Dramatic in situ conformational dynamics of the transmembrane protein bacteriorhodopsin

Abstract

The conformational dynamic capabilities of the in situ bacteriorhodopsin (bR) can be studied by determination of the changes of the bR net helical segmental tilt angle (the angle between the polypeptide segments and the membrane normal) induced by various perturbations of the purple membrane (PM). The analysis of the far-UV oriented circular dichroism (CD) of the PM provides one means of achieving this. Previous CD studies have indicated that the tilt angle can change from approximately 10 degrees to 39 degrees depending on the perturbants used with no changes in the secondary structure of the bR. A recent study has indicated that the bleaching-induced tilt angle can be enhanced from approximately 24 degrees to 39 degrees by cross-linkage and papain-digestion perturbations which by themselves do not alter the tilt angle. To add further credence, this study has been repeated using midinfrared (IR) linear dichroic spectral analysis. In contrast to the CD method, analysis by the IR method depends on the orientation of the amide plane of the helix assumed. Excellent consistency is achieved between the two methods only when it is assumed that the structural characteristics of the alpha-helices of the bR are equally alpha I and alpha II in nature. Furthermore, the analysis of the IR data becomes essentially independent of the three amide transitions utilized. The net tilt angle of segments completely randomized relative to the incident light must be 54.736 in view of helix symmetry. A value of 54.735 degrees +/- 0.001 degree was achieved by the IR method for the ethanol-treated PM film, establishing this kind of film as an ideal random state standard and demonstrating the accuracy potential of the IR method.

Evidence for an .alpha.II-type helical conformation for bacteriorhodopsin in the purple membrane

“…It is apparent that the randomized films exhibit this effect to a lesser degree than does the PM suspension, probably due to the absence of bulk water in the former. As would be expected, addition of glycerol has also been shown to eliminate this depression at 208-215 nm (Draheim et al, 1988b). These results are at variance with the conclusions of a number of laboratories (Jap et al, 1983;Mao & Wallace, 1984;Nabedryk et al, 1985) that light scattering is insignificant at all wavelengths from 240 to 190 nm if large acceptance angles are used during CD measurements.…”

“…This arrangement made it possible to quickly seal them without having to remove them from the hydration chamber, minimizing possible drying effects of ambient humidity. It was also observed that these films were sufficiently thin to lack noticeable light-scattering effects resulting from hydration state (Draheim et al, 1988b). For solution studies, membrane preparations were suspended in double-distilled water, filtered, and degassed prior to spectral measurements.…”

Evidence for unbenignant nature of glucose as a replacement for water in purple membranes

“…In the case of the bR, this reduction was limited to only 15%. This provides strong support for the hypothesis that the planes ofthe amide groups in the bR are tilted an appreciable amount away from the helix axis, since the planes of the amide groups in an a,,-type helix are expected to be tilted within the range of 35 to 450 with respect to the helix axis based on molecular models (12,17). (e) The net segmented tilt angle O,, (the average angle between the seven transmembrane polypeptide segments of the bR and the membrane normal of the PM) determined for the native state of the bR by the analysis of the far-UV oriented circular dichroism (OCD) of the PM (for a detailed discussion of this method, se -the following section) has been shown to be consistent with the one determined by an independent analytical spectral method, mid-infrared linear dichroism (IRLD), only when it is assumed in the IRLD analysis that the characteristics of the a-helices of the bR consist, on the average, of 60% a1 and 40% all.…”

“…(e) The net segmented tilt angle O,, (the average angle between the seven transmembrane polypeptide segments of the bR and the membrane normal of the PM) determined for the native state of the bR by the analysis of the far-UV oriented circular dichroism (OCD) of the PM (for a detailed discussion of this method, se -the following section) has been shown to be consistent with the one determined by an independent analytical spectral method, mid-infrared linear dichroism (IRLD), only when it is assumed in the IRLD analysis that the characteristics of the a-helices of the bR consist, on the average, of 60% a1 and 40% all. The change in the magnitude of the net angle (AO,) resulting from the transformation of the bR from the native to the bleached state has also been shown to be consistent when determined by these two independent methods, but only if based on the same assumption of the nature of the bR helices (17)(18)(19).…”

“…Oa has been shown to be most likely 00 in hydrated PM films by OCD and IRLD analyses as discussed above ( 17,19,20). That is, the helical segments of the native bR must all be oriented nearly parallel to the PM normal.…”