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Detection of fast light-activated H+ release and M intermediate formation from proteorhodopsin.

Krebs RA, Alexiev U, Partha R, DeVita AM, Braiman MS - BMC Physiol. (2002)

Bottom Line: At pH 9.5 and in the presence of octylglucoside and diheptanoylphosphotidylcholine, flash photolysis results in fast H+ release and a 400-nm absorbing (M-like) photoproduct.Both of these occur with a similar rise time (4-10 micros) as reported for monomeric bR in detergent.The presence of fast H+ release in pR indicates that either different groups are responsible for fast H+ release in pR and bR (i.e. that the H+ release group is not highly conserved); or, that the H+ release group is conserved and is therefore likely Arg-94 itself in pR (and Arg-82 in bR, correspondingly).

View Article: PubMed Central - HTML - PubMed

Affiliation: Chemistry Department, Syracuse University, Syracuse, NY 13244-4100, USA. rakrebs@syr.edu

ABSTRACT

Background: Proteorhodopsin (pR) is a light-activated proton pump homologous to bacteriorhodopsin and recently discovered in oceanic gamma-proteobacteria. One perplexing difference between these two proteins is the absence in pR of homologues of bR residues Glu-194 and Glu-204. These two residues, along with Arg-82, have been implicated in light-activated fast H+ release to the extracellular medium in bR. It is therefore uncertain that pR carries out its physiological activity using a mechanism that is completely homologous to that of bR.

Results: A pR purification procedure is described that utilizes Phenylsepharose and hydroxylapatite columns and yields 85% (w/w) purity. Through SDS-PAGE of the pure protein, the molecular weight of E.-coli-produced pR was determined to be 36,000, approximately 9,000 more than the 27,000 predicted by the DNA sequence. Post-translational modification of one or more of the cysteine residues accounts for 5 kDa of the weight difference as measured on a cys-less pR mutant. At pH 9.5 and in the presence of octylglucoside and diheptanoylphosphotidylcholine, flash photolysis results in fast H+ release and a 400-nm absorbing (M-like) photoproduct. Both of these occur with a similar rise time (4-10 micros) as reported for monomeric bR in detergent.

Conclusions: The presence of fast H+ release in pR indicates that either different groups are responsible for fast H+ release in pR and bR (i.e. that the H+ release group is not highly conserved); or, that the H+ release group is conserved and is therefore likely Arg-94 itself in pR (and Arg-82 in bR, correspondingly).

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Comparison of the kinetics of M formation and decay with kinetics of ET release and uptake. The time trace of the M-like intermediate was measured at 400 nm (upper panel). Time-resolved H+ concentration changes (lower panel) were measured with the pH indicator dye Cresol Red. A negative Cresol Red absorbance change at 580 nm is indicative of a transient decrease in the pH of the solution, i.e. of H+ release by pR. Solid lines represent multiexponential fits, with the main rise and decay times indicated for the M intermediate. The H+ release and uptake time constants obtained from the fit are marked with arrows pointing down for release and pointing up for uptake. Sample and excitation conditions are as in fig. 4.
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Figure 5: Comparison of the kinetics of M formation and decay with kinetics of ET release and uptake. The time trace of the M-like intermediate was measured at 400 nm (upper panel). Time-resolved H+ concentration changes (lower panel) were measured with the pH indicator dye Cresol Red. A negative Cresol Red absorbance change at 580 nm is indicative of a transient decrease in the pH of the solution, i.e. of H+ release by pR. Solid lines represent multiexponential fits, with the main rise and decay times indicated for the M intermediate. The H+ release and uptake time constants obtained from the fit are marked with arrows pointing down for release and pointing up for uptake. Sample and excitation conditions are as in fig. 4.

Mentions: Figure 5 shows a different type of time-resolved measurement, probing not the pR chromophore, but rather pH changes in the protein environment. Proton concentration changes in the aqueous bulk phase were measured with the pH sensitive dye cresol red, which has a pKa of 8.2–8.5. The bottom trace in Figure 5 shows the absorbance change of the indicator during the pR photocycle. The negative signal is indicative of a pH decrease, corresponding to transient H+ release from the protein into the solution. The best-fit time constant for the release phase is 6 μs. The positive 400 nm trace in fig. 5 (reproduced from fig. 3) shows that the proton release and uptake follow kinetics very similar to the apparent formation and decay of M, as is typically seen in bR near neutral pH [6,7,21]. However, no proton release signal could be observed for pR at pH 6 or 8 (data not shown).


Detection of fast light-activated H+ release and M intermediate formation from proteorhodopsin.

Krebs RA, Alexiev U, Partha R, DeVita AM, Braiman MS - BMC Physiol. (2002)

Comparison of the kinetics of M formation and decay with kinetics of ET release and uptake. The time trace of the M-like intermediate was measured at 400 nm (upper panel). Time-resolved H+ concentration changes (lower panel) were measured with the pH indicator dye Cresol Red. A negative Cresol Red absorbance change at 580 nm is indicative of a transient decrease in the pH of the solution, i.e. of H+ release by pR. Solid lines represent multiexponential fits, with the main rise and decay times indicated for the M intermediate. The H+ release and uptake time constants obtained from the fit are marked with arrows pointing down for release and pointing up for uptake. Sample and excitation conditions are as in fig. 4.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC103662&req=5

Figure 5: Comparison of the kinetics of M formation and decay with kinetics of ET release and uptake. The time trace of the M-like intermediate was measured at 400 nm (upper panel). Time-resolved H+ concentration changes (lower panel) were measured with the pH indicator dye Cresol Red. A negative Cresol Red absorbance change at 580 nm is indicative of a transient decrease in the pH of the solution, i.e. of H+ release by pR. Solid lines represent multiexponential fits, with the main rise and decay times indicated for the M intermediate. The H+ release and uptake time constants obtained from the fit are marked with arrows pointing down for release and pointing up for uptake. Sample and excitation conditions are as in fig. 4.
Mentions: Figure 5 shows a different type of time-resolved measurement, probing not the pR chromophore, but rather pH changes in the protein environment. Proton concentration changes in the aqueous bulk phase were measured with the pH sensitive dye cresol red, which has a pKa of 8.2–8.5. The bottom trace in Figure 5 shows the absorbance change of the indicator during the pR photocycle. The negative signal is indicative of a pH decrease, corresponding to transient H+ release from the protein into the solution. The best-fit time constant for the release phase is 6 μs. The positive 400 nm trace in fig. 5 (reproduced from fig. 3) shows that the proton release and uptake follow kinetics very similar to the apparent formation and decay of M, as is typically seen in bR near neutral pH [6,7,21]. However, no proton release signal could be observed for pR at pH 6 or 8 (data not shown).

Bottom Line: At pH 9.5 and in the presence of octylglucoside and diheptanoylphosphotidylcholine, flash photolysis results in fast H+ release and a 400-nm absorbing (M-like) photoproduct.Both of these occur with a similar rise time (4-10 micros) as reported for monomeric bR in detergent.The presence of fast H+ release in pR indicates that either different groups are responsible for fast H+ release in pR and bR (i.e. that the H+ release group is not highly conserved); or, that the H+ release group is conserved and is therefore likely Arg-94 itself in pR (and Arg-82 in bR, correspondingly).

View Article: PubMed Central - HTML - PubMed

Affiliation: Chemistry Department, Syracuse University, Syracuse, NY 13244-4100, USA. rakrebs@syr.edu

ABSTRACT

Background: Proteorhodopsin (pR) is a light-activated proton pump homologous to bacteriorhodopsin and recently discovered in oceanic gamma-proteobacteria. One perplexing difference between these two proteins is the absence in pR of homologues of bR residues Glu-194 and Glu-204. These two residues, along with Arg-82, have been implicated in light-activated fast H+ release to the extracellular medium in bR. It is therefore uncertain that pR carries out its physiological activity using a mechanism that is completely homologous to that of bR.

Results: A pR purification procedure is described that utilizes Phenylsepharose and hydroxylapatite columns and yields 85% (w/w) purity. Through SDS-PAGE of the pure protein, the molecular weight of E.-coli-produced pR was determined to be 36,000, approximately 9,000 more than the 27,000 predicted by the DNA sequence. Post-translational modification of one or more of the cysteine residues accounts for 5 kDa of the weight difference as measured on a cys-less pR mutant. At pH 9.5 and in the presence of octylglucoside and diheptanoylphosphotidylcholine, flash photolysis results in fast H+ release and a 400-nm absorbing (M-like) photoproduct. Both of these occur with a similar rise time (4-10 micros) as reported for monomeric bR in detergent.

Conclusions: The presence of fast H+ release in pR indicates that either different groups are responsible for fast H+ release in pR and bR (i.e. that the H+ release group is not highly conserved); or, that the H+ release group is conserved and is therefore likely Arg-94 itself in pR (and Arg-82 in bR, correspondingly).

Show MeSH
Related in: MedlinePlus