technologies, such as automation, miniaturization, integration and third-generation synchrotrons, has enhanced membrane protein structure determination rate. X-ray crystallography is still the only method capable of providing detailed information on how ligands, cofactors, and ions interact with proteins, and is therefore a powerful tool in biochemistry and drug discovery. Yet the growth of membrane protein crystals suitable for X-ray diffraction studies amazingly remains a fine art and a major bottleneck in the field. It is often necessary to apply as many innovative approaches as possible. In this review we draw attention to the latest methods and strategies for the production of suitable crystals for membrane protein structure determination. In addition we also highlight the impact that third-generation synchrotron radiation has made in the field, summarizing the latest strategies used at synchrotron beamlines for screening and data collection from such demanding crystals. This article is part of a Special Issue entitled: Structural and biophysical characterisation of membrane protein-ligand binding.