Evidence for charge-controlled conformational changes in the photocycle of bacteriorhodopsin

The existence of two different M-state structures in the photocycle of the bacteriorhodopsin mutant ASP38ARG was proved. At pH 6.7 (0 to -6 degrees C) a spectroscopic M intermediate (M,) that does not differ significantly in its tertiary structure from the light-adapted ground state accumulates under illumination. At pH > 9 another state (M-2), characterized by additional pronounced changes in the Fourier transform infrared difference spectrum in the region of the amide I and II bands, accumulates. The M-2 intermediate trapped at pH 9.6 displays the same changes in the x-ray diffraction intensities under continuous illumination as previously described for x-ray experiments with the mutant ASP96ASN. These observations indicate that in this mutant the altered charge distribution at neutral pH controls the tertiary structural changes that seem to be necessary for proton translocation.