On the two forms of bacteriorhodopsin

Abstract

In our previous work [(1993) FEBS Lett. 313,248-250; Biochem. Int. 30,[461][462][463][464][465][466][467][468][469] M-intermediate formation of wild-type bacteriorhodopsin was shown to involve two components differing in time constants (z, = 6&7Ops and z2 = 22&25Ops), which were suggested to reflect two independent pathways of M-intermediate formation. The contribution of the fast M was 4-times higher than the slow one. Our present research on M-intermediate formation in the Dll5N bacteriorhodopsin mutant revealed the same components but at a contribution ratio of 1:l. Upon lowering the pH, the slow phase of M-formation vanished at a pK of 6.2, and in the pH region 3.G5.5 only the M-intermediate with a rise time of 60 pus was present. A 5-6 h incubation of D115N bacteriorhodopsin at pH 10.6 resulted in the irreversible transformation of 50% of the protein into a form with a difference absorbance maximum at 460 nm. This form was stable at pH 7.5 and had no photocycle, including M-intermediate formation. The remaining bactenorhodopsin contained 100% fast M-intermediate. The disappearance of the 250;~s phase concomitant with bR460 formation indicates that at neutral pH bacteriorhodopsin exists as two spectroscopically indistinguishable forms.

Inhibition of the M1→M2 (Mclosed→Mopen) transition in the D96N mutant photocycle and its relation to the corresponding transition in wild‐type bacteriorhodopsin

“…In spite of the fact that all the inhibitors used decrease the equilibrium concentration of M open by 2–3 orders, none of them abolishes the multiphase kinetics of the M formation in either the D96N mutant or the wild‐type bR (note, that the fastest component reflects the K→L transition and that glycerol eliminates this component due to its effect [4, 6]). Thus, multiphase formation of the M intermediate could be explained by the presence of different conformational states in bR [6, 28, 29], or by the consecutive formation of different M forms belonging to the M closed pool (in the latter, the similar scheme L↔M′↔M″...↔M i may be valid, but all these M states seem to belong to the M closed pool). The rate of the M closed (M1)↔M open (M2) equilibration is a matter of future experiments.…”

The Ability of Actinic Light to Modify the Bacteriorhodopsin Photocycle. Heterogeneity and/or Photocooperativity?

“…In addition to the work of Eisfeld et al, there have been many other reports of heterogeneity in purple membranes (Hanamoto et al, 1994;Diller and Stockburger, 1988;Dancshazy et al, 1988;Bitting et al, 1990;Balashov et al, 1991;Hendler et al, 1994;Komrakov and Kaulen, 1994).…”

Photovoltage evidence that Glu‐204 is the intermediate proton donor rather than the terminal proton release group in bacteriorhodopsin

“…This decrease is accompanied by the similar lowering of the M level due to most probably a pH dependence of the MIL equilibrium. An equilibrium sensitive to the pK of the proton release group in the M state is quite obvious in the D115N and D96N mutants [9] but is also revealed in the wild-type bR [27].…”