Guanidinium restores the chromophore but not rapid proton release in bacteriorhodopsin mutant R82Q.
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.
Renthal
R
R
Division of Earth and Physical Sciences, University of Texas at San Antonio, 78249, USA. rrenthal@lonestar.utsa.edu
Chung
Y J
YJ
Escamilla
R
R
Brown
L S
LS
Lanyi
J K
JK
D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019791:Guanidine / Q000378:metabolism / Q000494:pharmacology*; D019617:Halobacterium salinarum / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D009154:Mutation; D011485:Protein Binding; D011522:Protons; D013053:Spectrophotometry; D013499:Surface Properties
D011522:Protons; D001436:Bacteriorhodopsins; D019791:Guanidine