Bacteriorhodopsin reconstituted from two individual helices and the complementary five-helix fragment is photoactive.
Bacteriorhodopsin (bR), a light-driven proton pump, consists of a bundle of seven membrane-spanning alpha-helices connected to each other by short extramembranous loops. Previously it has been shown that bR can be reconstituted from three fragments corresponding to the first helix, the second helix, and the remaining five helices, and that this reconstituted material reforms the native structure of bR. In this study, it is shown that the native function is also recovered. Low-temperature spectroscopy was used to examine the photochemical properties of bR reconstituted from three fragments. At room temperature at pH 6, the reconstituted material shows essentially the same absorption spectrum as native bR, while upon raising the pH at room temperature or cooling the sample in glycerol, a second, blue-shifted peak appears. The pH and temperature dependence of the absorption spectrum indicates that the reconstituted bR is in an equilibrium between two pigments, which we call P560 and P480. Both pigments convert to their own K intermediates, which differ in absorption maxima, upon illumination with green light at -180 degrees C. Each K intermediate can be reverted to its initial state by light. Similarly, both pigments convert to their own M intermediates upon irradiation with yellow light at -77 degrees C. The M intermediate of both species can be reverted only to P560 by light. Both pigments are therefore photoactive. These unique photochemical properties of bR reconstituted from three fragments may be attributable to the lack of a covalent linkage in the loop connecting the A and B helices, and thus possibly to a change in the orientation of the B helix.
Kataoka
M
M
Department of Physics, Faculty of Science, Tohoku University, Sendai, Japan.
Kahn
T W
TW
Tsujiuchi
Y
Y
Engelman
D M
DM
Tokunaga
F
F
D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000528:radiation effects; D010446:Peptide Fragments / Q000737:chemistry; D010777:Photochemistry; D011487:Protein Conformation
D010446:Peptide Fragments; D001436:Bacteriorhodopsins