Effects of Asp-96 .fwdarw. Asn, Asp-85 .fwdarw. Asn, and Arg-82 .fwdarw. Gln single-site substitutions on the photocycle of bacteriorhodopsin

Thorgeirsson, Thorgeir E.; Milder, Steven J.; Miercke, Larry J. W.; Betlach, Mary C.; Shand, Richard F.; Stroud, Robert M.; Kliger, David S.

doi:10.1021/bi00102a003

Cited by 65 publications

(60 citation statements)

References 43 publications

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“…As described earlier (Otto et al, 1990;Thorgeirsson et al, 1991;Cao et al, 1993a;Balashov et al, 1993), replacement of Arg 82 with glutamine or alanine causes distinct changes in the chromophore and proton kinetics. accumulates.…”

Section: Resultsmentioning

confidence: 55%

“…The first three and coordinated water constitute a diffuse counter-ion to the Schiff base (De Groot et al, 1989Dér et al, 1991). Interaction of the anionic Asp 85 with Arg 82 is indicated by the fact that the pK a of Asp 85 is increased by about 5 pH units in the R82Q and R82A mutants (Subramaniam et al, 1990;Thorgeirsson et al, 1991;Balashov et al, 1992;Brown et al, 1993), and interaction of Asp 85 with the Schiff base is inferred from the fact that the pK a of the Schiff base is decreased by about 4 pH units in the D85N mutant (Otto et al, 1990;Marti et al, 1992;Brown et al, 1993;Tittor et al, 1994;Kataoka et al, 1994). The participation of Asp 212 in these interactions is indicated by the observations that in D212N the Schiff base does not deprotonate after photoisomerization of the retinal near neutral pH (Otto et al, 1990;Needleman et al, 1991;Cao et al, 1993b), and in the D212N/ R82Q mutant the pK a of Asp 85 is not raised as in R82Q, but the pK a of the Schiff base appears to be several pH units higher than in the wild type (Brown et al, 1995b).…”

Section: Through a Network That Contains Water Dipolesmentioning

confidence: 99%

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Glutamic Acid 204 is the Terminal Proton Release Group at the Extracellular Surface of Bacteriorhodopsin

Brown

Sasaki

Kandori

et al. 1995

Journal of Biological Chemistry

289

342

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We have measured proton release into the medium after proton transfer from the retinal Schiff base to Asp 85 in the photocycle and the C‫؍‬O stretch bands of carboxylic acids in wild type bacteriorhodopsin and the E204Q and E204D mutants. In E204Q, but not in E204D, the normal proton release is absent. Consistent with this, a negative band in the Fourier transform infrared difference spectra at 1700 cm ؊1 in the wild type, which we now attribute to depletion of the protonated E204, is also absent in E204Q. In E204D, this band is shifted to 1714 cm

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Section: Resultsmentioning

confidence: 55%

Section: Through a Network That Contains Water Dipolesmentioning

confidence: 99%

Glutamic Acid 204 is the Terminal Proton Release Group at the Extracellular Surface of Bacteriorhodopsin

Brown

Sasaki

Kandori

et al. 1995

Journal of Biological Chemistry

289

342

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“…Replacement of D85 by N shows dramatic effects on the visible spectrum and proton-pumping activity [8,9,12]. Recently, it is shown that D85N exists as three distinct spectroscopic species in equilibrium [ 131.…”

Section: I~roductionmentioning

confidence: 99%

“…The role of each amino acid residue in the proton pumping activity has been revealed by the site-directed mutagenesis (for review, see [7]). Studies using mutagenic techniques have revealed the importance of aspartates in the proton pumping activity and spectroscopic properties of bR 17-111.Replacement of D85 by N shows dramatic effects on the visible spectrum and proton-pumping activity [8,9,12]. Recently, it is shown that D85N exists as three distinct spectroscopic species in equilibrium [ 131.…”

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confidence: 99%

Trimeric mutant bacteriorhodopsin, D85N, shows a monophasic CD spectrum

et al. 1993

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The structure of mutant bacteriorhodopsin (bR), D85N, was examined by CD and X-ray diffraction at pH 7. The absorption maximum of D85N at pH 7 is located at 605 nm, which is similar to the acid-biue form of wild-type bR. D85N shows a monophasic CD band, the maximum of which is at 575 nm, although the crystalline arrangement and the trimeric structure is rn~nt~n~. The acid-blue form of wild-type bR shows a biphasic CD despite the similarity in absorption spectra.Bacteriorhodopsin; Circular dichroism; Exciton coupling; Site-specific mutant; X-ray diffraction 1. I~RODUCTION Bacteriorhodopsin (bR), the sole protein of purple membrane of Halobacterium halobium, is a light-driven proton pump which generates a trans-membrane electroche~cal gradient [l]. bR molecules are arranged in trimers which form a two-dimensional hexagonal lattice [2,3]. The three-dimensional structure of bR at moderate resolution has been solved to clarify the locations and orientations of some amino acid residues [3]. The structural changes that occur upon formation of the M inte~ediate have been determined [4-61. The role of each amino acid residue in the proton pumping activity has been revealed by the site-directed mutagenesis (for review, see [7]). Studies using mutagenic techniques have revealed the importance of aspartates in the proton pumping activity and spectroscopic properties of bR 17-111.Replacement of D85 by N shows dramatic effects on the visible spectrum and proton-pumping activity [8,9,12]. Recently, it is shown that D85N exists as three distinct spectroscopic species in equilibrium [ 131. Turner et al. argued that these three species are closely related to M, N and 0 intermediates 1131. D85N is dominated by a blue species at pH 7, which would be related to 0, and a yellowish pigment at pH 12, which *Corresponding authors. Present address: Department of Earth and Space Science, Faculty of Science, Osaka University, Toyonaka 560, Japan. Fax: (81) (6) 845 7966. Fub~~shed by Elsevier Science publishers 3. i! 111would be related to M [13]. Structural studies of D85N is of great interest in the context of the structural analyses of the photo-intermediates.We have started intensive investigation of the structure of D85N using CD and X-ray diffraction. CD has been widely used to help understand the chromophoric properties of bR [14-171. The CD spectrum in the visible region of native bR is composed of a pair of negative and positive bands [14,15]. The origin of this biphasic shape is understood to be an exciton coupling between the chromophores in the trimeric structure of bR 116,171, although some ar~ents against the exciton model have been raised [l&19]. During the structural studies of DUN, we found that D85N shows a monophasic CD spectrum despite the fact that the crystalline structure is maintained. Here, we report the CD spectrum of D85N as compared with that of native bR. MATERIALS AND METHODS Sample preparationPurple membrane containing wild-type bR was isolated from H. haiob~~ strain RlMl by a standard procedure [20]. H. haZob...

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“…2 and 3). The proton pathway in the extracellular half channel involves the Schiff base (covalently bonding the retinal and Lys-216), Asp-85, and the region of Glu-204 and Glu-194, with water molecules also playing an important role (4)(5)(6)(7)(8)(9)(10)(11)(12)(13). In the cytoplasmic half channel, Asp-96 is the proton donor to the transiently deprotonated Schiff base (4,5,14,15).…”

mentioning

confidence: 99%

Intermediate spectra and photocycle kinetics of the Asp96 → Asn mutant bacteriorhodopsin determined by singular value decomposition with self-modeling

Zimányi

Kulcsár

Lanyi

et al. 1999

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

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Singular value decomposition with selfmodeling is applied to resolve the intermediate spectra and kinetics of the Asp96 3 Asn mutant bacteriorhodopsin. The search for the difference spectra of the intermediates is performed in eigenvector space on the stoichiometric plane. The analysis of data at pH values ranging from 4 to 8 and temperatures between 5 and 25°C reveals significant, early partial recovery of the initial state after photoexcitation. The derived spectra are not biased by assumed photocycles. The intermediate spectra derived in the initial step differ from spectra determined in prior analyses, which results in intermediate concentrations with improved stoichiometric properties. Increasingly more accurate photocycles follow with increasing assumed complexity, of which parallel models are favored, consistent with recent, independent experimental evidence.Application of singular value decomposition with selfmodeling (SVD-SM) to the determination of the pure intermediate spectra and kinetics of a simulated bacteriorhodopsin photocycle was demonstrated in the preceding paper (1). SVD-SM is applied to real data in this paper. Bacteriorhodopsin (BR) functions as a light-driven proton pump in the cell membrane of Halobacterium salinarium. Photoexcitation results in isomerization of the all-trans-retinal chromophore and sequential structural changes of the protein moiety (photocycle), characterized in the visible range by the distinct spectra of the metastable intermediates J, K, L, M, N, and O. At the end of the photocycle, the initial state, BR, recovers. The net result is the transfer of a proton from the cytoplasmic to the extracellular side (for reviews, see refs. 2 and 3). The proton pathway in the extracellular half channel involves the Schiff base (covalently bonding the retinal and Lys-216), Asp-85, and the region of , with water molecules also playing an important role (4-13). In the cytoplasmic half channel, Asp-96 is the proton donor to the transiently deprotonated Schiff base (4,5,14,15). Replacement of Asp-96 with asparagine decelerates this step by orders of magnitude, effectively preventing the accumulation of any intermediate after M, while retaining the proton pumping activity (16-18). The photocycle of the Asp96 3 Asn (D96N) mutant has been the target of a number of investigations because of its relative simplicity (19)(20)(21)(22).Understanding the proton pump requires knowledge of the exact time evolution of the intermediates. This information can be obtained from multichannel kinetic absorption spectroscopy once the spectra of the pure intermediates are determined. Determination of the spectra is difficult, however, because of the strong temporal and spectral overlap of the intermediates. Global model fits are designed to yield the spectra and the kinetics simultaneously, but experience, in the case of BR, shows that optimization routines are plagued by local minima that hamper selection among assumed kinetics models (23).Similar problems are general in spectroscopy and have bee...

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Effects of Asp-96 .fwdarw. Asn, Asp-85 .fwdarw. Asn, and Arg-82 .fwdarw. Gln single-site substitutions on the photocycle of bacteriorhodopsin

Cited by 65 publications

References 43 publications

Glutamic Acid 204 is the Terminal Proton Release Group at the Extracellular Surface of Bacteriorhodopsin

Glutamic Acid 204 is the Terminal Proton Release Group at the Extracellular Surface of Bacteriorhodopsin

Trimeric mutant bacteriorhodopsin, D85N, shows a monophasic CD spectrum

Intermediate spectra and photocycle kinetics of the Asp96 → Asn mutant bacteriorhodopsin determined by singular value decomposition with self-modeling

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