A neutron diffraction study on the location of the polyene chain of retinal in bacteriorhodopsin.

Abstract

We report on the location of the chain part of the retinylidene chromophore in the projected density of bacteriorhodopsin as determined by neutron diffraction from the two-dimensional purple membrane lattice. For this purpose, partially deuterated retinal was synthesized containing 10 deuterons at positions C-8, C-10, C-12, C-14, C-19(3), and C-20(3) of the polyene chain. Fig. 1). Since the retinal molecule is -15 A long in the all-trans form and makes an angle of =20°with the plane of the membrane (9), we labeled a site in the projected density of the membrane, which differs from that in the previous work. This site is, moreover, closer to the Schiff base linkage to lysine-216 of helix G, thus facilitating the assignment of helix G to one of the seven helices of the structure. Samples were prepared in two different ways. In the first procedure, the method of King was used (4) with one important modification: unlabeled retinal oxime was completely removed by washing with bovine serum albumin. In the second method, the partially deuterated retinal was added to the growth medium of the Halobacterium halobium mutant JW5, which is deficient in the synthesis of retinal. In this way, the labeled retinal was incorporated biosynthetically without the necessity of any of the possibly deleterious steps involved in the first procedure. With both methods, only a single major peak was observed in the density and the same position was found for the center of the polyene chain-close to helix 6 in the interior of bacteriorhodopsin. The problem of assigning the seven helical stretches in the sequence (A-G) to the seven helices observed in the structure (1-7; see Fig. 4) is still unsolved (10-13). At first sight, it appears that because of the lengths of the projected retinal chain and the lysine side chain to which it is attached, a unique assignment of helix G cannot be made. If we combine our result with that of previous diffraction work (7), however, the number of possibilities is reduced to two. It appears likely that G is either 2 or 6. MATERIALS AND METHODSMaterials. All-trans-retinal (Fluka), hydroxylamine hydrochloride (Merck) and bovine serum albumin (fraction V, fatty acid-free; Serva, Heidelberg) were used without further purification.

Small-angle scattering and its interplay with crystallography, contrast variation in SAXS and SANS

“…The surface of a particle is described by a single-valued function of the angles and ' (Stuhrmann, 1970b,c Phospholipid distribution (grey) in the plane diffraction. The difference Fourier map is based on the known structure of bacteriorhodopsin (Seiff et al, 1985) and the intensity differences from anomalous diffraction of X-rays. The unit cell contains three bacteriorhopsin molecules (contour lines) (Biou et al, 2005).…”

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.…”

“…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.…”

“…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).…”