J Biol Chem, Vol. 275, Issue 18, 13431-13440, May 5, 2000
Evidence for Long Range Allosteric Interactions between the
Extracellular and Cytoplasmic Parts of Bacteriorhodopsin from the
Mutant R82A and Its Second Site Revertant R82A/G231C*
Ulrike
Alexiev
§,
Ramin
Mollaaghababa¶
,
H. Gobind
Khorana¶, and
Maarten P.
Heyn
From the Biophysics Group, Department of Physics,
Freie Universität Berlin, Arnimallee 14, D-14195 Berlin, Germany and the ¶ Department of Biology,
Massachusetts Institute of Technology,
Cambridge, Massachusetts 02139
Evidence is presented for long range interactions
between the extracellular and cytoplasmic parts of the heptahelical
membrane protein bacteriorhodopsin in the mutant R82A and its second
site revertant R82A/G231C. (i) In the double mutants R82A/G72C and R82A/A160C, with the cysteine mutation on the extracellular or cytoplasmic surface, respectively, the photocycle is the same as in the
single mutant R82A with an accelerated deprotonation of the Schiff base
and a reversed order of proton release and uptake. Proton release and
uptake kinetics were measured directly at either surface by using the
unique cysteine residue as attachment site for the pH indicator
fluorescein. Whereas in wild type proton uptake on the cytoplasmic
surface occurs during the M-decay (
~ 8 ms), in R82A it
occurs already during the first phase of the M-rise ( < 1 µs). (ii) The introduction of a second mutation at the cytoplasmic
surface in position 231 (helix G) restores wild type ground state
absorption properties, kinetics of photocycle and of proton release,
and uptake in the mutant R82A/G231C. In addition, kinetic H/D isotope
effects provide evidence that the proton release mechanism in
R82A/G231C and in wild type is similar. These results suggest the
existence of long range interactions between the cytoplasmic and
extracellular surface domains of bacteriorhodopsin mediated by salt
bridges and hydrogen-bonded networks between helices C (Arg-82) and G
(Asp-212 and Gly-231). Such long range interactions are expected to be
of functional significance for activation and signal transduction in
heptahelical G-protein-coupled receptors.
*
This work was supported by Grant Sfb 312-B1 from the
Deutsche Forschungsgemeinschaft (to M. P. H.) and by Grant
GM28289 from the National Institutes of Health (to H. G. K.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
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