Current trends in α-helical membrane protein crystallization: an update.
ABSTRACT α-Helical membrane proteins (MPs) are the targets for many pharmaceutical drugs and play important roles in human physiology. In recent years, significant progress has been made in determining their atomic structure using X-ray crystallography. However, a major bottleneck in MP crystallography still remains, namely, the identification of conditions that give crystals that are suitable for structural determination. In 2008, we undertook an analysis of the crystallization conditions for 121 α-helical MPs to design a rationalized sparse matrix crystallization screen, MemGold. We now report an updated analysis that includes a further 133 conditions. The results reveal the current trends in α-helical MP crystallization with notable differences since 2008. The updated information has been used to design new crystallization and additive screens that should prove useful for both initial crystallization scouting and subsequent crystal optimization.
- SourceAvailable from: Patrick Douglas Shaw Stewart[Show abstract] [Hide abstract]
ABSTRACT: 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 proteins 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, miniaturisation, integration and third-generation synchrotrons, have 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.Biochimica et Biophysica Acta 07/2013; · 4.66 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Protein crystallization is known since 1840 and can be straightforward but in most cases constitutes a real bottleneck. This stimulated the birth of the biocrystallogenesis field with both 'practical' and 'basic' science aims. In the early time of biochemistry, crystallization was a tool for the preparation of biological substances. Today biocrystallogenesis aims to provide efficient methods for crystal fabrication and means to optimize crystal quality for X-ray crystallography. The historical development of crystallization methods for structural biology occurred first in conjunction with that of biochemical and genetic methods for macromolecule production, then with the development of structure determination methodologies, and recently with the routine access to synchrotron X-ray sources. In the past the finding of conditions sustaining crystal growth occurred mostly empirically but in the last decades is moving progressively towards more rationality as a result of a deeper understanding of the physical-chemistry of protein crystal growth and the use of idea-driven screening and high-throughput procedures. Protein and nucleic acid engineering procedures to facilitate crystallization as well as crystallization methods in gelled-media or by counter-diffusion are recent important achievements, although the underlying concepts are old. The new nano-technologies brought significant improvement in the practice of protein crystallization. Today, the increasing number of crystal structures deposited in Protein Data Banks, could mean that crystallization no more is a bottleneck. This is not the case, since structural biology projects become more challenging and thereby require adapted methods to grow the appropriate crystals, notably of macromolecular assemblages. This article is protected by copyright. All rights reserved.FEBS Journal 10/2013; · 4.25 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: In this report we highlight the latest trends in phasing methods used to solve alpha helical membrane protein structures and analyze the use of heavy atom metals for the purpose of experimental phasing. Our results reveal that molecular replacement is emerging as the most successful method for phasing alpha helical membrane proteins, with the notable exception of the transporter family, where experimentally derived phase information still remains the most effective method. To facilitate selection of heavy atoms salts for experimental phasing an analysis of these was undertaken and indicates that organic mercury salts are still the most successful heavy atoms reagents. Interestingly the use of seleno-L-methionine incorporated protein has increased since earlier studies into membrane protein phasing, so too the use of SAD and MAD as techniques for phase determination. Taken together this study provides a brief snapshot of phasing methods for alpha helical membrane proteins and suggests possible routes for heavy atom selection and phasing methods based on currently available data.Protein Science 08/2013; · 2.74 Impact Factor