Infrared spectroscopic demonstration of a conformational change in bacteriorhodopsin involved in proton pumping.

Abstract

Infrared spectral changes in bacteriorhodopsin (bR) were followed during the slow decay of the M intermediate in the temperature region 240-260 K. The decay of the M form is characterized by the disappearance of the ethylenic bands and the bands indicating the reprotonation of the Schiff base. The route of Schiff-base reprotonation completely changes between 240 K and 260 K. At 240 K reprotonation occurs from Asp-85, the group to which the proton was released during M formation, and there is no pumping. At 260 K Schiff-base reprotonation takes place through Asp-96 from the cytoplasmic side, in the normal sequence assumed for proton pumping. The dramatic change in the route of Schiffbase reprotonation is coupled to a protein conformational change characterized by the change of the ratio of the two amide I bands at 1658 cm-' and 1669 cm-'. This conformational change is interpreted as the conformational switch crucial for proton pumping: a protein relaxation following M formation results in a local rearrangement of the group, in the vicinity of the Schiff base. The rearrangement changes the accessibility of the Schiff base and provides that its deprotonation and reprotonation occur on different sides. The conformational change has characteristics typical for relaxations in proteins. In addition, it is shown that at 260 K an equilibrium exists between the M and N forms.

Trapping the M sub 1 and M sub 2 substrates of bacteriorhodopsin for electron diffraction studies

“…gain the C conformation, it is proposed that the proton originally on the Schiff base, but now donated to Asp-85 (L -M 1 transition), is returned to the Schiff base (M 1 -bR56 8) and no pumping occurs (Ormos, 1991). The upshot of this, as far as our approach is concerned, is that by regulating the temperature, M1 can be "frozen"…”

“…Recently, a procedure was reported that describes the means whereby bR can be trapped in either the M 1 or the M2 state (Ormos, 1991). The conformational marker bands in the Amide I region of the lR, M -bR difference spectrum differ markedly between M1 and M2 (There is some N present with M2.…”

“…Since the absorbance peak of M (412 nm) lies in the visible spectrum far removed from the peaks of other intermediates, (ali 550 nm or higher) it is straightforward to determine the , concentration of M. Visible spectroscopy cannot differentiate between M1 and M2, of course, since both have a peak at 412 nm, but FTIR difference spectroscopy can (Ormos, 1991). For spectroscopic studies, it is not crucial to form a high percentage of M. However, difference Fourier maps require a high level of specimen conformational homogeneity because every bR molecule in the crystal that is not in the M conformation adds noise to the map.…”

“…The shift to the C conformation is most likely to occur d between M1 and M2 (Ormos, 1991). Movement of residues may also be needed to allow water, both on the surface and inside the protein, to organize around newly exposed hydrophobic areas of the protein.…”

Functionally Relevant Coupled Dynamic Profile of Bacteriorhodopsin and Lipids in Purple Membranes

“…The decay of the M-intermediate in the photocycle and the regeneration of the ground state are strongly slowed below 80% rh (46). The decay of the multiple M-states can also be locked around 220-260 K (88). It is interesting to note that the T 2 values at 243 and 213 K of the transmembrane R-helices for the fully hydrated PM were longer than those at 293 and 273 K, and the T 2 values of loops increased between 243 and 213 K (Table 3, Figure 7).…”