The Influence of a Transmembrane pH Gradient on Protonation Probabilities of Bacteriorhodopsin: The Structural Basis of the Back-Pressure Effect

Abstract

Bacteriorhodopsin pumps protons across a membrane using the energy of light. The proton pumping is inhibited when the transmembrane proton gradient that the protein generates becomes larger than four pH units. This phenomenon is known as the back-pressure effect. Here, we investigate the structural basis of this effect by predicting the influence of a transmembrane pH gradient on the titration behavior of bacteriorhodopsin. For this purpose we introduce a method that accounts for a pH gradient in protonation probability calculations. The method considers that in a transmembrane protein, which is exposed to two different aqueous phases, each titratable residue is accessible for protons from one side of the membrane depending on its hydrogen-bond pattern. This method is applied to several ground-state structures of bacteriorhodopsin, which residues already present complicated titration behaviors in the absence of a proton gradient. Our calculations show that a pH gradient across the membrane influences in a non-trivial manner the protonation probabilities of six titratable residues which are known to participate in the proton transfer: D85, D96, D115, E194, E204, and the Schiff base. The residues connected to one side of the membrane are influenced by the pH on the other side because of their long-range electrostatic interactions within the protein. In particular, D115 senses the pH at the cytoplasmic side of the membrane and transmits this information to D85 and the Schiff base. We propose that the strong electrostatic interactions found between D85, D115, and the Schiff base as well as the interplay of their respective protonation states under the influence of a transmembrane pH gradient are responsible for the back-pressure effect on bacteriorhodopsin.

Factors That Differentiate the H-bond Strengths of Water Near the Schiff Bases in Bacteriorhodopsin and Anabaena Sensory Rhodopsin

“…Electrostatic calculations resulted in a pK a (Asp-85) of 1.5 in BR and a pK a (Asp-75) of Ϫ5.1 in ASR (Table 6). The pK a difference is mainly due to differences in the influences of Ala-53, Arg-82, Glu-194, and Asp-212 on Asp-85 in BR and the corresponding residues Ser-47, Arg-72, Ser-188, and Pro-206 on Asp-75 in ASR, indicating the presence of long range electrostatic interactions along the proton transfer pathways (31). A strongly H-bonded water near the Schiff base, which results from electrostatic interactions between Asp-85 and these acidic residues in BR, could play a key role in proton transfer.…”

Effect of a pH Gradient on the Protonation States of Cytochrome c Oxidase: A Continuum Electrostatics Study

“…23 Bacteriorhodopsin is perhaps the most well studied proton pumping system. It is an archaeal light-driven proton pump, composed of seven transmembrane helices and with a retinal chromophore, which, when stimulated by light, triggers a series of conformational events that lead to a proton being transferred from the N-to the P-side of the membrane.…”

“…The assignment of titratable sites to a side of the membrane was performed as described for CcO, which resulted in all sites being assigned in agreement with the method used by Ullmann and co-workers. 23 MC calculations were then performed for all combinations of pH values in the 0-16 range, every 0.1 pH units, using 10 3 equilibration steps and 10 4 MC steps.…”

“…Ullmann and co-workers 23 reported that six out of the total 39 titratable residues in bacteriorhodopsin exhibited an irregular titration behavior that was dependent on the pH values on both sides of the membrane. Our method was able to correctly identify the same six irregularly titrating residues: D85, D96, D115, E194, E204, and the retinal group ( Figure S2 in the supporting information).…”

“…Trial moves were evaluated with a Metropolis scheme 48 using the Poisson-Boltzmann protonation energy terms and the pH of the solution, as previously described. 23,26 …”