Constraints on the conformation of the cytoplasmic face of dark-adapted and light-excited rhodopsin inferred from antirhodopsin antibody imprints.
Rhodopsin is the best-understood member of the large G protein-coupled receptor (GPCR) superfamily. The G-protein amplification cascade is triggered by poorly understood light-induced conformational changes in rhodopsin that are homologous to changes caused by agonists in other GPCRs. We have applied the "antibody imprint" method to light-activated rhodopsin in native membranes by using nine monoclonal antibodies (mAbs) against aqueous faces of rhodopsin. Epitopes recognized by these mAbs were found by selection from random peptide libraries displayed on phage. A new computer algorithm, FINDMAP, was used to map the epitopes to discontinuous segments of rhodopsin that are distant in the primary sequence but are in close spatial proximity in the structure. The proximity of a segment of the N-terminal and the loop between helices VI and VIII found by FINDMAP is consistent with the X-ray structure of the dark-adapted rhodopsin. Epitopes to the cytoplasmic face segregated into two classes with different predicted spatial proximities of protein segments that correlate with different preferences of the antibodies for stabilizing the metarhodopsin I or metarhodopsin II conformations of light-excited rhodopsin. Epitopes of antibodies that stabilize metarhodopsin II indicate conformational changes from dark-adapted rhodopsin, including rearrangements of the C-terminal tail and altered exposure of the cytoplasmic end of helix VI, a portion of the C-3 loop, and helix VIII. As additional antibodies are subjected to antibody imprinting, this approach should provide increasingly detailed information on the conformation of light-excited rhodopsin and be applicable to structural studies of other challenging protein targets.
Department of Chemistry and Biochemistry, Montana State University, Bozeman, Montana 59717-3520, USA.
Brian W
BW
Bailey
Brendan
B
Mumey
Paul A
PA
Hargrave
Anatol
A
Arendt
Oliver P
OP
Ernst
Klaus Peter
KP
Hofmann
Patrik R
PR
Callis
James B
JB
Burritt
Algirdas J
AJ
Jesaitis
Edward A
EA
Dratz
D000465:Algorithms; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002417:Cattle; D016384:Consensus Sequence; D018360:Crystallography, X-Ray; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism; D003624:Darkness; D018604:Epitope Mapping / Q000379:methods; D008027:Light; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry* / Q000378:metabolism / Q000528:radiation effects; D012243:Rhodopsin / Q000737:chemistry* / Q000276:immunology / Q000528:radiation effects; D012374:Rod Cell Outer Segment / Q000737:chemistry / Q000378:metabolism
D000911:Antibodies, Monoclonal; D043562:Receptors, G-Protein-Coupled; D012243:Rhodopsin