Conformational characterization of a peptide mimetic of the third cytoplasmic loop of the G-protein coupled parathyroid hormone/parathyroid hormone related protein receptor.
The third-cytoplasmic loop of the G-protein coupled receptor responsible for the activity of parathyroid hormone and parathyroid hormone-related protein has been structurally characterized in aqueous solution in the presence of sodium dodecylsulfate and dodecylphosphocholine micelles. The high-resolution conformation of the 29-amino acid peptide containing the sequence of the cytoplasmic loop was obtained by CD and nmr. The structure was refined using a two-step distance geometry based method that first includes the removal of all side chains to quickly locate the globular fold of the peptide. After a simulated annealing protocol, the side chains are added in a random fashion. The resulting conformation was further refined with nuclear Overhauser enhancement restrained molecular dynamics using a two-phase simulation cell consisting of carbon tetrachloride and water as a mimetic of the biphasic, hydrophobic/hydrophilic character of the micelles in which the experimental measurements were carried out. The topological orientation of the cycloplasmic loop within the micelle was determined by addition of 5-doxylstearate and monitoring the decrease of nmr signal intensities from the radical-induced relaxation. The conformation and relative orientation of the peptide provided insight into the mechanism by which the cytoplasmic portion of the receptor activates the heterotrimeric, guanine nucleotide-binding regulatory protein, one of the first steps in signal transduction.
Pellegrini
M
M
Gustaf H. Carlson School of Chemistry, Clark University, Worcester, MA 01610, USA.
Royo
M
M
Chorev
M
M
Mierke
D F
DF
D000595:Amino Acid Sequence; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D010281:Parathyroid Hormone / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D018016:Receptors, Parathyroid Hormone / Q000737:chemistry* / Q000378:metabolism*
D010281:Parathyroid Hormone; D018016:Receptors, Parathyroid Hormone; D019204:GTP-Binding Proteins