HEAVY-ATOM LABELED RETINAL ANALOGS LOCATED IN BACTERIORHODOPSIN BY X-RAY-DIFFRACTION

The location of the heavy-atom label of three different retinal analogues in the plane of the purple membrane was determined by x-ray diffraction. The three analogues, i.e. 9-bromoretinal and 13-bromoretinal labelled in the polyene chain as well as the ring-labelled HgCl-retinal, were incorporated into bacteriorhodopsin (BR) either biosynthetically using a retinal-deficient mutant strain of Halobacterium halobium or with photochemically bleached bacterioopsin. All BR samples regenerated with retinal analogues were functionally active as proton pumps. The diffraction data show that the cyclohexene ring of retinal is situated in the corner formed by helices 4E and 5D, the 13-methyl group adjacent to helix 6C and therefore the Schiff's base nitrogen about midway between helices 6C and 2G. The 9-bromo label is found slightly off the line connecting the two other labels, directed towards helix 3F, suggesting torsion of the polyene chain or slight incline of the ring. The position and orientation of retinal obtained in our experiments are in agreement with data from neutron diffraction and high-resolution clectron microscopy. This indicates that the heavy-atom labelled chromophores, 9-bromo- and 13-bromo- as well as HgCl-retinal, are isomorphously incorporated into BR. These samples will allow kinetic investigation of light-induced structural changes of retinal during BR's pumping cycle using x-ray diffraction as well as extended x-ray absorption fine structure experiments probing changes in the retinal neighbourhood between different intermediates of the photocycle.