Stabilization of Membrane Proteins: the Case of G‐Protein‐Coupled Receptors

Abstract

G-protein-coupled receptors are integral membrane proteins which constitute the largest family of signal transduction molecules participating in the majority of normal physiological processes. G-protein-coupled receptors are responsible for the control of enzyme activity, ion channels and vesicle transport, and they respond to a wide variety of stimuli, like signals involved in sensory systems such as vision, taste and olfaction, but also to a diverse set of chemical signals such as lipids, hormones, neurotransmitters, amino acids, nucleotides, peptides and proteins. This family of receptors is being widely studied because of its potential use as pharmacological targets in drug development, and recently also for its potential use in the development of novel biosensors. G-protein-coupled receptors are specifically designed to fold and function in a lipid bilayer environment, where these membrane proteins are remarkably stable and achieve their optimal performance. The currently used technology for the purification of G-protein-coupled receptors consists in their extraction from the cell membrane and solubilization into detergent micelles. A common drawback of this strategy is that G-proteincoupled receptors solubilized in typical detergents show rather poor conformational stability, which may result in relatively rapid inactivation. The poor stability of detergent-solubilized samples renders many membrane proteins biochemically intractable. This precludes the determination of a high-resolution structure and imposes severe limitations for the development of applications. Thus, the enhancement of the stability of G-protein-coupled receptors is a major issue in order to facilitate structural determination and to unravel their potential in biotechnological applications. This work provides a brief overview of some current advances in the experimental methods for stabilizing G-protein-coupled receptors that can also be extended to other types of membrane proteins. Structure of G-protein-Coupled Receptors (GPCRs)The distinctive structural feature of GPCRs is the bundle of seven transmembrane (7TM) a-helices that constitutes a relevant signature in structural biology. This conformational architecture, and its functional implications, is central to pharmacology and therapeutics. Nearly 2000 GPCRs have been reported since bovine opsin was cloned in 1983 and the b-adrenergic receptor in 1986. They are classified into over 100 subfamilies as a function of their sequence homology, ligand structure and receptor function. Significant differences exist among the members of the GPCRs superfamily, in spite of the high degree of amino acid homology found among the members of a particular subfamily. The visual photoreceptor rhodopsin (Rho) was the first GPCR whose crystal structure was determined at high-resolution [1][2][3][4][5][6][7]. The natural abundance and availability of Rho has made of this protein the prototypical GPCR for structural studies. As a consequence, Rho is often selected as a model protein for biochemical and b...

Toward Rational Design of Protein Detergent Complexes: Determinants of Mixed Micelles That Are Critical for the In Vitro Stabilization of a G-Protein Coupled Receptor

“…These heuristics implicitly assume that molecular chemistry is the most, if not the only, important variable in selecting a micellar system to retain the in vitro stability of membrane proteins. This typically leads to strategies in which pure micelles of a particularly mild surfactant are used as a first attempt (e.g., dodecyl maltoside), and the micellar system is fine-tuned through the use of other detergents or additives until a suitable system is found for the protein and biophysical method of interest (16,18). Such trial-and-error methodology is time-consuming and resource-intensive, especially considering the low yields that are typically achieved for expression and purification of most membrane proteins, and does not lead to information that is readily translatable across different systems.…”

Salt Effects on the Conformational Stability of the Visual G-Protein-Coupled Receptor Rhodopsin

“…Understanding the molecular interactions that stabilize or destabilize membrane proteins is essential to our understanding of their function (11,22,23). High stability of GPCRs in solution is desirable for biotechnological applications (24), biochemical assays, and structural studies. It has been previously shown that inorganic salts play a key role in the stability of proteins (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35), and whereas a large number of studies are available for globular proteins, less information is available in the case of membrane proteins.…”