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.
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
Fred
F
Naider
Fa-Xiang
FX
Ding
Nathan C
NC
VerBerkmoes
Boris
B
Arshava
Jeffrey M
JM
Becker
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