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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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Dependence on pH of the M-like intermediate of pR. Time courses of flash-induced absorbance changes measured at 400 nm and 22°C for pR in 1% DHPC/100 mM NaCl solution at pH 6.5, 8.0 and 9.5. A positive differential absorbance at 400 nm is indicative of the presence of the M intermediate. The logarithmic time scale ranges from 100–107 μs after photolysis by a 10-ns laser pulse at 500 nm, with an energy of 3–6 mJ.
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Figure 3: Dependence on pH of the M-like intermediate of pR. Time courses of flash-induced absorbance changes measured at 400 nm and 22°C for pR in 1% DHPC/100 mM NaCl solution at pH 6.5, 8.0 and 9.5. A positive differential absorbance at 400 nm is indicative of the presence of the M intermediate. The logarithmic time scale ranges from 100–107 μs after photolysis by a 10-ns laser pulse at 500 nm, with an energy of 3–6 mJ.

Mentions: Photocycle kinetics were measured at 400, 500. and 580 nm in the presence of the short-chain lipid DHPC. This lipid does not support the formation of closed bilayer vesicles, but rather forms micelles like a detergent. The time-resolved measurements showed no positive 400-nm absorbance signals at pH 8.0 or lower (Fig. 3). This is somewhat in disagreement with Béjà et al [1], who detected small 400-nm transient absorbance increases upon photolysis at pH 8.0. However, we observed a transient 400-nm absorbance increase at an elevated pH of 9.5 (fig. 3).


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)

Dependence on pH of the M-like intermediate of pR. Time courses of flash-induced absorbance changes measured at 400 nm and 22°C for pR in 1% DHPC/100 mM NaCl solution at pH 6.5, 8.0 and 9.5. A positive differential absorbance at 400 nm is indicative of the presence of the M intermediate. The logarithmic time scale ranges from 100–107 μs after photolysis by a 10-ns laser pulse at 500 nm, with an energy of 3–6 mJ.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC103662&req=5

Figure 3: Dependence on pH of the M-like intermediate of pR. Time courses of flash-induced absorbance changes measured at 400 nm and 22°C for pR in 1% DHPC/100 mM NaCl solution at pH 6.5, 8.0 and 9.5. A positive differential absorbance at 400 nm is indicative of the presence of the M intermediate. The logarithmic time scale ranges from 100–107 μs after photolysis by a 10-ns laser pulse at 500 nm, with an energy of 3–6 mJ.
Mentions: Photocycle kinetics were measured at 400, 500. and 580 nm in the presence of the short-chain lipid DHPC. This lipid does not support the formation of closed bilayer vesicles, but rather forms micelles like a detergent. The time-resolved measurements showed no positive 400-nm absorbance signals at pH 8.0 or lower (Fig. 3). This is somewhat in disagreement with Béjà et al [1], who detected small 400-nm transient absorbance increases upon photolysis at pH 8.0. However, we observed a transient 400-nm absorbance increase at an elevated pH of 9.5 (fig. 3).

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