Abstract

One of the central aims of vision research is to elucidate the molecular changes photoreceptor membrane undergoes during bleaching. While a variety of biophysical methods have been developed which are capable of probing rhodopsin structure, only in special cases can specific information be obtained about individual molecular groups. We have recently demonstrated that Fourier transform infrared (FTIR) difference spectroscopy can detect and characterize single group alterations inside intact photoreceptor membrane during each step in the bleaching sequence. We now propose to utilize this new method in combination with a variety of biochemical and molecular genetic techniques to more fully elucidate how photoreceptor membrane functions. This includes rhodopsin regeneration with specifically labelled retinals, reconstitution with various lipids and site-specific mutation of individual amino acids in the rhodopsin sequence. The development of such a combined spectroscopic and biochemical approach should also help provide a general method for studying other membrane based systems.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY005499-08
Application #
3260620
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Project Start
1984-08-01
Project End
1993-07-31
Budget Start
1991-08-01
Budget End
1993-07-31
Support Year
8
Fiscal Year
1991
Total Cost
Indirect Cost

  Institution

Name
Boston University
Department
Type
Schools of Arts and Sciences
DUNS #
604483045
City
Boston
State
MA
Country
United States
Zip Code
02118

  Publications

Bergo, Vladislav; Amsden, Jason J; Spudich, Elena N et al. (2004) Structural changes in the photoactive site of proteorhodopsin during the primary photoreaction. Biochemistry 43:9075-83
Bergo, Vladislav; Spudich, Elena N; Spudich, John L et al. (2002) A Fourier transform infrared study of Neurospora rhodopsin: similarities with archaeal rhodopsins. Photochem Photobiol 76:341-9
Rath, P; Bovee-Geurts, P H; DeGrip, W J et al. (1994) Photoactivation of rhodopsin involves alterations in cysteine side chains: detection of an S-H band in the Meta I-->Meta II FTIR difference spectrum. Biophys J 66:2085-91
Rath, P; Olson, K D; Spudich, J L et al. (1994) The Schiff base counterion of bacteriorhodopsin is protonated in sensory rhodopsin I: spectroscopic and functional characterization of the mutated proteins D76N and D76A. Biochemistry 33:5600-6
Baenziger, J E; Miller, K W; Rothschild, K J (1993) Fourier transform infrared difference spectroscopy of the nicotinic acetylcholine receptor: evidence for specific protein structural changes upon desensitization. Biochemistry 32:5448-54
He, Y; Krebs, M P; Fischer, W B et al. (1993) FTIR difference spectroscopy of the bacteriorhodopsin mutant Tyr-185-->Phe: detection of a stable O-like species and characterization of its photocycle at low temperature. Biochemistry 32:2282-90
Sonar, S; Krebs, M P; Khorana, H G et al. (1993) Static and time-resolved absorption spectroscopy of the bacteriorhodopsin mutant Tyr-185-->Phe: evidence for an equilibrium between bR570 and an O-like species. Biochemistry 32:2263-71
Rath, P; Krebs, M P; He, Y et al. (1993) Fourier transform Raman spectroscopy of the bacteriorhodopsin mutant Tyr-185-->Phe: formation of a stable O-like species during light adaptation and detection of its transient N-like photoproduct. Biochemistry 32:2272-81
Rath, P; DeCaluwe, L L; Bovee-Geurts, P H et al. (1993) Fourier transform infrared difference spectroscopy of rhodopsin mutants: light activation of rhodopsin causes hydrogen-bonding change in residue aspartic acid-83 during meta II formation. Biochemistry 32:10277-82
Bousche, O; Sonar, S; Krebs, M P et al. (1992) Time-resolved Fourier transform infrared spectroscopy of the bacteriorhodopsin mutant Tyr-185-->Phe: Asp-96 reprotonates during O formation;Asp-85 and Asp-212 deprotonate during O decay. Photochem Photobiol 56:1085-95

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