Guanidinium restores the chromophore but not rapid proton release in bacteriorhodopsin mutant R82Q

Abstract

Replacement of the Arg residue at position 82 in bacteriorhodopsin by Gln or Ala was previously shown to slow the rate of proton release and raise the pK of Asp 85, indicating that R82 is involved both in the proton release reaction and in stabilizing the purple form of the chromophore. We now find that guanidinium chloride lowers the pK of D85, as monitored by the shift of the 587-nm absorbance maximum to 570 nm (blue to purple transition) and increased yield of photointermediate M. The absorbance shift follows a simple binding curve, with an apparent dissociation constant of 20 mM. When membrane surface charge is taken into account, an intrinsic dissociation constant of 0.3 M fits the data over a range of 0.2-1.0 M cation concentration (Na+ plus guanidinium) and pH 5.4-6.7. A chloride counterion is not involved in the observed spectral changes, as chloride up to 0.2 M has little effect on the R82Q chromophore at pH 6, whereas guanidinium sulfate has a similar effect to guanidinium chloride. Furthermore, guanidinium does not affect the chromophore of the double mutant R82Q/D85N. Taken together, these observations suggest that guanidinium binds to a specific site near D85 and restores the purple chromophore. Surprisingly, guanidinium does not restore rapid proton release in the photocycle of R82Q. This result suggests either that guanidinium dissociates during the pump cycle or that it binds with a different hydrogen-bonding geometry than the Arg side chain of the wild type.

Evidence for a Perturbation of Arginine-82 in the Bacteriorhodopsin Photocycle from Time-Resolved Infrared Spectra

“…The presence of TEG shifted the color of the membrane fragments from blue to purple near neutral pH, indicating that the exogenous guanidino group had substituted for Arg-82 and was successful in reducing the pK a of Asp-85 (41). Previous experiments with the R82Q protein had shown that a variety of guanidino compounds could exert such an effect (50). Upon washing the membranes to remove any unreacted TEG, the membrane fragments remained purple, indicating that the TEG had become covalently attached to the protein.…”

Biomolecular Electronics: Protein-Based Associative Processors and Volumetric Memories

“…However, a majority of mutants for which the O state yield is enhanced increase the lifetime of the function. 117,[119][120][121][122][123][124][125][126][127][128][129] We have reexamined the R82 mutants [125][126][127] to investigate the role of Arg-82 motion during the latter stages of the photocycle (see Figure 9 These mutations appear to enhance both the yield of O and the quantum efficiency for O f P photochemistry, but no mutant has performed as well as the chemically enhanced protein system described in section 6.2.…”

“…[Selected amino acid abbreviations are alanine (Ala, A); arginine (Arg, R); asparagine (Asn, N); aspartic acid (Asp, D); cysteine (Cys, C); glutamine (Gln, Q); leucine (Leu, L); lysine (Lys, K); methionine (Met, M); phenylalanine (Phe, F); threonine (Thr, T); tyrosine (Tyr, Y).] A majority of these mutants have been prepared and studied previously, although the goals were not data storage but rather a better understanding of structure and function. ,− We have reexamined the R82 mutants 125-127 to investigate the role of Arg-82 motion during the latter stages of the photocycle (see Figure of ref ), and to determine the extent to which Arg-82 mutants (with the addition of guanidine where necessary to maintain function) can control O state properties. To date, no Arg-82 mutant has been found that enhances O state yield significantly.…”