Independent photocycles of the spectrally distinct forms of bacteriorhodopsin

Abstract

Time-resolved, flash-induced difference absorbance spectra (300-700 am) at pH 10.5 and 5C for the bacteriorhodopsin photocycle fast and slow decaying forms of the M intermediate (M' and MS, respectively) MATERIALS AND METHODS Purple membranes were isolated from Halobacterium halobium, S9 strain, and suspended in 30 mM piperazine/30 mM glycylglycine, pH 10.5. All samples contained 40%o (vol/vol) glycerol except those used for the flash-induced absorbance changes in Fig. 4. All measurements were carried out on light-adapted BR.Two spectrophotometers were used to obtain the lightinduced difference spectra. For the faster measurements, the data were taken and analyzed as described (10,11). For the slower measurements a Hewlett-Packard model 8452A diode array spectrophotometer was used to take complete lightinduced difference spectra from 300 to 700 nm with 2-nm spectral resolution. (The absorption spectrum of the lightadapted sample was used as the baseline.) The actinic source was a commercial photoflash with Coming long-pass filter (CS 3-67, wavelength >550 nm). The half-duration time was 150 tUs. Since the measuring beam of the diode array spectrophotometer was relatively strong white light, special care was taken to check and avoid a possible actinic effect of the measuring beam. Sample OD570 was between 1.3 and 1.5. RESULTSSeparation of the Difference Spectra of Intermediates M', ME, and R. From the time-dependent series of difference spectra, we can determine the difference spectra of the late intermediates involved in the BR recovery at high pH (Mf, MS, and R). We have chosen conditions for which the differences in the lifetimes and yields of these three components are maximal. We assume that the intermediates before M can be neglected because of their much shorter lifetimes and that no significant amount of the late intermediate 0 is formed because of the low temperatures and high pH values (refs. 5, 6, and 10 and unpublished data). Fig. 1 6358The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Simultaneous monitoring of light-induced changes in protein side-group protonation, chromophore isomerization, and backbone motion of bacteriorhodopsin by time-resolved Fourier-transform infrared spectroscopy.

“….) (22,28,31,32); (ii) at least two parallel photocycles of slightly different bR molecules (23,28,(33)(34)(35). The present results cannot unequivocally distinguish between the two proposed models.…”

Bacteriorhodopsin photocycle at cryogenic temperatures reveals distributed barriers of conformational substates

“…The reason for heterogeneity or branching would be, for example, partial protonation states of critical residues depending on the pH (38), nonequivalence of the monomers in the bacteriorhodopsin trimer (39), or a neighbor/cooperativity effect of photocycling molecules on one another in the membrane (40,41). However, the temperature up-shift experiment (Fig.…”

The quantum yield of bacteriorhodopsin

“…BR equilibrates with different isoforms at different pH values [22,23]. Below pH 2.7 an acidic form absorbing maximally at 605 nm becomes dominant.…”