Structural Studies on Large Fragments of G Protein Coupled Receptors

Abstract

Introduction The use of peptide fragments to study the biophysical and structural properties of regions of polytopic membrane proteins is widespread [1,2]. In most cases these studies have been limited to synthetic peptides containing 20-40 amino acid residues. Moreover, although some high resolution structures of fragments corresponding to single transmembrane regions of membrane proteins in organicaqueous membrane mimetic solvents have appeared, there are few such structures from studies in detergent micelles. Although important information is obtained from studies on membrane peptides in media such as trifluoroethanol-water and dimethylsulfoxide, it is highly desirable to compare such structures to those obtained in micelles and bicelles. Furthermore, since long range interhelical interactions can influence the structures of individual transmembrane domains, investigations on peptides containing multiple transmembrane regions of integral membrane proteins are necessary. We have been studying structural characteristics of regions of the α-factor receptor, Ste2p a G protein coupled receptor from the yeast Saccharomyces cerevisiae [3]. Previously, we presented structures of peptides with 30 to 73 residues corresponding to each of the seven putative transmembrane domains of Ste2p [4,5]. Recently, we succeeded in expressing isotopically labeled peptides of Ste2p containing two transmembrane domains connected by the contiguous intracellular loop. One of these peptides TM1-TM2 Ste2p(G31-T130) was found to form stable solutions at 0.2 mM concentration in a variety of lipids including SDS and 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-RAC-(1-glycerol)] (LPPG) Here we report initial results from a detailed NMR analysis of TM1-TM2 in LPPG.