Mapping of contact sites in complex formation between light-activated rhodopsin and transducin by covalent crosslinking: use of a chemically preactivated reagent.
Contact sites in interaction between light-activated rhodopsin and transducin (T) have been investigated by using a chemically preactivated crosslinking reagent, N-succinimidyl 3-(2-pyridyldithio)propionate. The 3 propionyl-N-succinimidyl group in the reagent was attached by a disulfide exchange reaction to rhodopsin mutants containing single reactive cysteine groups in the cytoplasmic loops. Complex formation between the derivatized rhodopsin mutants and T was carried out by illumination at lambda > 495 nm. Subsequent increase in pH (from 6 to 7.5 or higher) of the complex resulted in crosslinking of rhodopsin to the T(alpha) subunit. Crosslinking to T(alpha) was demonstrated for the rhodopsin mutants K141C, S240C, and K248C, and the crosslinked sites in T(alpha) were identified for the rhodopsin mutant S240C. The peptides carrying the crosslinking moiety were isolated from the trypsin-digested peptide mixture, and their identification was carried out by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The main site of crosslinking is within the peptide sequence, Leu-19-Arg-28 at the N-terminal region of T(alpha). The total results show that both the N and the C termini of T(alpha) are in close vicinity to the third cytoplasmic loop of rhodopsin in the complex between rhodopsin and T.
Itoh
Y
Y
Departments of Biology and Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Cai
K
K
Khorana
H G
HG
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001386:Azides / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D015153:Blotting, Western; D002417:Cattle; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008027:Light; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D021122:Protein Subunits; D011725:Pyridines / Q000737:chemistry / Q000378:metabolism; D012243:Rhodopsin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013388:Succinimides / Q000737:chemistry / Q000378:metabolism; D015293:Transducin / Q000737:chemistry / Q000378:metabolism*
D001386:Azides; D003432:Cross-Linking Reagents; C425928:N-((2-pyridyldithio)ethyl)-4-azidosalicylamide; D010446:Peptide Fragments; D021122:Protein Subunits; D011725:Pyridines; D013388:Succinimides; C018151:N-succinimidyl 3-(2-pyridyldithio)propionate; D012243:Rhodopsin; D015293:Transducin; D003545:Cysteine