Antibody imprinting studies of rhodopsin: A model G protein-coupled receptor

Determination of three-dimensional structures of membrane proteins and their different functional states has been elusive. After years of attempts, a high resolution x-ray structure of dark-adapted rhodopsin was recently reported (Palczewski, et al., 2000). However, structures of light-activated rhodopsin states, which trigger the G protein amplification cascade and are homologous to agonist-stimulated conformations of other GPCRs, remain poorly understood.; “Antibody imprinting” was used to study the structures of the metarhodopsin-I and active metarhodopsin-II photostates. Peptide consensus sequences for each antibody in a panel of anti-rhodopsin monoclonal antibodies were found by bio-panning against phage-displayed random peptide libraries. These consensus peptides mimic structural epitopes on rhodopsin.; To understand the structural information contained in antibody epitopes, consensus sequences were mapped to the rhodopsin primary sequence manually, or with “Findmap”, an algorithm which scores all possible mappings of peptide sequences to target proteins. Most consensus peptides mimicked discontinuous epitopes, on rhodopsin, implicating sequentially distant residues as proximate in the structures of different rhodopsin photointermediates. Distinctions were found between the patterns of proximity constraints from antibodies that prefer metarhodopsin-I or metarhodopsin-II, allowing construction of networks of distance constraints for metarhodopsin-I and metarhodopsin-II. The proximity of the central part of the N-terminal tail and the I-3 loop found by Findmap are consistent with the x-ray structure of the dark-adapted form of the protein An in silico study demonstrated the feasibility of simultaneously applying multiple structural constraints to a rhodopsin model with explicit phospholipid bilayer and explicit water.; A detailed structural understanding of metarhodopsin-I and metarhodopsin-II, must await the availability of significantly larger numbers of antibodies with photostate preference. Many aspects of this work agree qualitatively with prior studies using a wide range of approaches. The ultimate level of structural detail that can be obtained from antibody imprinting is by co-crystallizing consensus epitopes peptides with the selecting FAb to determine the folded peptide structure in the Fab binding site by x-ray crystallography, and a start was made in this approach. Insights gained into rhodopsin photostate structures constitute a foundation of methodology which can be built upon by future studies.