Effects of tryptophan mutation on the deprotonation and reprotonation kinetics of the Schiff base during the photocycle of bacteriorhodopsin

Abstract

The rates of deprotonation and reprotonation of the protonated Schiff base (PSB) are determined during the photocycle of nine bacteriorhodopsin mutants in which Trp-10, 12, 80, 86, 137, 138, 182 and 189 are individually substituted by either phenylalanine or cysteine. Of all the mutants, the replacement of Trp-86, Trp-182, and Trp-189 by phenylalanine and Trp-137 by cysteine is found to significantly alter the rate of the deprotonation, but not that of the reprotonation process. As compared with ebR, the Trp-86 mutation dramatically increases the rate of deprotonation of the PSB while the Trp-182 mutation greatly decreases this rate. Temperature dependence studies on the rate constants of the deprotonation demonstrate that the different energetic and entropic effects of the mutation are responsible for the observed different kinetic behavior of the Trp-86 and Trp-182 mutants as compared with that of ebR. In the case of Trp-86 mutant, a large decrease in both energy and entropy of activation suggests that the mutation of this tryptophan residue opens up the protein structure as a result of eliminating the hydrogen-bonding group on its side chain by a phenylalanine substitution. A correlation is observed between the proton pumping yield and the relative amplitudes of the slow deprotonation component but not with rate constants of the rise or decay process at constant pH. These results are best discussed in terms of the heterogeneity model (with parallel cycle) rather than back reaction model.

Tryptophan perturbation in the L intermediate of bacteriorhodopsin: Fourier transform infrared analysis with indole-nitrogen-15 shift

“…Also, Trp182 is the only residue responsible for the UV change in the photocycle (Wu et al, 1991). Mutation of Trp182 decreases the rate of the L-to-M reaction, suggesting its importance for the instability of L (Wu et al, 1992). Mutation of Asp96 or Asp115, which is close to Trp182 causes 1-2-cm™1 shifts of the 3486-cm™1 band.…”

“…The indole nitrogen of tryptophan is frequently involved in H-bonding in proteins (Ippolito et al, 1990) and in some cases takes part in substrate binding, for example, in lysozyme (Phillips, 1967), and maltodextrin receptor (Spurlino et al, 1992). In bacteriorhodopsin, several tryptophan residues surround the chromophore (Henderson et al, 1990), and the mutation of some of these residues drastically affects the L-to-M conversion (Wu et al, 1992) and proton pumping (Mogi et al, 1989).…”

Replacement effects of neutral amino acid residues of different molecular volumes in the retinal binding cavity of bacteriorhodopsin on the dynamics of its primary process

“…To answer this question we have studied the dynamics of photoisomerization in mutants in which the neutral amino acids Val-49, Ala-53, or Trp-182 residues in the retinal-binding pocket are replaced by Ala, Gly, or Phe, respectively. These amino acids and others are proposed to constitute the retinal binding pocket (Wu et al, 1992). It was proposed (Brown et al, 1995) that in V49A and A53G mutants the geometrical arrangement of the Schiff base and Asp85 is changed.…”

Electrostatic and Steric Interactions Determine Bacteriorhodopsin Single-Molecule Biomechanics

“…Trp residues play an important role in bR stability (18,43,44) and activity (45)(46)(47) and are the only large bR residues located exclusively in PM extracellular leaflet (14,41) (see Fig. 5).…”