Membrane Protein Crystallography Research Articles

Overexpression of membrane proteins from higher eukaryotes in yeasts.

Heterologous expression and characterisation of the membrane proteins of higher eukaryotes is of paramount interest in fundamental and applied research. Due to the rather simple and well-established methods for their genetic modification and cultivation, yeast cells are attractive host systems for recombinant protein production. This review provides an overview on the remarkable progress, and discusses pitfalls, in applying various yeast host strains for high-level expression of eukaryotic membrane proteins. In contrast to the cell lines of higher eukaryotes, yeasts permit efficient library screening methods. Modified yeasts are used as high-throughput screening tools for heterologous membrane protein functions or as benchmark for analysing drug-target relationships, e.g., by using yeasts as sensors. Furthermore, yeasts are powerful hosts for revealing interactions stabilising and/or activating membrane proteins. We also discuss the stress responses of yeasts upon heterologous expression of membrane proteins. Through co-expression of chaperones and/or optimising yeast cultivation and expression strategies, yield-optimised hosts have been created for membrane protein crystallography or efficient whole-cell production of fine chemicals.

Femtosecond crystallography of membrane proteins in the lipidic cubic phase.

Despite recent technological advances in heterologous expression, stabilization and crystallization of membrane proteins (MPs), their structural studies remain difficult and require new transformative approaches. During the past two years, crystallization in lipidic cubic phase (LCP) has started gaining a widespread acceptance, owing to the spectacular success in high-resolution structure determination of G protein-coupled receptors (GPCRs) and to the introduction of commercial instrumentation, tools and protocols. The recent appearance of X-ray free-electron lasers (XFELs) has enabled structure determination from substantially smaller crystals than previously possible with minimal effects of radiation damage, offering new exciting opportunities in structural biology. The unique properties of LCP material have been exploited to develop special protocols and devices that have established a new method of serial femtosecond crystallography of MPs in LCP (LCP-SFX). In this method, microcrystals are generated in LCP and streamed continuously inside the same media across the intersection with a pulsed XFEL beam at a flow rate that can be adjusted to minimize sample consumption. Pioneering studies that yielded the first room temperature GPCR structures, using a few hundred micrograms of purified protein, validate the LCP-SFX approach and make it attractive for structure determination of difficult-to-crystallize MPs and their complexes with interacting partners. Together with the potential of femtosecond data acquisition to interrogate unstable intermediate functional states of MPs, LCP-SFX holds promise to advance our understanding of this biomedically important class of proteins.

Protein and lipid interactions driving molecular mechanisms of in meso crystallization.

The recent advances in the in meso crystallization technique for the structural characterization of G-protein coupled receptor (GPCR) proteins have established the usefulness of the lipidic-cubic phases (LCPs) in the field of crystallography of membrane proteins. It is surprising that despite the success of the approach, the molecular mechanisms of the in meso method are still not well understood. Therefore, the approach must rely on extensive screening for a suitable protein construct, for host and additive lipids, and for the appropriate precipitants and temperature. To shed light on the in meso crystallization mechanisms, we used extensive coarse-grained molecular dynamics simulations to study, in molecular detail, LCPs under different conditions (compositions and temperatures relevant to crystallogenesis) and their interactions with different types of GPCR constructs. The results presented show how the modulation of the lattice constant of the LCP (triggered by the addition of precipitant during the in meso assay), or of the host lipid type, can destabilize monomeric proteins in the bilayer of the LCP and thus drive their aggregation into the stacked lamellae, where the residual hydrophobic mismatch between the protein and the membrane can drive the formation of lateral contacts leading to nucleation and crystal growth. Moreover, we demonstrate how particular protein designs (such as transmembrane proteins engineered to contain large polar regions) can promote protein stacking interactions in the third, out-of-plane, dimension. The insights provided by the new aspects of the specific molecular mechanisms responsible for protein–protein interactions inside the cubic phase presented here should be helpful in guiding the rational design of future in meso trials with successful outcomes.

Detergent quantification in membrane protein samples and its application to crystallization experiments

The structural characterization of membrane proteins remains a challenging field, largely because the use of stabilizing detergents is required. Researchers must first select a suitable detergent for the solubility and stability of their protein during in vitro studies. In addition, an appropriate concentration of detergent in membrane protein samples can be essential for protein solubility, stability, and experimental success. For example, in membrane protein crystallography, detergent concentration in the crystallization drop can be a critical parameter influencing crystal growth. Over the past decade, multiple techniques have been developed for the measurement of detergent concentration using a wide variety of strategies. These methods include colorimetric reactions, which target specific detergent classes, and analytical techniques applicable to a wide variety of detergents. This review will summarize and discuss the available options. It will be a useful resource to those selecting a strategy that best fits their experimental requirements and available instruments.

NMR crystallography of membrane proteins

NMR crystallography of membrane proteins

Serial femtosecond crystallography of membrane proteins with a lipidic cubic phase injector

Serial femtosecond crystallography of membrane proteins with a lipidic cubic phase injector

Generation of functional antibodies for mammalian membrane protein crystallography

Membrane proteins act as gateways to cells, and they are responsible for much of the communication between cells and their environments. Crystallography of membrane proteins is often limited by the difficulty of crystallization in detergent micelles. Co-crystallization with antibody fragments has been reported as a method to facilitate the crystallization of membrane proteins; however, it is widely known that the generation of mouse monoclonal antibodies that recognize the conformational epitopes of mammalian integral membrane proteins is typically difficult. Here, we present our protocols to generate functional mouse antibodies for the membrane protein crystallography, which have enabled us to solve crystal structures of mammalian receptors and transporters complexed with antibody fragments.

A Toolbox of Fluorescence Microscopic Approaches Reveals Dynamics and Assembly of a Membrane-Associated Protein

A Toolbox of Fluorescence Microscopic Approaches Reveals Dynamics and Assembly of a Membrane-Associated Protein

Characterization of the dynamic events of GPCRs by automated computational simulations

The recent advances in membrane protein crystallography have provided extremely valuable structural information of the superfamily of GPCRs (G-protein-coupled receptors). This has been particularly true for a few receptors whose structure was solved several times under different biochemical conditions. It follows that the mechanisms of receptor conformational equilibrium and related dynamic events can be explored by computational simulations. In the present article, we summarize our recent understanding of several dynamic features of GPCRs, accomplished through the use of MD (molecular dynamics) simulations. Our pipeline for the MD simulations of GPCRs, implemented in the web service http://gpcr.usc.es, is updated in the present paper and illustrated by recent applications. Special emphasis is put on the A2A adenosine receptor, one of the selected cases where crystal structures in several conformations and conditions exist, and on the dimerization process of the CXCR4 (CXC chemokine receptor 4).

Evaluation of cell‐free protein synthesis for the crystallization of membrane proteins – a case study on a member of the glutamate transporter family from Staphylothermus marinus

Cell-free in vitro synthesis of proteins using coupled transcription/translation is considered to be a powerful alternative to the use of traditional cell-based expression systems. Recently, promising developments have been reported applying cell-free production to membrane proteins for structural biology and in particular for NMR spectroscopy. However, the general applicability of this system to produce large amounts of stable, functional and homogeneous membrane proteins as required for X-ray crystallography remains to be determined. Here, we present a systematic study comparing structural and functional properties of membrane proteins produced using Escherichia coli derived in vitro and in vivo expression systems. The function of the target membrane protein, a previously uncharacterized bacterial glutamate transporter homolog from Staphylothermus marinus, was analyzed using ligand binding and transport assays. In addition, the protein structure was investigated with respect to its overall fold and oligomeric state in different detergents. We found that the protein synthesized in vitro is highly stable and monodisperse. However, in contrast to the protein produced using an in vivo system, it was not able to assemble into the native trimeric state nor to bind substrate. We thus conclude that cell-free expression systems can compromise folding and function of such complex secondary active transporters. The expression product has to be carefully characterized prior to biophysical investigations like crystallography of membrane proteins.

A new high-performance thin layer chromatography-based assay of detergents and surfactants commonly used in membrane protein studies

The hydrophobic nature of membrane proteins (MPs) necessitates the use of detergents for their extraction, solubilization and purification. Because the concentration of amphiphiles is crucial in the crystallization process, detergent quantification is essential to routine analysis. Here we describe a quantitative high-performance thin-layer chromatography (HPTLC) method we developed for the detection of small quantities of detergent bound to solubilized MPs. After optimization of aqueous deposit conditions, we show that most detergents widely used in membrane protein crystallography display distinctive mobilities in a mixture of dichloromethane, methanol and acetic acid 32:7.6:0.4 (v/v/v). Migration and derivatization conditions were optimized with n-dodecyl-β-d-maltoside (DDM), the most popular detergent for membrane protein crystallization. A linear calibration curve very well fits our data from 0.1 to 1.6μg of DDM in water with a limit of detection of 0.05μg. This limit of detection is the best achieved to date for a routine detergent assay, being not modified by the addition of NaCl, commonly used in protein buffers. With these chromatographic conditions, no prior treatment is required to assess the quantities of detergent bound to purified MPs, thus enabling the quantification of close structure detergents via a single procedure. This HPTLC method, which is fast and requires low sample volume, is fully suitable for routine measurements.

Current trends in α‐helical membrane protein crystallization: An update

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.

Membrane protein structure determination by electron crystallography

During the past year, electron crystallography of membrane proteins has provided structural insights into the mechanism of several different transporters and into their interactions with lipid molecules within the bilayer. From a technical perspective there have been important advances in high-throughput screening of crystallization trials and in automated imaging of membrane crystals with the electron microscope. There have also been key developments in software, and in molecular replacement and phase extension methods designed to facilitate the process of structure determination.

Membrane Protein Crystallography is Going to Be an Owa-Con!?

Membrane Protein Crystallography is Going to Be an Owa-Con!?

創薬ターゲット膜タンパク質の高速構造解析

創薬ターゲット膜タンパク質の高速構造解析

New amphiphiles for membrane protein structural biology

A challenging requirement for X-ray crystallography or NMR structure determination of membrane proteins (MPs), in contrast to soluble proteins, is the necessary use of amphiphiles to mimic the hydrophobic environment of membranes. A number of new detergents, lipids and non-detergent-like amphiphiles have been developed that stabilize MPs, and these have contributed to increased success in MP structural determinations in recent years. Despite some successes, currently available reagents are inadequate and there remains a pressing need for new amphiphiles. Literature examples and some new developments are selected here as a framework for discussing desirable properties of new amphiphiles for MP structural biology.

High-Efficiency Screening of Monoclonal Antibodies for Membrane Protein Crystallography

Determination of crystal structures of membrane proteins is often limited by difficulties obtaining crystals diffracting to high resolution. Co-crystallization with Fab fragments of monoclonal antibodies has been reported to improve diffraction of membrane proteins crystals. However, it is not simple to generate useful monoclonal antibodies for membrane protein crystallography. In this report, we present an optimized process for efficient screening from immunization to final validation of monoclonal antibody for membrane protein crystallography.

1SI-05 X-ray FELを用いた膜蛋白質構造解析(1SI 第4世代光源、X線自由電子レーザーが拓く生物物理学,日本生物物理学会第49回年会(2011年度))

1SI-05 X-ray FELを用いた膜蛋白質構造解析(1SI 第4世代光源、X線自由電子レーザーが拓く生物物理学,日本生物物理学会第49回年会(2011年度))

The potential for two-dimensional crystallography of membrane proteins at future x-ray free-electron laser sources

Ultrashort pulses from x-ray free-electron laser (XFEL) sources promise to assist in obtaining the structures of membrane proteins at high resolution. We have reconstructed the electron density distribution of a two-dimensional (2D) aquaporin crystal from simulated XFEL data using ptychography, a diffractive imaging technique based on multiple exposures. Increasing the number of exposures compensates for Poisson noise, indicating that the achievable resolution is limited by the reproducibility of the crystals. This technique should therefore be applicable at all future ultrashort-pulsed hard x-ray sources.

Fluorescence Detection of Heavy Atom Labeling (FD-HAL): A rapid method for identifying covalently modified cysteine residues by phasing atoms

Membrane protein crystallography frequently stalls at the phase determination stage due to poor crystal diffraction and the inability to identify heavy atom derivatization prior to data collection. Thus, a majority of time, effort and resources are invested preparing potential derivatized crystals for synchrotron data collection and analysis without knowledge of heavy atom labeling. To remove this uncertainty, we introduce Fluorescence Detection of Heavy Atom Labeling (FD-HAL) using tetramethylrhodamine-5-maleimide (a fluorescent maleimide compound) to monitor in-gel cysteine residue accessibility and ascertain covalent modification by mercury, platinum and gold compounds. We have tested this technique on three integral membrane proteins (LacY, vSGLT and mVDAC1) and can quickly assess the optimal concentrations, time and heavy atom compound to derivatize free cysteine residues in order to facilitate crystal phasing. This, in conjunction with cysteine scanning for incorporating heavy atoms at strategic positions, is a useful tool that will considerably assist in phasing membrane protein structures.