Synthesis and spectroscopic characterization of photo-affinity peptide ligands to study rhodopsin-G protein interaction.
G protein-coupled receptors (GPCRs) are involved in the control of virtually all aspects of our behavior and physiology. Activated receptors catalyze nucleotide exchange in heterotrimeric G proteins (composed of alpha.GDP, beta and gamma subunits) on the inner surface of the cell membrane. The GPCR rhodopsin and the G protein transducin (G(t)) are key proteins in the early steps of the visual cascade. The main receptor interaction sites on G(t) are the C-terminal tail of the G(t)alpha-subunit and the farnesylated C-terminal tail of the G(t)gamma-subunit. Synthetic peptides derived from these C-termini specifically bind and stabilize the active rhodopsin conformation (R*). Here we report the synthesis of R*-interacting peptides containing photo-reactive groups with a specific isotope pattern, which can facilitate detection of cross-linked products by mass spectrometry. In a preliminary set of experiments, we characterized such peptides derived from the farnesylated G(t)gamma C-terminus (G(t)gamma(60-71)far) in terms of their capability to bind R*. Here, we describe novel peptides with photo-affinity labels that bind R* with affinities similar to that of the native G(t)gamma(60-71)far peptide. Such peptides will enable an improved experimental strategy to probe rhodopsin-G(t) interaction and to map so far unknown interaction sites between both proteins.
Chen
Yihui
Y
Department of Chemistry, Columbia University, New York, NY, USA.
Herrmann
Rolf
R
Fishkin
Nathan
N
Henklein
Peter
P
Nakanishi
Koji
K
Ernst
Oliver P
OP
D000595:Amino Acid Sequence; D004355:Drug Stability; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D010777:Photochemistry; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D012243:Rhodopsin / Q000737:chemistry* / Q000378:metabolism*
D010455:Peptides; D021122:Protein Subunits; D012243:Rhodopsin; D019204:GTP-Binding Proteins