Building a Thermostable Membrane Protein

406

416

There are Ϸ350 non-odorant G protein-coupled receptors (GPCRs) encoded by the human genome, many of which are predicted to be potential therapeutic targets, but there are only two structures available to represent the whole of the family. We hypothesized that improving the detergent stability of these receptors and simultaneously locking them into one preferred conformation will greatly improve the chances of crystallization. We developed a generic strategy for the isolation of detergent-solubilized thermostable mutants of a GPCR, the ␤1-adrenergic receptor. The most stable mutant receptor, ␤AR-m23, contained six point mutations that led to an apparent Tm 21°C higher than the native protein, and, in the presence of bound antagonist, ␤AR-m23 was as stable as bovine rhodopsin. In addition, ␤AR-m23 was significantly more stable in a wide range of detergents ideal for crystallization and was preferentially in an antagonist conformation in the absence of ligand.G protein-coupled receptor ͉ membrane protein ͉ stabilization O ver the past 20 years the rate of determination of membrane protein structures has gradually increased, but most success has been in crystallizing membrane proteins from bacteria rather than from eukaryotes (1). Bacterial membrane proteins have been easier to overexpress using standard techniques in Escherichia coli than eukaryotic membrane proteins (2, 3), and the bacterial proteins are often far more stable in detergent, detergent stability being an essential prerequisite to purification and crystallization. However, of the 125 unique membrane protein structures that have been solved to date, there are only eight structures of mammalian integral membrane proteins; five of these membrane proteins were purified from natural sources and are stable in detergent solutions. Apart from the difficulties in overexpressing eukaryotic membrane proteins, they often have poor stability in detergent solutions, which severely restricts the range of crystallization conditions that can be explored without their immediate denaturation or precipitation. Ideally, membrane proteins should be stable for many days in any given detergent solution, but the detergents that are best suited to growing diffraction-quality crystals tend to be the most destabilizing detergents, i.e., those with short aliphatic chains and small or charged head groups. It is also the structures of human membrane proteins that we would like to solve, because these are required to help the development of therapeutic agents by the pharmaceutical industry; often there are substantial differences in the pharmacology of receptors, channels, and transporters from different mammals, whereas yeast and bacterial genomes may not include any homologous genes. There is thus an overwhelming need to develop a generic strategy that will allow the production of detergent-stable eukaryotic integral membrane proteins for crystallization and structure determination.Membrane proteins have evolved to be sufficiently stable in the membrane to ensure cell viability, b...

mentioning

confidence: 99%

Conformational thermostabilization of the β1-adrenergic receptor in a detergent-resistant form

Serrano-Vega

Magnani

Shibata

et al. 2008

406

416

“…Accurate assessments of their thermodynamic stability will also aid in the design of more stable membrane proteins for therapeutic applications and for structural studies of this structurally underrepresented class of proteins. For example, overcoming conformational dynamics has been a major breakthrough in the recent determination of the structure of lactose permease (10) and superstable mutants of diacylglycerolkinase have dramatically improved crystallization and NMR conditions (11,12). Unfortunately, quantitative studies of membrane protein stability have been hampered by difficulties to completely and reversibly unfold these proteins (13,14).…”

mentioning

confidence: 99%

Elastic coupling of integral membrane protein stability to lipid bilayer forces

Hong

Tamm

2004

217

247

It has been traditionally difficult to measure the thermodynamic stability of membrane proteins because fully reversible protocols for complete folding these proteins were not available. Knowledge of the thermodynamic stability of membrane proteins is desirable not only from a fundamental theoretical standpoint, but is also of enormous practical interest for the rational design of membrane proteins and for optimizing conditions for their structure determination by crystallography or NMR. Here, we describe the design of a fully reversible system to study equilibrium folding of the outer membrane protein A from Escherichia coli in lipid bilayers. Folding is shown to be two-state under appropriate conditions permitting data analysis with a classical folding model developed for soluble proteins. The resulting free energy and m value, i.e., a measure of cooperativity, of unfolding are ⌬G T he material elastic properties of biological membranes such as curvature stress and bilayer deformation from hydrophobic mismatch have emerged as important functional modulators of ion channels, receptors, and other integral membrane proteins (1-5). These properties are also expected to modulate the thermodynamic stability of membrane proteins because the membrane environment imposes very different mechanical constraints (6, 7) on membrane proteins than water does on soluble proteins (8). A thorough understanding of the factors that determine the stability of membrane proteins is of fundamental theoretical interest to understand the forces that shape these proteins (9). Accurate assessments of their thermodynamic stability will also aid in the design of more stable membrane proteins for therapeutic applications and for structural studies of this structurally underrepresented class of proteins. For example, overcoming conformational dynamics has been a major breakthrough in the recent determination of the structure of lactose permease (10) and superstable mutants of diacylglycerolkinase have dramatically improved crystallization and NMR conditions (11,12). Unfortunately, quantitative studies of membrane protein stability have been hampered by difficulties to completely and reversibly unfold these proteins (13,14). We have now designed a fully reversible system to study equilibrium folding of membrane proteins in lipid bilayers, by using the outer membrane protein A (OmpA) from Escherichia coli as a model. OmpA is an abundant protein of the outer membranes of Gram-negative bacteria, where it serves a structural role and also functions as a phage and colicin receptor. The eight-stranded ␤-barrel structure of the N-terminal transmembrane domain (residues 1-177) has been solved by x-ray crystallography (15) and NMR (16). Denatured OmpA in solution spontaneously refolds into lipid bilayer membranes after dilution of denaturants (17), and the kinetics of refolding of OmpA have been shown to depend on the composition, thickness, and overall curvature of the lipid bilayer (18). We demonstrate here that OmpA folding into lipid bilayers is a ...

“…Site-directed mutagenesis was used by Bowie and coworkers to improve the stability of two bacterial membrane proteins (11)(12)(13). Rhodopsin has also been stabilized by 10°C, although in this case a disulfide bond was engineered between the N terminus and the third extracellular loop based on structural data (14).…”

mentioning

confidence: 99%

Co-evolving stability and conformational homogeneity of the human adenosine A _2a receptor

Magnani

Shibata

Serrano-Vega

et al. 2008

209

278

Structural studies on mammalian integral membrane proteins have long been hampered by their instability in detergent. This is particularly true for the agonist conformation of G protein-coupled receptors (GPCRs), where it is thought that the movement of helices that occurs upon agonist binding results in a looser and less stable packing in the protein. Here, we show that mutagenesis coupled to a specific selection strategy can be used to stabilize the agonist and antagonist conformations of the adenosine A2a receptor. Of the 27 mutations identified that improve the thermostability of the agonist conformation, only three are also present in the 17 mutations identified that improve the thermostability of the antagonist conformation, suggesting that the selection strategies used were specific for each conformation. Combination of the stabilizing mutations for the antagonist-or agonist-binding conformations resulted in mutants that are more stable at higher temperatures than the wild-type receptor by 17°C and 9°C, respectively. The mutant receptors both showed markedly improved stability in short-chain alkyl-glucoside detergents compared with the wild-type receptor, which will facilitate their structural analysis.conformational thermostabilization ͉ G protein-coupled receptor ͉ membrane protein G protein-coupled receptors (GPCRs) represent one of the largest single families of integral membrane proteins in the human genome and bind multifarious ligands that mediate many physiological processes, which explains why GPCRs represent a major proportion of drug targets (1). The binding of an extracellular ligand to a GPCR promotes coupling of the receptor to trimeric G proteins, situated on the intracellular side of the plasma membrane, triggering a signaling cascade. Despite sharing common features such as seven transmembrane helices and conserved signatures in their amino acid sequence (2), the sequence homology between different GPCRs is rather low. Detailed structure determination of GPCRs is therefore required to elucidate the mechanism of receptor activation to improve the design of both agonist and antagonist ligands of medical relevance.For many years, crystallographic studies of GPCRs were limited to rhodopsin because of its abundance in native sources (retina) and its intrinsic stability in the dark state (3, 4). However, even rhodopsin has been shown to be structurally more dynamic in detergent solution than in lipid bilayers (5), and such flexibility is more pronounced for GPCRs that bind to diffusible ligands. Another obstacle to structural analysis, and especially to the formation of well ordered crystals, arises from the conformational heterogeneity of these receptors (6, 7). The active agonist-bound state of GPCRs is normally found to be intrinsically less stable than the inactive antagonist-bound state, probably reflecting the receptor requirement for higher flexibility in its active state (8). Agonist binding to the receptor triggers the recruitment of G ␣ protein binding to the intracellular side of the...