Location of the cyclohexene ring of the chromophore of bacteriorhodopsin by neutron diffraction with selectively deuterated retinal

Abstract

We report on the location of the cyclohexene ring of the retinylidene chromophore of bacteriorhodopsin projected onto the plane of the membrane. For this purpose, partially deuterated retinal was synthesized containing 11 deuterons at the following positions ofthe cyclohexene ring: one at C-2, two at C-4, three at C-16, three at C-17, and two at C-18. The partially deuterated retinal was incorporated biosynthetically during growth of the bacteria by using the mutant JW5, which is deficient in the synthesis of retinal. Undeuterated samples were prepared in the same way. Characterization by x-ray diffraction and absorption spectroscopy showed that these samples are identical to native purple membranes as judged by these criteria. A Fourier difference map was calculated from the differences in in-plane diffraction intensities between the deuterated and undeuterated darkadapted membrane samples. Model calculations showed that the observed difference density had the amplitude expected for a label containing 11 deuterons. At 8.7 A resolution, the map shows one major peak with the center of mass of the deuterated ring in the interior of the molecule between helices 3, 4, 5, and 6. Based on this result and on our previous work on the location of the middle of the polyene chain, we conclude that the COOH-terminal helix G, to which retinal is attached at lysine-216, is either helix 2 or helix 6.The chromophore of bacteriorhodopsin is of central importance in the photochemistry and in the charge cycle of this light-driven proton pump (1). Fluorescence energy transfer (2, 3) and neutron diffraction with perdeuterated retinal (4,5) have been used in attempts to determine the location of retinal in the projected density of bacteriorhodopsin. Since retinal is 15 A long in its all-trans form and since its polyene chain makes an angle of =20°with the plane of the membrane (6), the mass density of retinal projected onto the plane of the membrane will be smeared out and elongated. Low-resolution neutron diffraction experiments with perdeuterated retinal (28 deuterons) will therefore determine only the center of deuteration of this delocalized mass distribution. More detailed structural information about the location of the various parts of retinal may be obtained by using partially deuterated retinal. This method was recently applied with a synthetic retinal containing 10 deuterons to determine the position of the middle of the polyene chain (C-11) (7). In the present work, we continue this approach with a synthetic retinal that was selectively labeled with 11 deuterons in the cyclohexene ring to find the location of the ring (Fig. 1). Taken together, the results of these two studies provide a low-resolution map of the arrangement and orientation of the chromophore within bacteriorhodopsin and allow an estimate to be made of the position of the Schiffs base nitrogen. The result limits the assignment of helix G of the sequence to which the chromophore is attached at lysine-216 to helix 2 or 6 of the structure. MATERIALS AND...

High-sensitivity neutron diffraction of membranes: Location of the Schiff base end of the chromophore of bacteriorhodopsin

“…From the lack of a proton signal at 2.33, 5.97, and 10.11 ppm and the mass peak at 289, we conclude that the end product of the synthesis corresponds to the structure shown in Fig. 1 fifth day of growth as described (11,12 inal-bacteriorhodopsin (48 hr). The intensity differences for the 24-hr runs were the same as for the 48-hr runs.…”

“…Only one major difference peak occurs, which is twice as large as the next highest peak. The approximations used in the data analysis have been discussed in detail (11,12,16,18). As in our previous work the phases (Table 1, last column) and the intensity ratios of nonequivalent reflections, which overlap in the powder pattern, were taken from the electron diffraction work (4,5).…”

“…Apart from one 1 were obtained with an entirely different sample during another machine run, this experiment shows convincingly that the observed intensity differences are reproducible and due to the partially deuterated retinals. The data were analyzed as described (11,12,16). As a result of the almost identical sample preparation, the sum of the intensities of the deuterated sample was only 0.6% larger than the corresponding sum for the undeuterated sample.…”

“…Since the angle between the polyene chain and the plane of the membrane is only about 20° (10), the projection of the chromophore onto this plane is elongated as well. Neutron diffraction provides an elegant method to obtain detailed structural information on the in-plane location and orientation of the various parts of the chromophore by using partially deuterated retinals (11,12 length between hydrogen (protium, 1H) and deuterium (2H).…”

“…1). Bacteriorhodopsin samples containing [2H5]retinal and unlabeled retinal were prepared as described (11,12), by adding them to the growth medium of the retinal-minus mutant JW5, which cannot synthesize retinal. The added retinals are incorporated spontaneously and form the normal pigment.…”