Conformation of a double-membrane-spanning fragment of a G protein-coupled receptor: Effects of hydrophobic environment and pH

Overcoming the problems associated with the expression, purification and in vitro handling of membrane proteins requires an understanding of the factors governing the folding and stability of such proteins in detergent solutions. As a sequel to our earlier report (Biochim. Biophys. Acta 1747(2005), 133–140), we describe an improved purification procedure and a detailed structural analysis of a fragment of the μ-opioid receptor (‘TM2–3’) that comprises the second and third transmembrane segments and the extracellular loop that connects them. Circular dichroism (CD) spectroscopy of TM2–3 in 2,2,2-trifluoroethanol gave a helical content similar to that predicted by published homology models, while spectra acquired in several detergents showed significantly lower helical contents. This indicates that this part of the μ-opioid receptor has an intrinsic propensity to be highly helical in membrane-like environments, but that in detergent solutions, this helical structure is not fully formed. Proteolysis of TM2–3 with trypsin showed that the helical portions of TM2 and TM3 are both shorter than their predicted lengths, indicating that helix–helix interactions in the full-length receptor are apparently important for stabilizing their conformation. Lengthening the alkyl chain of the detergent led to a small but significant increase in the helicity of TM2–3, suggesting that hydrophobic mismatch could play an important role in the stabilization of transmembrane helices by detergents. Protonation of aspartic acid residues in detergent-solubilized TM2–3 also caused a significant increase in helicity. Our results thus suggest that detergent alkyl chain-length and pH may influence membrane protein stability by modulating the stability of individual transmembrane segments.