Synthesis and Biophysical Characterization of a Multidomain Peptide from a Saccharomyces cerevisiae G Protein-coupled Receptor

Abstract

We attached peptides corresponding to the seventh transmembrane domain (TMD7) of the ␣-mating factor receptor (Ste2p) of Saccharomyces cerevisiae to a hydrophilic, 40-residue fragment of the carboxyl terminus of this G protein-coupled receptor. Peptides corresponding to (a) the 40-residue portion of the carboxyl tail (T-40), (b) the tail plus a part of TMD7 (M7-12-T40), and (c) to the tail plus the full TMD7 (M7-24-T40) were chemically synthesized and purified. The molecular mass and primary sequence of these peptides were confirmed by mass spectrometry and tandem mass spectrometry procedures. Circular dichroism (CD) revealed that T-40 was disordered in phosphate buffer and in the presence of 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-[phospho-racemic-(1-glycerol)] bilayers. In contrast, M7-12-T40 and M7-24-T40 peptides were partially helical in the presence of vesicles, and difference CD spectroscopy showed that the transmembrane regions of these peptides were 42 and 94% helical, respectively. CD analysis also demonstrated that M7-24-T40 retained its secondary structure in the presence of 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-racemic-(1-glycerol)] micelles at 0.5 mM concentration. Thus, the tail and the transmembrane domain of the multidomain 64-amino acid residue peptide manifest individual conformational preferences. Measurement of tryptophan fluorescence indicated that the transmembrane domain integrated into bilayers in a manner similar to that expected for this region in the native state of the receptor. This study demonstrated that the tail of Ste2p can be used as a hydrophilic template to study transmembrane domain structure using techniques such as CD and NMR spectroscopy.The determination of the structure of G protein-coupled receptors remains an unfulfilled goal of structural biologists with the notable exception of the solution of a high resolution structure of rhodopsin by x-ray crystallography (1). These membrane-bound, information-transducing proteins are acknowledged as a major family of proteins with thousands of representatives encoded by human genes (2, 3). Moreover, the impressive successes in the development of pharmaceuticals targeting G protein-coupled receptors (GPCRs) 1 have created much interest in their structure and mechanism of action (4). Because of the difficulty in obtaining refraction grade crystals of most membrane proteins and their large size, neither x-ray diffraction nor NMR approaches are readily applied to intact receptors. As a route to gain information relevant to these and other membrane proteins such as transporters, many laboratories have taken a reductionist approach and are examining relatively short peptides corresponding to loops and single transmembrane domains (TMDs; Refs. 5-7). A problem encountered in these studies is the poor solubility exhibited by TMDs that leads to difficulty in purifying and conducting biophysical investigations on these molecules. Moreover, although important insights into the conformational preference...

α-Helical transmembrane peptides: A “Divide and Conquer” approach to membrane proteins

“…They allow for structure elucidation of hydrophobic TM sequences in the absence of hydrophilic flanks (Tomich et al, 1998;Naider et al, 2003). In at least one case , helix formation by an isolated TM segment could hence be shown to be independent of flanking hydrophilic residues (however, see Sections 3.2 and 5.2 for examples where this is not the case).…”

Synthetic peptides as probes for conformational preferences of domains of membrane receptors

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Biosynthesis and NMR Analysis of a 73-Residue Domain of aSaccharomyces cerevisiaeG Protein-Coupled Receptor

“…Recently, we chemically synthesized a 64-residue multidomain peptide containing the seventh transmembrane domain and 40 residues of the cytosolic terminus of Ste2p and showed that this peptide retained distinct structural characteristics for each of these domains in organic-aqueous medium and various detergents using circular dichroism analyses. Moreover, CD patterns in detergents were nearly identical at peptide concentrations from 50 to 500 µM indicating that NMR analyses were feasible (40).…”