Three important events in the functional cycle of bacteriorhodopsin occur at the chromophore: the primary absorption of light, the isomerization from the alltrans to the 13-cis form, and the deprotonation and reprotonation of its Schiff base. The protonated Schiff base linkage of the chromophore with lysine-216 plays an essential role in the color regulation of the pigment and is most likely directly involved in the charge translocation of this light-driven proton pump. Although much is known about the structure of the protein, the position of this key functional group has not yet been determined. We have synthesized a retinal in which the five protons closest to the Schiff base are replaced by deuterons. The labeled retinal was spontaneously incorporated into bacteriorhodopsin by using a mutant of Halobacterium halobium that is deficient in the synthesis of retinal. The position of the labeled Schiff base end of the chromophore was determined in the two-dimensional projected density of darkadapted bacteriorhodopsin by neutron diffraction. The result fits very well with our previous work using retinals that were selectively deuterated in the middle of the polyene chain or in the cyclohexene ring. A coherent structure emerges with the three labeled positions on one line, separated by distances that are in good agreement with the tilt angle of the polyene chain (about 200). The chromophore is located in the interior of the protein with the nitrogen of the Schiff base between helices 2 and 6 and with its ring in the vicinity of helix 4. Our results show that it is possible to locate a small group containing as few as five deuterons in a membrane protein of molecular weight 27,000.The chromophore of bacteriorhodopsin consists of retinal that is attached via a protonated Schiff base to lysine-216, a residue in the membrane-spanning helix G (1). Because of its essential role in the function of bacteriorhodopsin, we have investigated the position of this part of the chromophore in the plane of the membrane, Within the purple membrane, bacteriorhodopsin is arranged in a two-dimensional hexagonal lattice allowing the use of diffraction methods (2-9). In this way a structural model was obtained at a resolution of 3.5 A in-plane (9). Due to its low electron density, however, the chromophore could not be resolved. In the light-adapted state of bacteriorhodopsin, the retinal is in the extended all-trans form having a length of approximately 15 A (see Fig. 1). 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 length between hydrogen (protium, 1H) and deuterium (2H).Replacing 'H by 2H has the great advantage that it does not affect the electronic structure, as would be the case with a heavy atom. In order to localize the end of ...

The transmembrane location of the chromophore of bacteriorhodopsin was obtained by neutron diffraction on oriented stacks of purple membranes. Two selectively deuterated retinals were synthesized and incorporated in bacteriorhodopsin by using the retinal- mutant JW5: retinal-d11 (D11) contained 11 deuterons in the cyclohexene ring, and retinal-d5 (D5) had 5 deuterons as close as possible to the Schiff base end of the chromophore. The membrane stacks had a lamellar spacing of 53.1 A at 86% relative humidity. Five orders were observed in the lamellar diffraction pattern of the D11, D5, and nondeuterated reference samples. The reflections were phased by D2O-H2O exchange. The absolute values of the structure factors were nonlinear functions of the D2O content, suggesting that the coherently scattering domains consisted of asymmetric membrane stacks. The centers of deuteration were determined from the observed intensity differences between labeled and unlabeled samples by using model calculations and Fourier difference methods. With the origin of the coordinate system defined midway between consecutive intermembrane water layers, the coordinates of the center of deuteration of the D11 and D5 label are 10.5 +/- 1.2 and 3.8 +/- 1.5 A, respectively. Alternatively, the label distance may be measured from the nearest membrane surface as defined by the maximum in the neutron scattering length density at the water/membrane interface. With respect to this point, the D11 and D5 labels are located at a depth of 9.9 +/- 1.2 and 16.6 +/- 1.5 A, respectively. The chromophore is tilted with the Schiff base near the middle of the membrane and the ring closer to the membrane surface. The vector connecting the two label positions in the chromophore makes an angle of 40 +/- 12 degrees with the plane of the membrane. Of the two possible orientations of the plane of the chromophore, which is perpendicular to the membrane plane, only the one in which the N----H bond of the Schiff base points toward the same membrane surface as the vector from the Schiff base to the cyclohexene ring is compatible with the known tilt angle of the polyene chain.

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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...

Purple membranes were prepared in which bacteriorhodopsin was labeled at lysine 41 with phenylisothiocyanate (PITC) and with perdeuterated PITC. The in-plane position of this small label containing only five deuterons was determined from the differences between the neutron diffraction intensities of the two samples. At 8.7-A resolution the Fourier difference map revealed a well-defined site between helices 3 and 4. This position was confirmed by a refinement procedure in reciprocal space. Model calculations showed that the observed difference density had the right amplitude for the label. Thus it is possible to locate a small group in a large protein structure by replacing as few as five hydrogens by deuterium. The observed location of PITC restricts the number of possibilities for the assignment of helix B in the sequence (to which lysine 41 is attached) to one of the seven helices of the structure. Taking into account the size of the label and the length of the lysine side chain our result excludes helices 1, 2, and 7 as candidates for B.