Location of an extrinsic label in the primary and tertiary structure of bacteriorhodopsin

Katre, Nandini V.; Finer-Moore, J.; Stroud, Robert M.; Hayward, Steven B.

doi:10.1016/s0006-3495(84)84013-8

Cited by 26 publications

(16 citation statements)

References 23 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%

See 1 more Smart Citation

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%

“…This procedure, which gets around some of the limitations of the Fourier difference approach, has been discussed and described in more detail elsewhere (27)(28)(29). The label is assumed to be a point.…”

Section: Cmentioning

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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“…However, it is important to note that not all polypeptide sequences that contain such features have membrane spanning helices ; some soluble proteins may include fe atures of this kind, (8,16,26,37,41,56,65,97,98). The prediction of helix B has two possible extremes ; that which is preferred on the basis of experimental observation is shown.…”

Section: Discussionmentioning

confidence: 98%

“…While the application of the initial scale suggested locations for all seven helices, the current scale more convincingly delineates the existence and positions of helices C and G. Subsequent experiments have been consistent with and thus support the use of the computer-generated model. While the exact sequence locations ofthe helices in the actual structure are not known, a number of recent chemical modification and protease-digestion studies narrow the possible locations substantially (8,16,26,37,41,56,65,97,98). There remains some ambiguity in the precise location of the short loop connecting helices F and G, but the water-accessible portions of the rest of the sequence are well defined.…”

Section: Structuresmentioning

confidence: 99%

Identifying Nonpolar Transbilayer Helices in Amino Acid Sequences of Membrane Proteins

Engelman¹,

Steitz²,

Goldman³

1986

Annu. Rev. Biophys. Biophys. Chem.

1,441

795

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“…The locations of the helix-connecting links are not known with complete certainty, although several attempts have been made to obtain this information by structural methods (Agard & Stroud, 1982;Wallace & Henderson, 1982;Katre et al, 1984;Jaffe & Glaeser, 1987). There have, however, been extensive studies of this question using combinations of low resolution diffraction data, hydropathy plots, and biochemical methods to assess connecting link accessibility, which give a reasonably consistent assignment of the helices.…”

Section: Bacteriorhodopsinmentioning

confidence: 99%

Observations concerning topology and locations of helix ends of membrane proteins of known structure

White

Jacobs²

1990

J. Membrain Biol.

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Hydropathy plots of amino acid sequences reveal the approximate locations of the transbilayer helices of membrane proteins of known structure and are thus used to predict the helices of proteins of unknown structure. Because the three-dimensional structures of membrane proteins are difficult to obtain, it is important to be able to extract as much information as possible from hydropathy plots. We describe an "augmented" hydropathy plot analysis of the three membrane proteins of known structure, which should be useful for the systematic examination and comparison of membrane proteins of unknown structure. The sliding-window analysis utilizes the floating interfacial hydrophobicity scale [IFH(h)] of Jacobs and White (Jacobs, R.E., White, S. H., 1989. Biochemistry 28:3421-3437) and the reverse-turn (RT) frequencies of Levitt (Levitt, M., 1977, Biochemistry 17:4277-4285). The IFH(h) scale allows one to examine the consequences of different assumptions about the average hydrogen bond status (h = 0 to 1) of polar side chains. Hydrophobicity plots of the three proteins show that (i) the intracellular helix-connecting links and chain ends can be distinguished from the extracellular ones and (ii) the main peaks of hydrophobicity are bounded by minor ones which bracket the helix ends. RT frequency plots show that (iii) the centers of helices are usually very close to wide-window minima of average RT frequency and (iv) helices are always bounded by narrow-window maxima of average RT frequency. The analysis suggests that side-chain hydrogen bonding with membrane components during folding may play a key role in insertion.

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Location of an extrinsic label in the primary and tertiary structure of bacteriorhodopsin

Cited by 26 publications

References 23 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

Identifying Nonpolar Transbilayer Helices in Amino Acid Sequences of Membrane Proteins

Observations concerning topology and locations of helix ends of membrane proteins of known structure

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