Conformational changes in rhodopsin. Movement of helix f detected by site-specific chemical labeling and fluorescence spectroscopy.
A recent proposal for the formation of functionally active rhodopsin has placed critical importance on a movement of one of its transmembrane helices (Farrens, D. L., Altenbach, C., Yang, K., Hubbell, W. L., and Khorana, H. G. (1996) Science 274, 768-770). We investigated this hypothesis using a series of eight rhodopsin mutants containing single reactive cysteine residues in the region (helix F) where movement was previously detected. The cysteine mutants were studied in two ways, by measuring their reactivity to a cysteine-specific reagent (PyMPO-maleimide), and by labeling the cysteines with a fluorescent label (monobromobimane) followed by fluorescence spectroscopic analysis. The chemical reactivity data showed sequence-specific variations in reactivity for the mutants in the dark state, resulting in a pattern suggestive of an alpha helix. Interestingly, only upon photoactivation to the MII form did residues found on the inner "face" of this helix react with the PyMPO-maleimide. The ability of the dark state mutants to react with the fluorescent label monobromobimane followed a similar pattern. Furthermore, fluorescence measurements indicate that a bimane label on the inner face of the helix (at V250C) detects changes in the polarity of its environment and accessibility to a fluorescence quenching agent upon MII formation. Viewed together, the data provide further direct evidence that rhodopsin activation involves a conformational change at helix F.
Dunham
T D
TD
Departments of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland, Oregon 97201-3098, USA.
Farrens
D L
DL
D000595:Amino Acid Sequence; D000818:Animals; D001643:Bridged Bicyclo Compounds; D019556:COS Cells; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D005456:Fluorescent Dyes; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012243:Rhodopsin / Q000031:analogs & derivatives / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence; D013113:Spin Labels
D001643:Bridged Bicyclo Compounds; D005456:Fluorescent Dyes; D013113:Spin Labels; C013110:metarhodopsins; D012243:Rhodopsin; D003545:Cysteine; C028381:monobromobimane