Surface pH controls purple-to-blue transition of bacteriorhodopsin. A theoretical model of purple membrane surface

Abstract

We have developed a surface model of purple membrane and applied it in an analysis of the purple-to-blue color change of bacteriorhodopsin which is induced by acidification or deionization. The model is based on dissociation and double layer theory and the known membrane structure. We calculated surface pH, ion concentrations, charge density, and potential as a function of bulk pH and concentration of mono- and divalent cations. At low salt concentrations, the surface pH is significantly lower than the bulk pH and it becomes independent of bulk pH in the deionized membrane suspension. Using an experimental acid titration curve for neutral, lipid-depleted membrane, we converted surface pH into absorption values. The calculated bacteriohodopsin color changes for acidification of purple, and titrations of deionized blue membrane with cations or base agree well with experimental results. No chemical binding is required to reproduce the experimental curves. Surface charge and potential changes in acid, base and cation titrations are calculated and their relation to the color change is discussed. Consistent with structural data, 10 primary phosphate and two basic surface groups per bacteriorhodopsin are sufficient to obtain good agreement between all calculated and experimental curves. The results provide a theoretical basis for our earlier conclusion that the purple-to-blue transition must be attributed to surface phenomena and not to cation binding at specific sites in the protein.

The binding site of the strongly bound Eu3+ in Eu3+‐regenerated bacteriorhodopsin

“…is the Scatchard plot [23] which should give a straight line with a slope of -K and an intercept that gives n. For any added concentration of Eu a+ to a sample of dlbR, if the concentration of the free Eu ~+ (L) is determined, the moles of Eu a÷ hound v can be calculated. We have used chelating agents to enhance the fluorescence [28] of the free Eu a+ to determine its concentration after separating it from the bR and its bound Eu ~÷.…”

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

“…It has been shown that this transition reflects the protonation state of the primary proton acceptor Asp-85 (22,31). The purple to blue transition is directly affected by the proton concentration at the protein surface, which depends on the surface charge (30,32). Therefore, the apparent pK a of this transition is strongly dependent on ionic strength.…”

PURPLE MEMBRANE: SURFACE CHARGE DENSITY and THE MULTIPLE EFFECT OF pH and CATIONS

“…Jang and El-Sayed also found an increased emission and degree of hydration of Eu3+ with the delipidated membrane vs the native membrane. They suggested that these differences were probably due to changes in binding site conformation and not to new sites forming upon delipidation, and were therefore protein-specific, but Szundi and Stoeckenius (1989) proposed that the Eu3+ could have been bound to phospholipids in the purple membrane and to protein carboxyls in the delipidated membrane. Jang and El-Sayed also suggested that a conformational change in the bR active site occurs (the conformation becomes more open) in the delipidated membrane such that cations are no longer required for the deprotonation of the Schiff base, as in Corcoran et al (1987) for the native membrane; the active site is more heterogeneous.…”