Abstract
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.