The spatial location and orientation of the retinal chromophore in bacteriorhodopsin were estimated from a fluorescence energy transfer study. The energy donor used in this study was a fluorescent retinal derivative, which was obtained by partial reduction of the purple membrane with sodium borohydride, and the energy acceptor was the native chromophore remaining in the same membrane. Since bacteriorhodopsin forms a two-dimensional crystal with P3 symmetry in the purple membrane, and the membrane structure is maintained after the reduction, the rate of energy transfer from a donor to any acceptor existing in the same membrane can be calculated as a function of the location and orientation of the chromophores in the unit cell. Quantitative analyses of the fluorescence decay curve and the quantum yield, with various extents of reduction, enabled us to determine the most probable location and orientation. The result suggested that the chromophore was situated near the centre of the protein in such an orientation that the dipole-dipole interaction with neighbouring chromophores was close to minimum.
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