Expression strategies for structural studies of eukaryotic membrane proteins (original) (raw)
Related papers
Unlocking the eukaryotic membrane protein structural proteome
Current Opinion in Structural Biology, 2010
Most of the 231 unique membrane protein structures (as of 3/2010) are of bacterial membrane proteins (MPs) expressed in bacteria, or eukaryotic MPs from natural sources. However eukaryotic membrane proteins, especially those with more than three membrane crossings rarely succumb to any suitable expression in bacterial cells. They typically require expression in eukaryotic cells that can provide appropriate endoplasmic reticulum, chaperones, targeting and posttranslational processing. In evidence, only $20 eukaryotic MP structures have resulted from heterologous expression. This is required for a general approach to target particular human or pathogen membrane proteins of importance to human health. The first of these appeared in 2005. Our review addresses the special issues that pertain to the expression of eukaryotic and human membrane proteins, and recent advances in the tool kit for crystallization and structure determination.
Biochemistry and Cell Biology, 2016
Membrane proteins are still heavily under-represented in the protein data bank (PDB), owing to multiple bottlenecks. The typical low abundance of membrane proteins in their natural hosts makes it necessary to overexpress these proteins either in heterologous systems or through in vitro translation/cell-free expression. Heterologous expression of proteins, in turn, leads to multiple obstacles, owing to the unpredictability of compatibility of the target protein for expression in a given host. The highly hydrophobic and (or) amphipathic nature of membrane proteins also leads to challenges in producing a homogeneous, stable, and pure sample for structural studies. Circumventing these hurdles has become possible through the introduction of novel protein production protocols; efficient protein isolation and sample preparation methods; and, improvement in hardware and software for structural characterization. Combined, these advances have made the past 10–15 years very exciting and eventf...
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.
Where do we go from here? Membrane protein research beyond the structure-function horizon
Biochimica Et Biophysica Acta - Biomembranes, 2018
Where do we go from here? Membrane protein research beyond the structure-function horizon Biology has evolved and thrives in an aqueous environment. The water-insolubility of membrane proteins is one of the biggest reasons why membrane protein research has been among the most challenging aspects of protein biology, with every study being plagued by uncertainties regarding the physiological relevance of its findings. But thanks to savvy technical innovations (see below), researchers are closer than ever to getting a firm handle on the biology of several membrane proteins, even to the point where bench-to-bedside research is becoming a consideration. This Special Issue is dedicated to such advances and is our humble attempt at highlighting some key areas. For a while now, the workflow of membrane protein research has proceeded as follows: (i) A select protein of interest is cloned out from its native genome, and tested in a battery of functional biochemical assays, either in a convenient heterologous host or in purified form. (ii) Mutations are made across the protein to assess their various effects, and predictions are made about its structure and mechanism(s). In the past few decades, this cycle was iterated several times over with many discoveries of novel classes of membrane proteins being made, based on topology mappings and functional nuances. The advances in biophysical tools in the form of high-resolution X-ray and cryo-electron microscopy (cryoEM) structures have allowed the addition of architecture to be ascribed to protein function. However, for the longest time, obtaining the structure of a researcher's favorite protein (especially if it happened to be a membrane protein) was the culmination of a lifetime's worth of workif it was ever attained. Membrane protein research certainly has changed, and although the first high-resolution structure of a membrane protein by cryoEM was only published four years ago [1], the resulting avalanche of high-resolution cryoEM membrane protein structures certainly profits from the fact that their structure-function relationships had been heavily investigated for decades. It is not a stretch, then, to expect to see protein structures for all major membrane protein families, and potentially all medically relevant targets in the next couple of years. But where do we go from there? What is left to do and what can structures fall short of revealing? In the first paper in this Special Issue, Seeger muses on how it is time for us to reflect on what we, as a field, can do in "times of countless structures" [2]. And encouragingly, he reminds us that structural biology will not become obsolete any time soon. Instead, a new species of structural biologist is emerging-that of an integrated scientist, who works with experts on the functional, computational, and biophysical facets of membrane proteins to arrive at a holistic understanding for each protein of interest. Advances in biophysical tools have, in close collaboration with high resolution structures and functional data, provided means to detect interatomic distance changes in membrane-embedded proteins, to assess their dynamics and now even allow to predict their behavior to a certain degree through molecular dynamic simulations of proteins in modelled native-like environments. But no matter how advanced our techniques become, ultimately, they still rely on a careful sample preparation. And while it may by now seem reasonably straight forward to pull a membrane protein out of its lipid environment to then reconstitute it back into a lipid bilayer, there has also been a huge demand for optimizing these approaches and obtain more native-like reconstitution conditions. The introduction of nanodiscs clearly revolutionized the field (e.g. [3-5]) as they allow to study membrane proteins in near-native conditions. Another novel technology for the reconstitution of membrane proteins for functional studies using styrene-maleic acid copolymer discs is discussed in Pollock et al. [6]. Here, a major advantage is that the protein can be purified in its native membrane, thus maintaining lipid composition and presumably, at least for a short amount of time, asymmetry. Together, combining advances in protein preparation with biophysical approaches do a fantastic job validating and accelerating functional experimentation much further than would otherwise have been possible with the individual techniques alone. As spectroscopic techniques become more advanced, they begin to shed light on intricate details of protein dynamics as described by Spadaccini et al., using solid-state NMR (nuclear magnetic resonance) in combination with DNP (dynamic nuclear polarization) [7], Zhogbi et al., using LRET (luminescence resonance electron transfer) [8] and Bordignon and Bleicken with EPR (electron paramagnetic resonance) [9]. EPR spectroscopy has long been recognized as a formidable tool to study membrane protein dynamics, and in the present review, the authors also give an overview over recent hardware, pulse sequence and spin label improvements. EPR studies are often combined with NMR studies [10,11], as global and local conformational changes can be studied in a complementary fashion. DNP-enhanced NMR additionally allows spectra to be recorded in a fraction of the previously required time, thus making lowly populated conformations or difficult to obtain samples accessible to inherently insensitive NMR studies. Spadaccini et al. show how substrate interactions with MsbA lead to specific changes in helix 4 and 6 of this ABC transporter [7]. Likewise, Zhogbi et al. use this ABC transporter to demonstrate the advantages of LRET in the study of membrane proteins, such as the long lifetime of excited states for the lanthanide donor and the line spectra emissions [8].
2010
The New York Consortium on Membrane Protein Structure (NYCOMPS) was formed to accelerate the acquisition of structural information on membrane proteins by applying a structural genomics approach. NY-COMPS comprises a bioinformatics group, a centralized facility operating a high-throughput cloning and screening pipeline, a set of associated wet labs that perform highlevel protein production and structure determination by xray crystallography and NMR, and a set of investigators focused on methods development. In the first three years of operation, the NYCOMPS pipeline has so far produced and screened 7,250 expression constructs for 8,045 target proteins. Approximately 600 of these verified targets were scaled up to levels required for structural studies, so far yielding 24 membrane protein crystals. Here we describe the overall structure of NYCOMPS and provide details on the high-throughput pipeline.
Journal of Molecular Biology, 2009
A medium throughput approach is used to rapidly identify membrane proteins from a eukaryotic organism that are most amenable to expression in amounts and quality adequate to support structure determination. The goal was to expand knowledge of new membrane protein structures based on proteome-wide coverage. In the first phase membrane proteins from the budding yeast Saccharomyces cerevisiae were selected for homologous expression in S. cerevisiae, a system that can be adapted to expression of membrane proteins from other eukaryotes. We performed mediumscale expression and solubilization tests on 351 rationally selected membrane proteins from the budding yeast Saccharomyces cerevisiae. These targets are inclusive of all annotated and unannotated membrane protein families within the organism's membrane proteome. 272 targets were expressed and of these 234 solubilized in the detergent n-dodecyl-β-D-maltopyranoside. Furthermore, we report the identity of a subset of targets that were purified to homogeneity to facilitate structure determinations. The extensibility of this approach is demonstrated with the expression of ten human integral membrane proteins from the solute carrier superfamily (SLC). This discovery-oriented pipeline provides an efficient way to select proteins from particular membrane protein classes, families, or organisms that may be more suited to structure analysis than others.
Expression of eukaryotic membrane proteins in eukaryotic and prokaryotic hosts
Methods, 2020
The production of membrane proteins of high purity and in satisfactory yields is crucial for biomedical research. Due to their involvement in various cellular processes, membrane proteins have increasingly become some of the most important drug targets in modern times. Therefore, their structural and functional characterization is a high priority. However, protein expression has always been more challenging for membrane proteins than for soluble proteins. In this review, we present four of the most commonly-used expression systems for eukaryotic membrane proteins. We describe the benefits and drawbacks of bacterial, yeast, insect and mammalian cells. In addition, we describe the different features (growth rate, yield, post-translational modifications) of each expression system, and how they are influenced by the construct design and modifications of the target gene. Cost-effective and fast-growing E. coli is mostly selected for the production of small, simple membrane proteins that, if possible, do not require post-translational modifications but has the potential for the production of bigger proteins as well. Yeast hosts are advantageous for larger and more complex proteins but for the most complex ones, insect or mammalian cells are used as they are the only hosts able to perform all the post-translational modifications found in human cells. A combination of rational construct design and host cell choice can dramatically improve membrane protein production processes.
Comparative and Functional Genomics, 2002
Membrane proteins (MPs) are responsible for the interface between the exterior and the interior of the cell. These proteins are implicated in numerous diseases, such as cancer, cystic fibrosis, epilepsy, hyperinsulinism, heart failure, hypertension and Alzheimer's disease. However, studies on these disorders are hampered by a lack of structural information about the proteins involved. Structural analysis requires large quantities of pure and active proteins. The majority of medically and pharmaceutically relevant MPs are present in tissues at very low concentration, which makes heterologous expression in large-scale production-adapted cells a prerequisite for structural studies. Obtaining mammalian MP structural data depends on the development of methods that allow the production of large quantities of MPs. This review focuses on the different heterologous expression systems, and the purification strategies, used to produce large amounts of pure mammalian MPs for structural prot...
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