FOLDING AND ASSEMBLY IN VISUAL RHODOPSIN

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This is a Shannon award providing partial support for the research projects that fall short of the assigned Institute's funding range but are in the margin of excellence. The Shannon award is intended to provide support to test the feasibility of the approach; develop further tests and refine research techniques; perform secondary analysis of available data sets; or conduct discrete projects that can demonstrate the PI's research capabilities or lend additional weight to an already meritorious application. The abstract below is taken from the original document submitted by the principal investigator. DESCRIPTION (Adapted from applicant's abstract): The goal of this proposal is to describe the folding and assembly of the rod cell photoreceptor rhodopsin by identifying and characterizing the independent folding domains of the opsin apoprotein. Opsin polypeptide fragments generated by genetic manipulation of the bovine opsin gene have been examined for functional assembly in COS-1 cells. Co-expression of two or three complementary opsin polypeptide fragments allows the formation of rhodopsins with spectral characteristics similar to the native pigment. These results suggest that the functional assembly of rhodopsin may be mediated by the association of multiple protein-folding domains. To further substantiate these findings, the localization of additional sites where discontinuity of the opsin polypeptide chain allows in vivo complementation will be pursued. The development of an in vitro complementing system will be facilitated by examining conditions which promote opsin polypeptide fragment complex formation and through characterization of their structural properties by circular dichroism and fluorescence spectroscopy as well as their state of association by analytical ultracentrifugation. In order to produce sufficient quantities of opsin polypeptide fragments for further complementation studies, overexpression in a bacterial system will be required. Although some of the fragments have been overexpressed successfully in E. coli, several modified approaches are proposed to express those fragments that initially have resisted expression. The nature and extent of the interactions accompanying fragment complementation and the effects of natural and site-directed mutations on this process will be examined by titration calorimetry. Analysis of the unfolding transitions in the opsin polypeptide fragments by differential scanning calorimetry should verify whether they are composed of one or more independent folding domains. The applicant postulates that these studies should provide new insights into the structural consequences associated with some naturally occurring opsin mutations that are associated with certain kinds of autosomal dominant retinitis pigmentosa. They are also expected to have relevance to the folding and assembly of structurally-related receptors which are coupled to guanine nucleotide-binding proteins.