Molecular Mechanism for the function of the Green Proteorhodopsin (GPR): Alteration of Asp97's pKa and the effect of anions binding on GPR propertie

Proteorhodopsin (PR) belongs to GPR (Green Proteorhodopsin) family, a retinal protein of marine proteobacteria which functions as a light-driven proton pump. Light excitation of GPR initiates a photocycle that triggers the translocation of a proton from the cytoplasmic to the extracellular side. Asp97 is located near the retinal-protonated Schiff base and serves as the proton acceptor during the photocycle. The pKa of Asp97 is unusually high (∼7.0), especially in comparison with that of its bR equivalent residue Asp85 (∼2.6). We have studied possible anions binding to GPR (produced from gene vector eBAC31A08 and expressed in Escherichia coli) and their effect on its absorption maxima, Asp97 pKa, and the photocycle. We have revealed that the pKa of Asp97 is sensitive to and is affected by the binding of anions to the protein. We propose that the protein has two binding sites for anions, and occupation of these binding sites affects the pKa of Asp97 differently. It appears that the effect depends on the nature of the anion. Whereas chloride and sulfate anions reduce the pKa following binding to the protein, acetate anions substituted for halogens (especially trichloroacetate and tribromoacetate) elevate significantly the pKa, once binding to the protein. Our results underscore the important role that chloride or sulfate anions play in reducing the pKa of Asp97 to a value (∼7) that may allow GPR to function in its native environment.;Additional interesting observation regarding anions binding to GPR was detected in pH range of 2-5. It was found that following incubation of GPR samples with sulfate anions, a blue shift of the absorption maximum from lambda max=545nm to lambdamax= 525nm with a pKa of ∼3.3 was detected. This pKa depends on sulfate anions concentration and was not observed with other anions. It is plausible that the effect is associated with protonation of Asp 227 which is substituted by a sulfate anion. Asp227 is the equivalent residue to Asp212 in bR which is known to be part of a counter-ion complex stabilizing the protonated Schiff base. In addition, we have found that sulfate anions influence and modify the pigment photocycle at low pH (2.6).;Previously, the titration process of Asp97 has been fitted to a single pKa value unusually high (∼7 in the present of chloride anions) especially in comparison to its bR equivalent residue Asp85 (pKa ca 2.6) [1-3]. However, the titration of Asp97 is quite broad and extended over a wide pH range of almost 4 pH units (5-9). We have revealed that the titration of GPR samples incubated with sodium acetate or trimethylacetate (TMA) yielded a pKa curve shape for Asp97 which was different from water or NaCl. The titration curve was spread over a wider pHs range and for the first time it is evident that the curve represents a complex titration of Asp97. The complex titration implies that the titration is associated with two pKa values which are attributed to Asp97 in which one has a value below 6. An additional support for the existence of two pKa values for Asp97 is gained by studying the GPR-E142Q mutant. GPR-E142Q mutant exhibits a large acid-induced red shift (∼45nm) in the absorption maxima, from pH ∼9 to ∼2. This shift in lambdamax is attributed to the titration of Asp97 which spread across a broad range of pHs (9-2) in the E142Q mutant relative to the WT (9-5). The mutant exhibits two well separated pKa values for Asp97 in the presence of chloride anions whereas in the wildtype (WT) it is not clearly noticeable. More over, it appears that E142Q mutant induces a significant decrease of pKa1 (∼2) by almost 4 units relative to its value detected in the WT (∼6). The E142Q mutant affects also pKa2 (∼6.0) and decreases it by ∼1 pKa unit compared with WT containing chloride (∼7). Our results suggest that an additional residue (Residue X) is titrated in the range of Asp97 titration. Residue X exists in two forms, a protonated and deprotonated form while each one interacts differently with Asp97. In addition, the anions affect the pKa of Asp97 and X differently in the GPR-WT. It appears that the absence of Glu142 affects the pKa of X and more significantly Asp97's pKa1 although this residue is located relatively far (∼12A) from Asp97 on the extracellular side of the membrane (based on the 1m0k bR structural model). Such a large effect may originate from alteration in hydrogen bonding network enforced by the mutation. This may suggest that either the protonated or the deprotonated form of Glu142 (WT) is involved in hydrogen bonding network. (Abstract shortened by UMI.).