Opening the Schiff base moiety of bacteriorhodopsin by mutation of the four extracellular Glu side chains

Abstract

The quadruple bacteriorhodopsin (BR) mutant E9Q+E74Q+E194Q+E204Q shows a V V max of about 500 nm in water at neutral pH and a great influence of pH and salts on the visible absorption spectrum. Accessibility to the Schiff base is strongly increased, as detected by the rapid bleaching effect of hydroxylamine in the dark as well as in light. Both the proton release kinetics and the photocycle are altered, as indicated by a delayed proton release after proton uptake and changed M kinetics. Moreover, affinity of the color-controlling cation(s) is found to be decreased. We suggest that the four Glu side chains are essential elements of the extracellular structure of BR.z 1999 Federation of European Biochemical Societies.

Thr-90 Plays a Vital Role in the Structure and Function of Bacteriorhodopsin

“…The construction and expression of T90A and D115A mutants in Halobacterium salinarum was carried out as described (14). The mutant T90V was a generous gift from Dr. J. K. Lanyi.…”

“…One example refers to the bleached membrane, where the retinal absence compels the helices to lose several interactions (12,13). Another case corresponds to the triple or quadruple mutants E9Q/E194Q/ E204Q and E9Q/E74Q/E194Q/E204Q, which exhibit a more relaxed conformation as compared with WT (14,15).…”

Contribution of Extracellular Glu Residues to the Structure and Function of Bacteriorhodopsin

“…The construction of the mutants and their expression in Halobacterium salinarum was done as described previously (12,36). The wild type and mutagenized membranes were isolated as purple membrane sheets according to Oesterhelt and Stoeckenius (41).…”

“…All experiments were performed in the dark using dark-adapted samples. Because of abnormal dark adaptation kinetics of the E194Q, 3Glu, and 4Glu mutants (36), the samples were kept in the dark for about 25 days. Absorbance changes at 630 nm as a function of pH were used to monitor the purple-to-blue transition.…”

“…Recently we have described some spectral and functional properties of the quadruple mutant E9Q/E74Q/E194Q/E204Q (4Glu), including high hydroxylamine and Cl Ϫ accessibility to the Schiff base environment and severalfold decreased effect of Ca 2ϩ on the regeneration of the purple color of deionized 4Glu (36). These results suggest a more open structure for the extracellular region of the 4Glu mutant and reduced affinity of cations for the color-controlling binding site(s) as compared with wild type.…”