Electric-field effects in dry films of D85N and D85,96N mutant bacteriorhodopsin

In the D85N mutant of the protein bacteriorhodopsin (BR), the Schiff base, by which the retinal chromophore is bound to the protein, exhibits an abnor mally low proton affinity (p K-a = 8.9). Recent experiments on thin films o f this protein have shown that this causes the protonation state of the Sch iff base, and thus the visible absorption spectrum, to be sensitive to exte rnal electric fields. In this paper, we explore the dependence of this effe ct on parameters such as pH, humidity, and film thickness. The results of t hese experiments point to the importance of water molecules bound in the ac ceptor part of the proton channel as sources and donors in field-induced pr oton-transfer reactions. We describe additional results obtained with the D 85,96N mutant, which also exhibits a low Schiff-base pK. The similar behavi or of the two mutants under applied electric fields at high pH implies that the residue Asp-96 plays no role in field-induced Schiff-base protonation. (C) 2000 Elsevier Science S.A. All rights reserved.