Assignment of segments of the bacteriorhodopsin sequence to positions in the structural map

Trewhella, Jill; Anderson, S.; Fox, Robert O.; Gogol, Edward P.; Khan, Shahid; Engelman, Donald M.; Zaccaı̈, Giuseppe

doi:10.1016/s0006-3495(83)84391-4

Cited by 66 publications

(38 citation statements)

References 17 publications

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“…8 (3,1,7) fall clearly outside the circle, it is unlikely that this conclusion will be altered. Three previous structural studies provide information on the location of helix G (23,28,31). Our result is in disagreement with the conclusion of one of these reports, in which G is assigned to helix 4 (31).…”

Section: Discussioncontrasting

confidence: 99%

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

Seiff

Westerhausen²,

Wallat³

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Section: Discussioncontrasting

confidence: 99%

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

Seiff

Westerhausen²,

Wallat³

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

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“…Its characteristic feature is the proximity of segments A and F, as deduced from the effect of Schiff's base reduction on signal 4 of Trp(5F) 10 or 12. If the tentative assignment of signal 3 is correct, the ,&ionone ring should have contacts with segment E, A comparison of our conclusions with the earlier proposed models for bR folding [13,14] and data cm spatial disposition of the retinal residue [ 15,361 reveals no contradiction between the bR tertiary structure in medium (I) and in the purple membrane. Thus further I% NMR and twodimensional 'H NMR studies of bR and its fragments solubilized in medium (I) appear promising for elucidating the folding motif and detailed secondary structure.…”

Section: Resultssupporting

confidence: 66%

¹⁹F NMR study of 5‐fluorotryptophan‐labeled bacteriorhodopsin

et al. 1987

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19F NMR and CD spectra reveal that bacteriorh~opsin as well as its S-fluorotryptophan-labeled analog solubjIized in a CH,OH-CHCI, mixture (i) retains a secondary structure of the fully active chromoprotein in the purple membrane and (ii) possesses a foided structure in which modi~cations at the Lys-216 bound retinal are sensed by sequentially remote tryptophan residues. Individual fragments isolated after limited proteolysis and NaBH&eavage of bacteriorhodopsin keep the spatial structure of the intact poiypeptide chain in the organic solvent.

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“…The intensity I(r) was split into the two squared structure factors F2 (h, k) with h2 + hk + 1 = r according to their ratios in the electron diffraction data (2). This approximation and the use of phase information from electron microscopy have been discussed in detail elsewhere (4,7,11). Model calculations show that the conditions under which these approximations are valid are satisfied in the present case (G. Buldt and H.-J.…”

Section: Resultsmentioning

confidence: 76%

“…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)(11)(12)(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.…”

mentioning

confidence: 99%

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

Seiff¹,

Wallat²,

Ermann³

et al. 1985

Proc. Natl. Acad. Sci. U.S.A.

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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.

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Assignment of segments of the bacteriorhodopsin sequence to positions in the structural map

Cited by 66 publications

References 17 publications

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

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

¹⁹F NMR study of 5‐fluorotryptophan‐labeled bacteriorhodopsin

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

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Assignment of segments of the bacteriorhodopsin sequence to positions in the structural map

Cited by 66 publications

References 17 publications

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

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

19F NMR study of 5‐fluorotryptophan‐labeled bacteriorhodopsin

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

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Product

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¹⁹F NMR study of 5‐fluorotryptophan‐labeled bacteriorhodopsin