title: The two pKa's of aspartate-85 and control of thermal isomerization and proton release in the arginine-82 to lysine mutant of bacteriorhodopsin.
abstract: To explore the role of Arg82 in the catalysis of proton transfer in bacteriorhodopsin, we replaced Arg82 with Lys, which is also positively charged at neutral pH but has an intrinsic pKa of about 1.7 pH units lower than that of Arg. In the R82K mutant expressed in Halobacterium salinarium, we found the following: (1) The pKa of the purple-to-blue transition at low pH (which reflects the pKa of Asp85) is 3.6 +/- 0.1. At high pH a second inflection in the blue-to-purple transition with pKa = 8.0 is found. The complex titration behavior of Asp85 indicates that the pKa of Asp85 depends on the protonation state of another amino acid residue, X', which has a pKa = 8.0 in R82K. The fit of the experimental data to a model of two interacting residues shows that deprotonation of X' at high pH causes a shift in the pKa of Asp85 from 3.7 to 6.0. In turn, protonation of Asp85 decreases the pKa of X' by 2.3 pH units. This suggests that X' can release a proton upon formation of the M intermediate and the concomitant protonation of Asp85 in the photocycle. (2) The rate constant of dark adaptation, kda, is proportional to the fraction of blue membrane between pH 2 and 10, indicating that thermal isomerization proceeds through the transient protonation of Asp85. The pH dependence of kda shows that two groups with pKal = 3.9 and pKa2 = 8.0 control the rate of dark adaptation in R82K. The 1.7 pH unit shift in pKa2 in R82K compared to the wild type (WT) (pKa2 = 9.7) supports the hypothesis that X' is Arg82 in WT and Lys82 in R82K (or at least that these groups are the principal part of a cluster of residues that constitute X'). (3) Under steady state illumination, the efficiency of proton transport in R82K incorporated in phosphatidylcholine vesicles is at least 40% of that in the WT. A flash-induced transient signal of the pH-sensitive dye pyranine is similar to that in the WT (proton release precedes uptake), but the amplitude is small in R82K (about 15% of that found in the WT), indicating that only a small fraction of protons is released fast in R82K. This supports the suggestions that Arg82 is associated with the proton release pathway (acts as a proton release group or part of a proton release complex) and that Lys cannot efficiently substitute for Arg in this process.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Department of Cell and Structural Biology, University of Illinois at Urbana--Champaign 61801, USA.
authors.forename: S P
authors.initials: SP
authors.lastname: Balashov
authors.forename: R
authors.initials: R
authors.lastname: Govindjee
authors.forename: E S
authors.initials: ES
authors.lastname: Imasheva
authors.forename: S
authors.initials: S
authors.lastname: Misra
authors.forename: T G
authors.initials: TG
authors.lastname: Ebrey
authors.forename: Y
authors.initials: Y
authors.lastname: Feng
authors.forename: R K
authors.initials: RK
authors.lastname: Crouch
authors.forename: D R
authors.initials: DR
authors.lastname: Menick
mesh_terms: D001120:Arginine / Q000737:chemistry*; D001224:Aspartic Acid / Q000737:chemistry*; D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000235:genetics; D001483:Base Sequence; D017931:DNA Primers; D003624:Darkness; D006217:Halobacterium / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007536:Isomerism; D007700:Kinetics; D008239:Lysine / Q000737:chemistry*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010777:Photochemistry; D011522:Protons; D013057:Spectrum Analysis
chemical_list: D017931:DNA Primers; D011522:Protons; D001224:Aspartic Acid; D001436:Bacteriorhodopsins; D001120:Arginine; D008239:Lysine