NMR studies in dodecylphosphocholine of a fragment containing the seventh transmembrane helix of a G-protein-coupled receptor from Saccharomyces cerevisiae.
The structure and dynamics of a large segment of Ste2p, the G-protein-coupled alpha-factor receptor from yeast, were studied in dodecylphosphocholine (DPC) micelles using solution NMR spectroscopy. We investigated the 73-residue peptide EL3-TM7-CT40 consisting of the third extracellular loop 3 (EL3), the seventh transmembrane helix (TM7), and 40 residues from the cytosolic C-terminal domain (CT40). The structure reveals the presence of an alpha-helix in the segment encompassing residues 10-30, which is perturbed around the internal Pro-24 residue. Root mean-square deviation values of individually superimposed helical segments 10-20 and 25-30 were 0.91 +/- 0.33 A and 0.76 +/- 0.37 A, respectively. 15N-relaxation and residual dipolar coupling data support a rather stable fold for the TM7 part of EL3-TM7-CT40, whereas the EL3 and CT40 segments are more flexible. Spin-label data indicate that the TM7 helix integrates into DPC micelles but is flexible around the internal Pro-24 site, exposing residues 22-26 to solution and reveal a second site of interaction with the micelle within a region comprising residues 43-58, which forms part of a less well-defined nascent helix. These findings are discussed in light of previous studies in organic-aqueous solvent systems.
Neumoin
Alexey
A
Institute of Organic Chemistry, University of Zurich, Zurich, Switzerland.
Arshava
Boris
B
Becker
Jeff
J
Zerbe
Oliver
O
Naider
Fred
F
D000595:Amino Acid Sequence; D055592:Biophysical Phenomena; D001703:Biophysics; D004578:Electron Spin Resonance Spectroscopy; D008823:Micelles; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D010767:Phosphorylcholine / Q000031:analogs & derivatives; D017433:Protein Structure, Secondary; D051964:Receptors, Mating Factor / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics; D012997:Solvents; D013113:Spin Labels; D013816:Thermodynamics
D008823:Micelles; D010446:Peptide Fragments; D051964:Receptors, Mating Factor; D011994:Recombinant Proteins; C498031:STE2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D012997:Solvents; D013113:Spin Labels; D010767:Phosphorylcholine; C028810:dodecylphosphocholine