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Constraints on the conformation of the cytoplasmic face of dark-adapted and light-excited rhodopsin inferred from antirhodopsin antibody imprints

¹ Department of Chemistry and Biochemistry,
² Department of Computer Science, and
³ Department of Microbiology, Montana State University, Bozeman, Montana 59717-3520, USA
⁴ Department of Ophthalmology, University of Florida College of Medicine, Gainesville, Florida 32610-0284, USA
⁵ Institut für Medizinische Physik und Biophysik, Charité, Medizinische Fakultät der Humboldt-Universität zu Berlin, 10098 Berlin, Germany

Reprint requests to: Edward A. Dratz, Department of Chemistry and Biochemistry, Montana State University, 108 Gaines Hall, Bozeman, MT 59717-3520; e-mail: dratz{at}chemistry.montana.edu; fax: (406) 994-5407.

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.

Keywords: Antibody epitopes; epitope mapping; G protein–coupled receptors; guanosine diphosphate; guanosine triphosphate; phage display; protein structure; rhodopsin

Abbreviations: CDR, complementary determining regions on an antibody • CNBr, cyanogen bromide • DTT, dithiothreitol • EDTA, ethylenediaminetetraacetic acid • ELISA, enzyme-linked immunosorbent assay • Fab, fragment antibody-binding domain • FTIR, Fourier transform infrared • GPCR, G protein–coupled receptor • IgG, Immunoglobulin G • ITC, isothermal titration calorimetry • LB, Luria-Bertani broth • mAb, monoclonal antibody • MI, metarhodopsin I • MII, metarhodopsin II • NOESY NMR, nuclear Overhauser effect spectroscopy nuclear magnetic resonance • PBS, phosphate buffered saline • PFU, plaque forming unit • SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis

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