Synthesis and biophysical characterization of a multidomain peptide from a Saccharomyces cerevisiae G protein-coupled receptor.
We attached peptides corresponding to the seventh transmembrane domain (TMD7) of the alpha-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.
Naider
Fred
F
Department of Chemistry, College of Staten Island and Institute for Macromolecular Assemblies, City University of New York, Staten Island, New York 10314, USA. naider@postbox.csi.cuny.edu
Ding
Fa-Xiang
FX
VerBerkmoes
Nathan C
NC
Arshava
Boris
B
Becker
Jeffrey M
JM
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D008051:Lipid Bilayers; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008823:Micelles; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010710:Phosphates / Q000737:chemistry; D017434:Protein Structure, Tertiary; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry; D014867:Water / Q000737:chemistry
D008051:Lipid Bilayers; D008823:Micelles; D010455:Peptides; D010710:Phosphates; D043562:Receptors, G-Protein-Coupled; D014867:Water; D014364:Tryptophan