REORIENTATIONAL MOTIONS OF THE D96N AND T46V D96N MUTANTS OF BACTERIORHODOPSIN IN THE PURPLE MEMBRANE/

The reorientational motions of the D96N and T46V/D96N mutants of bacte riorhodopsin in purple membrane have been investigated by time-resolve d linear dichroism measurements. The reorientations in the early stage s of the photocycle are identical to those observed in wild-type bacte riorhodopsin: anisotropies of photocycle intermediates in both D96N an d T46V/D96N are r(K) = 0.38 +/- 0.01, r(L) = 0.35 +/- 0.01, r(M) = 0.3 5 +/- 0.01. The anisotropy of the initial state, r(BR), exhibits decay s to zero in D96N and to negative values in T46V/D96N on the time scal e of tens of milliseconds. This anisotropy decay can be explained by a model that involves the motion of unexcited or spectator proteins adj acent to a photocycling protein. The amplitude and time constants of s pectator reorientational motion are similar to those that have been ob served in the wild type, Contributions from the anisotropy of the N-st ate were detected in measurements of the T46V/D96N mutant, in which a large N-state population accumulates. The value of r(N) is estimated t o be 0.30 +/- 0.05 in T46V/D96N.