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.
Highlights
Allosteric interactions are well known and of great importance for water soluble enzymes, where ligand or substrate binding to a specific site alters the conformation and changes the affinity at a different site of the protein
In the double mutant R82A/G231C, in which the second mutation is located at the cytoplasmic surface at the end of helix G, the pKa of the purple to blue transition is shifted completely back to the wild type value, even to a somewhat lower number
The effect of the arginine 82 to alanine mutation on dark adaptation, proton release, and photochemical cycle in membrane fragments was analyzed by Balashov et al [21]
Summary
Release and uptake, detected with pH indicator dyes in the aqueous solution, were already observed [20, 21]. To detect proton concentration changes separately on each surface of the protein, the attachment site (single cysteine residue) for the pH indicator dye at the surface of the mutant protein R82A was varied. The second site revertant produced by the replacement of glycine by cysteine at the cytoplasmic surface of the protein far away from the location of the first mutation (R82A) can shed light on the molecular mechanisms that promote long range interactions in 7-helix transmembrane proteins. Such allosteric interactions are expected to be important for ligand binding and signal transduction in G-protein-coupled receptors [24]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
