Optical picosecond studies of bacteriorhodopsin containing a sterically fixed retinal

Abstract

The photochemical behaviour of an analogous bacteriorhodopsin (9,12-Ph-BR) which contains the sterically fixed 9,12-phenylretinal has been investigated with picosecond spectroscopy. The following results have been obtained. No ground-state intermediate photoproduct is found in agreement with the previous observation that 9,12-Ph-BR does not exhibit proton pumping under illumination. The excited singlet state has a lifetime of ¢s-10 + 2 ps. This lifetime agrees favourably with the value calculated from the radiative lifetime ¢~d ----6.2 ns and the fluorescence quantmn efficiency of 1.2 • 10-3 Excited-state absorption occurs which results in fluorescence in the ultraviolet region. These various observations differ drastically from the corresponding findings on bacteriorhodopsin. Most important for an understanding of the differences is the fact that 9,12-phenylretinal does not isomerize in the protein's binding site in contrast to retinal. Our data therefore suggest that the formation of the intermediate K observed in bacteriorhodopsin is accompanied by the all-trans to 13-c/s isomerization.

Femtosecond and picosecond fluorescence of native bacteriorhodopsin and a nonisomerizing analog

“…In contrast to what was believed earlier,2 I‐460 is found to be all‐trans. It was also found that the torsional flexibility in native bR is responsible for the short lifetime of I‐460,7 in agreement with earlier work 10…”

Energy transfer from retinal to amino acids — a time-resolved study of the ultraviolet emission of bacteriorhodopsin

“…9. The apomembrane (bacterioopsin) obtained was either reconstituted with 9,12-phenylretinal [10] or with all-trans and 13-cis retinal purified by high-pressure liquid chromatography. Reconstitution of the chromoproteins was followed spectroscopically in an Aminco DW2 spectrophotometer [11] and fluorescence emission spectra ()~ excitation 280 nm) recorded in a Perkin Elmer MPF 3 spectrofluorimeter.…”

“…First, retinal was extracted from the purple membrane and by doing this a chromophore-free suspension of bacterioopsin prepared [10]. Exciting this sample with light at 527 nm at the same intensity as used above did not give an ultraviolet emission within the dynamic range of our experiment.…”

Photoelectric Measurements of Membranes

“…To test the current hypothesis that it is the all-trans~ 13-cis isomerization that triggers the proton pumping mechanism, Polland et a/. (1984) carried out picosecond absorption spectroscopy and fluorescence measurements with a sterically fixed "9, 12-phenyl-retinal" reconstituted with bacterioopsin.…”