ASP(96) DEPROTONATION AND TRANSMEMBRANE ALPHA-HELICAL STRUCTURAL-CHANGES IN BACTERIORHODOPSIN

The M-->N transition in the photocycle of bacteriorhodopsin involves t he transfer of a proton from Asp96 to the retinylidene Schiff base, po ssibly through a network of hydrogen-bonded amino acid residues and wa ter molecules (Rothschild, K. J., He, Y. W., Sonar, S., Marti, T., and Khorana, H. G. (1992) J. Biol. Chem. 267, 1615-1622). A conformationa l change of the protein backbone is also observed during this transiti on. In this work, we have investigated the effects of replacing the re sidue Thr46, which might be part of this chain, with an aspartic acid. Both Fourier transform infrared and resonance Raman spectroscopy show that the chromophore structure of this mutant (T46D) is normal. Howev er, N formation is accelerated and N decay is significantly slowed com pared to wild-type bacteriorhodopsin. This effect causes the N interme diate to accumulate under steady-state illumination thereby facilitati ng spectroscopic studies under normal pH conditions. Fourier transform infrared difference spectroscopy reveals that like native bacteriorho dopsin, N formation in T46D involves deprotonation of Asp96, reprotona tion of the Schiff base, and a change in the backbone secondary struct ure. However, in contrast to bacteriorhodopsin, bands assigned to the C=O stretch mode of the carboxylic acid group of Asp96 are upshifted b y 10 cm-1 reflecting a change in the Asp96 environment and a drop in i ts effective pK(a) throughout the photocycle. This change in the pK(a) can directly account for changes in the photocycle kinetics and indic ates that Asp96 deprotonation/protonation are the rate limiting steps in the formation and decay of the N intermediate. By studying the effe cts of H/D exchange, evidence is found that the backbone structural ch anges involve transmembrane alpha-helices. It is proposed that these s tructural changes serve to modulate the local environment and protonat ion state of Asp96 during the photocycle and are also essential for fo rmation of the proton conducting hydrogen bonded network which functio ns during Schiff base reprotonation.