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

Evidence is presented for long range interactions between the extracellular and cytoplasmic parts of the heptahelical membrane protein bacteriorhodops in 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 th e 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 rele ase and uptake kinetics were measured directly at either surface by using t he unique cysteine residue as attachment site for the pH indicator fluoresc ein. Whereas in wild type proton uptake on the cytoplasmic surface occurs d uring the M-decay (tau similar to 8 ms), in R82A it occurs already during t he first phase of the M-rise (tau < 1 mu s). (ii) The introduction of a sec ond 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 HID isotope effects provide evidence that the proton release mechanism in R 82A/G231C and in wild type is similar. These results suggest the existence of long range interactions between the cytoplasmic and extracellular surfac e 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 activ ation and signal transduction in heptahelical G-protein-coupled receptors.