skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Light activates the reaction of bacteriorhodopsin aspartic acid-115 with dicyclohexylcarbodiimide

Abstract

Conditions for a light-induced reaction between the carboxyl-modifying reagent N,N'-dicyclohexylcarbodiimide (DCCD) and bacteriorhodopsin in Triton X-100 micelles were previously reported. The authors have now located the DCCD site in the bacteriorhodopsin amino acid sequence. ( UC)DCCD-bacteriorhodopsin was cleaved with CNBr. The resulting peptides were purified by gel filtration and reverse-phase high-performance liquid chromatography (HPLC). One major UC peptide (50%) and two minor fractions were obtained. Amino acid analysis and sequence analysis showed that the major fraction contained residues 69-118. This region includes six carboxyl side chains. The major UC peptide was also subjected to pepsin hydrolysis. HPLC analysis of the product gave only a single major radioactive subfragment. Amino acid analysis of the peptic peptide showed that it contained residues 110-118. The only carboxyl side chain in this region is Asp-115. Thus, the authors conclude that Asp-115 is the major DCCD site. The light sensitivity of this reaction suggests that Asp-115 becomes more exposed or that its environment becomes more acidic during proton pumping. The DCCD reaction blue-shifts the retinal chromophore. Such a result would be expected if Asp-115 is the negative point charge predicted to be near the cyclohexene ring of retinal.

Authors:
; ; ; ;
Publication Date:
Research Org.:
Univ. of Texas, San Antonio
OSTI Identifier:
6045375
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemistry; (United States); Journal Volume: 16
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; ASPARTIC ACID; CHEMICAL REACTIONS; CARBON 14 COMPOUNDS; AMINO ACID SEQUENCE; IMIDES; RHODOPSIN; BACTERIA; CAROTENOIDS; LIQUID COLUMN CHROMATOGRAPHY; PEPSIN; RETINA; ACID PROTEINASES; AMINO ACIDS; BODY; BODY AREAS; CARBOXYLIC ACIDS; CHROMATOGRAPHY; ENZYMES; EYES; FACE; HEAD; HYDROCARBONS; HYDROLASES; LABELLED COMPOUNDS; MICROORGANISMS; MOLECULAR STRUCTURE; ORGANIC ACIDS; ORGANIC COMPOUNDS; ORGANIC NITROGEN COMPOUNDS; ORGANS; PEPTIDE HYDROLASES; PIGMENTS; PROTEINS; SENSE ORGANS; SEPARATION PROCESSES; TERPENES; 550201* - Biochemistry- Tracer Techniques

Citation Formats

Renthal, R., Cothran, M., Espinoza, B., Wall, K.A., and Bernard, M.. Light activates the reaction of bacteriorhodopsin aspartic acid-115 with dicyclohexylcarbodiimide. United States: N. p., 1985. Web. doi:10.1021/bi00337a004.
Renthal, R., Cothran, M., Espinoza, B., Wall, K.A., & Bernard, M.. Light activates the reaction of bacteriorhodopsin aspartic acid-115 with dicyclohexylcarbodiimide. United States. doi:10.1021/bi00337a004.
Renthal, R., Cothran, M., Espinoza, B., Wall, K.A., and Bernard, M.. 1985. "Light activates the reaction of bacteriorhodopsin aspartic acid-115 with dicyclohexylcarbodiimide". United States. doi:10.1021/bi00337a004.
@article{osti_6045375,
title = {Light activates the reaction of bacteriorhodopsin aspartic acid-115 with dicyclohexylcarbodiimide},
author = {Renthal, R. and Cothran, M. and Espinoza, B. and Wall, K.A. and Bernard, M.},
abstractNote = {Conditions for a light-induced reaction between the carboxyl-modifying reagent N,N'-dicyclohexylcarbodiimide (DCCD) and bacteriorhodopsin in Triton X-100 micelles were previously reported. The authors have now located the DCCD site in the bacteriorhodopsin amino acid sequence. ( UC)DCCD-bacteriorhodopsin was cleaved with CNBr. The resulting peptides were purified by gel filtration and reverse-phase high-performance liquid chromatography (HPLC). One major UC peptide (50%) and two minor fractions were obtained. Amino acid analysis and sequence analysis showed that the major fraction contained residues 69-118. This region includes six carboxyl side chains. The major UC peptide was also subjected to pepsin hydrolysis. HPLC analysis of the product gave only a single major radioactive subfragment. Amino acid analysis of the peptic peptide showed that it contained residues 110-118. The only carboxyl side chain in this region is Asp-115. Thus, the authors conclude that Asp-115 is the major DCCD site. The light sensitivity of this reaction suggests that Asp-115 becomes more exposed or that its environment becomes more acidic during proton pumping. The DCCD reaction blue-shifts the retinal chromophore. Such a result would be expected if Asp-115 is the negative point charge predicted to be near the cyclohexene ring of retinal.},
doi = {10.1021/bi00337a004},
journal = {Biochemistry; (United States)},
number = ,
volume = 16,
place = {United States},
year = 1985,
month = 7
}
  • The molecular events during the photocycle of bacteriorhodopsin have been studied by the method of time-resolved and static infrared difference spectroscopy. Characteristic spectral changes involving the C=O stretching vibration of protonated carboxylic groups were detected. To identify the corresponding groups with either glutamic or aspartic acid, BR was selectively labeled with (4-/sup 13/C)aspartic acid. An incorporation of ca. 70% was obtained. The comparison of the difference spectra in the region of the CO/sub 2/- stretching vibrations of labeled and unlabeled BR indicates that ionized aspartic acids are influenced during the photocycle, the earliest effect being observed already at the K610more » intermediate. Taken together, the results provide evidence that four internal aspartic acids undergo protonation changes and that one glutamic acid, remaining protonated, is disturbed. The results are discussed in relation to the various aspects of the proton pumping mechanism, such as retinal isomerization, charge separation, pK changes, and proton pathway.« less
  • Fourier transform infrared (FTIR) difference spectra have been obtained for the bR----K, bR----L, and bR----M photoreactions in bacteriorhodopsin mutants in which Asp residues 85, 96, 115, and 212 have been replaced by Asn and by Glu. Difference peaks that had previously been attributed to Asp COOH groups on the basis of isotopic labeling were absent or shifted in these mutants. In general, each COOH peak was affected strongly by mutation at only one of the four residues. Thus, it was possible to assign each peak tentatively to a particular Asp. From these assignments, a model for the proton-pumping mechanism ofmore » bR is derived, which features proton transfers among Asp-85, -96, and -212, the chromophore Schiff base, and other ionizable groups within the protein. The model can explain the observed COOH peaks in the FTIR difference spectra of bR photointermediates and could also account for other recent results on site-directed mutants of bR.« less
  • Above pH 8 the decay of the photocycle intermediate M of bacteriorhodopsin splits into two components: the usual millisecond pH-independent component and an additional slower component with a rate constant proportional to the molar concentration of H+, (H+). In parallel, the charge translocation signal associated with the reprotonation of the Schiff base develops a similar slow component. These observations are explained by a two-step reprotonation mechanism. An internal donor first reprotonates the Schiff base in the decay of M to N and is then reprotonated from the cytoplasm in the N----O transition. The decay rate of N is proportional tomore » (H+). By postulating a back reaction from N to M, the M decay splits up into two components, with the slower one having the same pH dependence as the decay of N. Photocycle, photovoltage, and pH-indicator experiments with mutants in which aspartic acid-96 is replaced by asparagine or alanine, which we call D96N and D96A, suggest that Asp-96 is the internal proton donor involved in the re-uptake pathway. In both mutants the stoichiometry of proton pumping is the same as in wild type. However, the M decay is monophasic, with the logarithm of the decay time (log (tau)) linearly dependent on pH, suggesting that the internal donor is absent and that the Schiff base is directly reprotonated from the cytoplasm. Like H+, azide increases the M decay rate in D96N. The rate constant is proportional to the azide concentration and can become greater than 100 times greater than in wild type. Thus, azide functions as a mobile proton donor directly reprotonating the Schiff base in a bimolecular reaction. Both the proton and azide effects, which are absent in wild type, indicate that the internal donor is removed and that the reprotonation pathway is different from wild type in these mutants.« less
  • To investigate how a photoactivated chromophore drives the proton pump mechanism of bacteriorhodopsin, the authors have observed how the chromophore rotates during the photocycle. To do this, they examined the dichroism induced in aqueous suspensions of purple membrane fragments by flashes of linearly polarized light. It was found that the flash stimulates both the photocycling chromophores and their noncycling neighbors to undergo large (> 10/sup 0/-20/sup 0/) rotations within the membrane during the photocycle, and that these two chromophore populations undergo distinctly different sequences of rotations. All these rotations could be eliminated by glutaraldehyde fixation as well as by embeddingmore » unfixed fragments in polyacrylamide or agarose gels. Thus, in these immbolizing preparations the chromophore can photocycle without rotating inside a bacteriorhodopsin monomer by more than our detection limit of 2/sup 0/-5/sup 0/. The large rotations observed in aqueous suspensions of purple membranes were probably due to rotations of entire protein monomers. The process by which a photocycyling monomer causes its noncycling neighbors to rotate may help explain the highly cooperative behavior bacteriorhodopsin exhibits when it is aggregated into crystalline arrays of trimers.« less