A local electrostatic change is the cause of the large-scale protein conformation shift in bacteriorhodopsin

Abstract

During light-driven proton transport bacteriorhodopsin shuttles between two protein conformations. A large-scale structural change similar to that in the photochemical cycle is produced in the D85N mutant upon raising the pH, even without illumination. We report here that (i) the pKa values for the change in crystallographic parameters and for deprotonation of the retinal Schiff base are the same, (ii) the retinal isomeric configuration is nearly unaffected by the protein conformation, and (iii) preventing rotation of the C13-C14 double bond by replacing the retinal with an all-trans locked analogue makes little difference to the Schiff base pKa. We conclude that the direct cause of the conformational shift is destabilization of the structure upon loss of interaction of the positively charged Schiff base with anionic residues that form its counter-ion.

Figure 1

Difference projection maps of structural changes in D85N bacteriorhodopsin upon raising the pH from 6.0 to 9.2 (a) or 10.4 (b). Bold contour lines indicate density increase, dashed lines density decrease. Although the changes in b are larger than in a, they are represented with equal amplitude. End-on views of transmembrane helices B, C, and D, and the smeared outline of the more tilted helices A, G, F, and E of one bacteriorhodopsin in the trimer are superimposed.

Figure 2

Integrated intensities of reflections (1,1), (3,2), and (4,1) in a, b, and c, respectively vs. pH. Conditions are the same as in Fig. 1.

Figure 3

FT-Raman spectra of wild-type, D85N, and D85N/D96N bacteriorhodopsins. Conditions are 100 mM NaCl, pH 6.6, at 22°C. Wild-type spectra are for all-trans (light-adapted) and 13-cis (from a dark-adapted sample that contains 33% all-trans) chromophores. These are essentially the same as reported before (39) and shown as controls only. For the mutants spectra of dark-adapted samples are shown. Amplitudes of the two wild-type spectra adjusted (together) for best comparison with the mutants.

Figure 4

Titration of the retinal Schiff base in D85N bacteriorhodopsin. Deprotonation was determined from the amplitudes of the shift of the maximum from near 600 nm to 410 nm. Reconstitution of the hydroxylamine-bleached chromophores was with either authentic (○) or 13-demethyl 12–14 ethano (•) retinal. Titration was in 2 M NaCl, where the apparent pK_a of the Schiff base is somewhat lower than at lower NaCl concentrations (4, 33, 37).