title: 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.
abstract: 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 (tau approximately 8 ms), in R82A it occurs already during the first phase of the M-rise (tau < 1 microseconds). (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.
authors.affiliation: Biophysics Group, Department of Physics, Freie Universität Berlin, Arnimallee 14, D-14195 Berlin, Germany. USA.
authors.forename: U
authors.initials: U
authors.lastname: Alexiev
authors.forename: R
authors.initials: R
authors.lastname: Mollaaghababa
authors.forename: H G
authors.initials: HG
authors.lastname: Khorana
authors.forename: M P
authors.initials: MP
authors.lastname: Heyn
mesh_terms: D000494:Allosteric Regulation; D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000235:genetics; D006217:Halobacterium / Q000737:chemistry*; D009154:Mutation; D011487:Protein Conformation
chemical_list: D001436:Bacteriorhodopsins