Bacteriorhodopsin, the single protein of the purple membrane in extremely halophilic bacteria, transduces light energy to chemical energy by pumping protons from inside to outside of the cell. The overall aim of this research is to study the structure, mechanism of action and biosynthesis of bacteriorhodopsin by using chemical, physicochemical, biochemical and genetic approaches. The primary structure and orientation of the protein in the purple membrane is now known. Attention will be focussed on the tertiary structure of the protein. There are presumed to be seven helices which traverse the membrane. Folding, interactions between individual helices, and polypeptidic environments around the retinal in different conformational states (e.g., light-adapted, dark-adapted, and the bleached form) of bacteriorhodopsin will be investigated. For studies of structure-function relationships, a major approach will involve the use of recombinant DNA carrying the gene for bacteriorhodopsin. This gene will be cloned and its expression will be studied in E. coli. If not expressed, synthetic E. coli promoters will be linked to the gene. Alternatively, coupled transcription and translation using a variety of systems will be investigated for production of bacteriorhodopsin in vitro. Site-directed mutagenesis of the cloned DNA will be used by using in vitro reconstitution of the proton pump which now has been worked out in this laboratory. In studies of biosynthesis of bacteriorhodopsin, the nature of the messenger RNA, the synthesis and insertion of the protein into membrane and different mechanisms of its regulation (by retinal, light and oxygen) will be studied.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM028289-05
Application #
3275573
Study Section
Biochemistry Study Section (BIO)
Project Start
1980-12-01
Project End
1985-11-30
Budget Start
1984-12-01
Budget End
1985-11-30
Support Year
5
Fiscal Year
1985
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
Ou, Wen-bin; Yi, Tingfang; Kim, Jong-Myoung et al. (2011) The roles of transmembrane domain helix-III during rhodopsin photoactivation. PLoS One 6:e17398
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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
Klein-Seetharaman, J; Reeves, P J; Loewen, M C et al. (2002) Solution NMR spectroscopy of [alpha -15N]lysine-labeled rhodopsin: The single peak observed in both conventional and TROSY-type HSQC spectra is ascribed to Lys-339 in the carboxyl-terminal peptide sequence. Proc Natl Acad Sci U S A 99:3452-7
Reeves, Philip J; Kim, Jong-Myoung; Khorana, H Gobind (2002) Structure and function in rhodopsin: a tetracycline-inducible system in stable mammalian cell lines for high-level expression of opsin mutants. Proc Natl Acad Sci U S A 99:13413-8
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
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
Loewen, M C; Klein-Seetharaman, J; Getmanova, E V et al. (2001) Solution 19F nuclear Overhauser effects in structural studies of the cytoplasmic domain of mammalian rhodopsin. Proc Natl Acad Sci U S A 98:4888-92

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