Biophysical Journal
February 1997.
Mutation of a Surface Residue, Lysine-129, Reverses the Order of Proton Release and
Uptake in Bacteriorhodopsin; Guanidine Hydrochloride Restores it
Rajni Govindjee, Eleonora S. Imasheva, Saurav Misra, Sergei P. Balashov,
Thomas G. Ebrey, Ning Chen#, Donald R. Menick#,
and Rosalie K. Crouch#
From the Center for Biophysics and Computational Biology,
and the Department of Cell and Structural Biology, University of Illinois at Urbana-Champaign,
Urbana, IL 61801;
# Medical University of South Carolina, Charleston,
29425
ABSTRACT
K129 is a residue located in the extracellular loop connecting transmembrane helices D and E of
bacteriorhodopsin. Replacement of K129 with a histidine alters the pKas
of two key residues in the proton transport pathway, D85 and the proton release group (probably E204);
the resulting pigment has properties which differ markedly from the wild type. 1) In the unphotolyzed
state of K129H mutant, the pKa of D85 is 5.1 ± 0.1 in 150 mM KCl
(compared to ~2.6 in the wild type bacteriorhodopsin), while the unphotolyzed-state
pKa of E204 decreases to 8.1 ± 0.1 (from ~9.5 in the wild type
pigment). 2) The pKa of E204 in the M state is 7.0 ± 0.1 in K129H,
compared to ~5.8 in the wild type pigment. 3) As a result of the change in the
pKa of E204 in M, the order of light-induced proton release and uptake
exhibits a dependence on pH in K129H differing from the wild type: at neutral pH and moderate salt
concentrations (150 mM KCl), light-induced proton uptake precedes proton release, while it
follows proton release at higher pH. This pumping behavior is similar to that seen in a related
bacterial rhodopsin, archaerhodopsin-1, which has a histidine in the position analogous to K129.
4) At alkaline pH, a substantial fraction of all-trans- K129H pigment (ca. 30%), undergoes
a conversion into a shorter wavelength species, P480, with pKa ~
8.1, close to the pKa of E204. 5) Guanidine hydrochloride lowers the
pKa's of D85 and E204 in the ground state and the
pKa of E204 in the M intermediate, and restores the normal order of
proton release before uptake at neutral pH. 6) In the K129H mutant the coupling between D85 and
E204 is weaker than in wild type bacteriorhodopsin. In the unphotolyzed pigment, the change in
the pKas of either residue when the other changes its protonation
state is only 1.5 units compared to 4.9 units in wild type bacteriorhodopsin. In the M state of
photolyzed K129H pigment, the corresponding change is 1 unit, compared to 3.7 units in the wild
type pigment. We suggest that K129 may be involved in stabilizing the hydrogen bonding network
that couples E204 and D85. Substitution of K129 with a histidine residue causes structural
changes which alter this coupling and affect the pKas of E204 and D85.
Received for publication on 7 August 1996 and in final form 12 November 1996.
Address reprint requests to Dr. Thomas G. Ebrey, Department of Cell and Structural Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801. Tel.: 217-333-2015; Fax: 217-244-6615; tebrey@ux1.cso.uiuc.edu
Copyright 1996 by the Biophysical Society
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