Two progressive substrates of the M-intermediate can be identified in glucose-embedded, wild-type bacteriorhodopsin

Abstract

Glucose-embedded bacteriorhodopsin shows M-intermediates with different Amide I infrared bands when samples are illuminated at 240 or 260 K, in contrast with fully hydrated samples where a single M-intermediate is formed at all temperatures. In hydrated, but not in glucose-embedded specimens, the N intermediate is formed together with M at 260 K. Both Fourier transform infrared and electron diffraction data from glucose-embedded bacteriorhodopsin suggest that at 260 K a mixture is formed of the M-state that is trapped at 240 K, and a different M-intermediate (MN) that is also formed by mutant forms of bacteriorhodopsin that lack a carboxyl group at the 96 position, necessary for the M to N transition. The fact that an MN species is trapped in glucose-embedded, wild-type bacteriorhodopsin suggests that the glucose samples lack functionally important water molecules that are needed for the proton transfer aspartate 96 to the Schiff base (and, thus, to form the N-intermediate); thus, aspartate 96 is rendered ineffective as a proton donor.

High resolution electron diffraction analysis of structural changes associated with the photocycle of bacteriorhodopsin

“…5-7, is similar to the ratio that also best accounts for the M 260K FTIR difference spectrum as a mixture of M and N FTIR spectra, confirming the previous conclusion that M260K sample is a mixture of M240K and MN (Vonck et al, 1994).…”

“…Using electron diffraction data from 'crystals tilted by only 10°, Subramaniam et al (1993) suggested that there might be a selective opening of the cytoplasmic half of the channel, which would require some tilting of helices. These differences between our experiment and that of Subramaniam now can be explained in terms of different proportions of the M and MN intermediates, however (Sasaki et aI., 1992;Perkins et al, 1992;Han et aI., 1994a;Vonck et al, 1994).…”

“…Sasaki et al (1992) should cycle between an inward facing and an outward facing conformation is well-established (Stoeckenius, 1979;Nagle and Mille, 1981;Chou, 1993, Vonck et al, 1994 (Mathies et al, 1991) or removal of one or two water molecules (Chou et al, 1993) will be enough to "close" the outward facing channels. As shown in Figure 7-6, it will be necessary to increase the tilt angles to 45°, and even further up to 60°, to improve the resolution of the map in the z-direction.…”

Kinetic isotope effects reveal an ice‐like and a liquid‐phase‐type intramolecular proton transfer in bacteriorhodopsin

“…Fax: (49) (234) 709-4238. recovery to another group which is also unidentified [6]. Molecular dynamics simulations and different experimental observations suggest the existence of several bound water molecules that may contribute to specific proton-translocating hydrogen bonded networks in the proton release and proton uptake pathways [7][8][9][10].…”

Time‐resolved surface charge change on the cytoplasmic side of bacteriorhodopsin

“…A change in the dielectric constant may be induced by a conformational change which brings the label into a more hydrophobic environment. Indeed diffraction data suggest that structural changes occur in the cytoplasmic half of bR in the M-and N-intermediates [13,14]. Recent time-resolved EPR signals on the ms time scale observed with a spin label bound to V101C were interpreted as being due to structural changes in the N-and/or O-intermediates [15].…”