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

Abstract

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...

Fluorine NMR‐Based Screening on Cell Membrane Extracts

“…Often, modification of membrane enzymes with fusion partners, such as maltose binding protein (MBP), or point mutations are required to enhance the level of expression of these proteins, their solubility and stability in solution. [13][14][15][16][17][18] Here, we show that n-FABS method can be extended to cell extracts, therefore using the enzyme of interest in more physiological conditions. As a model protein, we used fatty acid amide hydrolase (FAAH), a membrane-bound serine hydrolase responsible for the catabolism of a class of endogenous bioactive lipids called fatty acid ethanolamides (FAEs).…”

Directed evolution of a G protein-coupled receptor for expression, stability, and binding selectivity

“…There have been recent advances in membrane protein engineering, including technologies for the screening of highexpressing members in a diverse pool of eukaryotic membrane proteins (24), identification of functionally critical amino acids in a GPCR (25)(26)(27), manual blot screening of randomly mutated membrane proteins for increased expression (28), and introduction of thermostabilizing mutations, individually identified by trial and error, in a GPCR (29). Complementary to such approaches, we present here a powerful, high-throughput platform for the directed evolution of a GPCR to enhance both expression level and stability while retaining function and to tailor ligand selectivity.…”

Manipulation des récepteurs couplés aux protéines G

“…Elle est couramment utilisée en biochimie et biophysique. Cette méthode a été employée avec succès, dans le cas des récepteurs 1AR, A 2A de l'adénosine et NTS1 de la neurotensine, notamment [29][30][31]. La seconde approche consiste à modifier l'environnement de la protéine.…”