Retinal location in purple membrane of Halobacterium halobium: a neutron diffraction study of membranes labelled in vivo with deuterated retinal.

Purple membranes were prepared by growing Halobacterium halobium in a medium containing nicotine (which inhibits biosynthesis of retinal) and the oxidation products of fully deuterated beta-carotene. This allowed the in vivo incorporation of deuterated retinal into the membranes. The labelled membranes were crystalline and isomorphous with native membrane as determined by X-ray diffraction, and their optical absorption spectra were very similar. Neutron diffraction data for the two dimensional in-plane lattice from labelled and native membranes were analysed by difference Fourier and direct methods to 8.6 A resolution. The difference Fourier shows the retinal to be located in the centre of the bacteriorhodopsin molecule. The best fit to the data was obtained with the projection of retinal as a 10 A long rod forming an angle of -40 degrees +/- 10 degrees with the x axis centred at x = -0.19 +/- 0.02, y = -0.35 +/- 0.02 in fractional unit cell coordinates. The main peak in the difference Fourier map is at x = -0.17, y = -0.33.