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.
The role of Thr-90 in the bacteriorhodopsin structure and function was investigated by its replacement with Ala and Val. The mutant D115A was also studied because Asp-115 in helix D forms a hydrogen bond with Thr-90 in helix C. Differential scanning calorimetry showed a decreased thermal stability of all three mutants, with T90A being the least stable. Light-dark adaptation of T90A was found to be abnormal and salt-dependent. Proton transport monitored using pyranine signals was ϳ10% of wild type for T90A, 20% for T90V, and 50% for D115A. At neutral or alkaline pH, the M rise of these mutants was faster than that of wild type, whereas M decay was slower in T90A. Overall, Fourier transform infrared (FTIR) difference spectra of T90A were strongly pH-dependent. Spectra recorded on films adjusted at the same pH at 243 or 277 K, dry or wet, showed similar features. The D115A and T90V FTIR spectra were closer to WT, showing minor structural differences. The band at 1734 cm ؊1 of the deconvoluted FTIR spectrum, corresponding to the carboxylate of Asp-115, was absent in all mutants. In conclusion, Thr-90 plays a critical role in maintaining the operative location and structure of helix C through three complementary interactions, namely an interhelical hydrogen bond with Asp-115, an intrahelical hydrogen bond with the peptide carbonyl oxygen of Trp-86, and a steric contact with the retinal. The interactions established by Thr-90 emerge as a general feature of archaeal rhodopsin proteins.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.