I. BACTERIORHODOPSIN: Bacteriorhodopsin, the single protein of the differentiated purple membrane in Halobacterium halobium, transduces light energy to chemical energy by pumping protons from inside to outside of the cell. The purpose of this work is to understand the mechanism of proton translocation by this membrane protein. Does the mechanism involve a proton channel or conductance along a proton """"""""wire""""""""? The experimental approach would involve specific amino acid replacements in the protein in order to ask specific questions. Mutant proteins will be prepared by site specific mutagenesis of the bacteriorhodopsin gene followed by expression. They will be examined for the following properties: (1) refolding, binding of retinal and regeneration of bacteriorhodopsin-like chromophore; (2) reconstitution into vesicles and ability to pump protons; (3) biophysical (Fourier transform infrared laser Raman spectroscopy) studies of the effects of mutations on the protein structure, on interactions between retinal and the protein and on different steps in the photochemical cycle; and (4) possible correlations between effects on photochemical cycle and on proton translocation. II. BIOCHEMISTRY OF LIGHT-TRANSDUCTION IN ROD OUTER SEGMENTS IN VERTEBRATE RETINA; STRUCTURE-FUNCTION STUDIES ON RHODOPSIN. A major objective is to understand the dynamics of rhodopsin; the structural change on bleaching, the interaction with GTPase and the regulation of phosphorylation and dephosphorylation. Studies of rhodopsin at membrane level would involve delipidation, denaturation, refolding and reconstitution. A major aim would be the study of structure-function relationships by site-specific mutagenesis of the gene and expression of the mutated gene products. Total synthesis of the rhodopsin gene has been undertaken so as to facilitate completely unrestricted mutagenesis throughout the gene. For this purpose a suitable number of unique restriction sites have been introduced at appropriate positions along the gene. Structures of the rod outer segment proteins involved in light transduction (GTPase, cGMP phosphodiesterase, the Na channel) are also being investigated by methods of recombinant DNA.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM028289-08
Application #
3484633
Study Section
Biochemistry Study Section (BIO)
Project Start
1980-12-01
Project End
1990-11-30
Budget Start
1987-12-01
Budget End
1988-11-30
Support Year
8
Fiscal Year
1988
Total Cost
Indirect Cost
Name
Massachusetts Institute of Technology
Department
Type
Schools of Arts and Sciences
DUNS #
City
Cambridge
State
MA
Country
United States
Zip Code
02139
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Kim, Jong-Myoung; Hwa, John; Garriga, Pere et al. (2005) Light-driven activation of beta 2-adrenergic receptor signaling by a chimeric rhodopsin containing the beta 2-adrenergic receptor cytoplasmic loops. Biochemistry 44:2284-92
Eilers, Markus; Ying, Weiwen; Reeves, Philip J et al. (2002) Magic angle spinning nuclear magnetic resonance of isotopically labeled rhodopsin. Methods Enzymol 343:212-22
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Niu, Li; Kim, Jong-Myoung; Khorana, H Gobind (2002) Structure and function in rhodopsin: asymmetric reconstitution of rhodopsin in liposomes. Proc Natl Acad Sci U S A 99:13409-12
Reeves, Philip J; Callewaert, Nico; Contreras, Roland et al. (2002) Structure and function in rhodopsin: high-level expression of rhodopsin with restricted and homogeneous N-glycosylation by a tetracycline-inducible N-acetylglucosaminyltransferase I-negative HEK293S stable mammalian cell line. Proc Natl Acad Sci U S A 99:13419-24
Klein-Seetharaman, J; Hwa, J; Cai, K et al. (2001) Probing the dark state tertiary structure in the cytoplasmic domain of rhodopsin: proximities between amino acids deduced from spontaneous disulfide bond formation between Cys316 and engineered cysteines in cytoplasmic loop 1. Biochemistry 40:12472-8
Altenbach, C; Klein-Seetharaman, J; Cai, K et al. (2001) Structure and function in rhodopsin: mapping light-dependent changes in distance between residue 316 in helix 8 and residues in the sequence 60-75, covering the cytoplasmic end of helices TM1 and TM2 and their connection loop CL1. Biochemistry 40:15493-500
Altenbach, C; Cai, K; Klein-Seetharaman, J et al. (2001) Structure and function in rhodopsin: mapping light-dependent changes in distance between residue 65 in helix TM1 and residues in the sequence 306-319 at the cytoplasmic end of helix TM7 and in helix H8. Biochemistry 40:15483-92

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