Studies of the protein folding process offer insights into the mechanisms, kinetics, and thermodynamics of polypeptide folding. The goal of this research is to provide a detailed analysis of the folding and assembly of the rod cell photoreceptor rhodopsin by focusing on the identification and characterization of independent folding domains in the bovine opsin apoprotein. Opsin polypeptide fragments generated by genetic manipulation of the bovine opsin gene have been examined for functional assembly in vivo. Remarkably, co-expression of two or three complementary opsin polypeptide fragments separated in the cytoplasmic region 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 is being 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 (CD) 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, bacterial overexpression is in progress. The nature and extent of the interactions accompanying fragment complementation and the effects of natural and site-directed mutations on the 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. In addition to identifying regions of bovine opsin which contain sufficient information to independently fold, insert into a membrane, and assemble into a functional molecule, these studies should provide insights into the structural consequences associated with some naturally-occurring opsin mutations seen in patients afflicted with retinitis pigmentosa. They are also expected to have relevance to the folding and assembly of a growing number of related receptors which are coupled to guanine nucleotide-binding proteins.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29EY011112-05
Application #
6179332
Study Section
Visual Sciences C Study Section (VISC)
Program Officer
Dudley, Peter A
Project Start
1996-06-01
Project End
2002-05-31
Budget Start
2000-06-01
Budget End
2002-05-31
Support Year
5
Fiscal Year
2000
Total Cost
$103,600
Indirect Cost
Name
University of MD Biotechnology Institute
Department
Type
Organized Research Units
DUNS #
City
Baltimore
State
MD
Country
United States
Zip Code
21202