Reducing the flexibility of retinal restores a wild-type-like photocycle in bacteriorhodopsin mutants defective in protein-retinal coupling.
The thermal re-isomerization of retinal from the 13-cis to the all-trans state is a key step in the final stages of the photocycle of the light-driven proton pump, bacteriorhodopsin. This step is greatly slowed upon replacement of Leu-93, a residue in van der Waals contact with retinal. The most likely role of this key interaction is that it restricts the flexibility of retinal. To test this hypothesis, we have exchanged native retinal in Leu-93 mutants with bridged retinal analogs that render retinal less flexible by restricting free rotation around either the C10-C11 (9,11-bridged retinal) or C12-C13 (11,13-bridged retinal) single bonds. The effect of the analogs on the photocycle was then determined spectroscopically by taking advantage of the previous finding that the decay of the O intermediate in the Leu-93 mutants provides a convenient marker for retinal re-isomerization. Time-resolved spectroscopic studies showed that both retinal analogs resulted in a dramatic acceleration of the photocycling time by increasing the rate of decay of the O intermediate. In particular, exchange of native retinal in the Leu-93 --> Ala mutant with the 9,11-bridged retinal resulted in an acceleration of the decay of the O intermediate to a rate similar to that seen in wild-type bacteriorhodopsin. We conclude that the protein-induced restriction of conformational flexibility in retinal is a key structural requirement for efficient protein-retinal coupling in the bacteriorhodopsin photocycle.
Delaney
J K
JK
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore MD 21205, USA.
Yahalom
G
G
Sheves
M
M
Subramaniam
S
S
D000409:Alanine; D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000378:metabolism*; D002151:Calorimetry; D007700:Kinetics; D007930:Leucine; D008027:Light; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012172:Retinaldehyde / Q000031:analogs & derivatives / Q000737:chemistry* / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D011994:Recombinant Proteins; D001436:Bacteriorhodopsins; D007930:Leucine; D000409:Alanine; D012172:Retinaldehyde