Membrane protein structure determination — The next generation

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 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.

Monitoring changes of paramagnetically-shifted 31P signals in phospholipid vesicles

“…The use of other, often more dilute, NMR-active nuclei can alleviate this problem, depending on their location in or around the vesicle bilayer. For example, the functioning of ion channels, and their inhibition by various compounds such as toxins or anaesthetics has been investigated using 7 Li and 23 Na NMR and the use of paramagnetic shift reagents based on dysprosium (III) [15,16]. The lipids themselves, can be observed directly using the single phosphorus atom located in the head group of each molecule.…”

“…The ability to prepare a vesicle comprising only key components of the membrane allows the role of individual parts to be investigated. Vesicles, and lipid bilayers, are also used to support membrane proteins allowing study in an environment enabling the adoption of their native folds [6][7][8]. This technique, and its variations, allows membrane proteins such as ion channels to be investigated [9].…”

The Styrene-Maleic Acid Copolymer: A Versatile Tool in Membrane Research

“…To achieve extraction of different MPs from membranes with varying properties, a wide range of detergents with high solubilizing efficiency has been utilized (for reviews see Moraes et al 2014;Privé 2007;Seddon et al 2004). Although this approach without any doubt has contributed much to our understanding of MPs, detergent solubilization has some inherent disadvantages.…”

Ab Initio Semi-Quantitative Analysis of Micro-Beam Grazing-Incidence Small-Angle X-Ray Scattering (Μ-GISAXS) during Protein Crystal Nucleation and Growth

“…Problems and difficulties due to the identification of the precise crystallization conditions and parameters [6] are often encountered in classical crystallography, despite its advancements and achievements [7].…”