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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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Related in: MedlinePlus

SDS-PAGE of pR (wild type and pR-triple cysteine mutant). Lane A contains bacteriorhodopsin (bR). Lanes B and F contain BioRad protein molecular weight markers including labeled bands at 21.5 (trypsin inhibitor), 31 (bovine carbonic anhydrase), and 45 (ovalbumin) kDa. Lane C is of the pR triple cysteine mutant (TCM). Lane D contains the Phenylsepharose™-purified pR wild type protein, corresponding to spectrum B of fig. 1. Lane E contains the hydroxylapatite-purified pR wild type protein, corresponding to spectrum C in fig. 1.
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Figure 2: SDS-PAGE of pR (wild type and pR-triple cysteine mutant). Lane A contains bacteriorhodopsin (bR). Lanes B and F contain BioRad protein molecular weight markers including labeled bands at 21.5 (trypsin inhibitor), 31 (bovine carbonic anhydrase), and 45 (ovalbumin) kDa. Lane C is of the pR triple cysteine mutant (TCM). Lane D contains the Phenylsepharose™-purified pR wild type protein, corresponding to spectrum B of fig. 1. Lane E contains the hydroxylapatite-purified pR wild type protein, corresponding to spectrum C in fig. 1.

Mentions: Relative to protein standards, the apparent molecular weight of bR is 25,000 while the apparent molecular weights of pR-wt and pR-TCM are 36,000 and 31,000, respectively (fig. 2, lanes E and C, respectively). SDS-PAGE (fig. 2) also confirms the estimates of purity level based on the assumed ε280/ε546 ratio identical with that of detergent solubilized bR. Interestingly, the pR appears to be a doublet band whose relative concentrations remain almost unchanged during purification. This doublet is also present in the less-purified sample of pR-TCM, with both bands shifted down by approximately the same amount (fig. 2, lane C).


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)

SDS-PAGE of pR (wild type and pR-triple cysteine mutant). Lane A contains bacteriorhodopsin (bR). Lanes B and F contain BioRad protein molecular weight markers including labeled bands at 21.5 (trypsin inhibitor), 31 (bovine carbonic anhydrase), and 45 (ovalbumin) kDa. Lane C is of the pR triple cysteine mutant (TCM). Lane D contains the Phenylsepharose™-purified pR wild type protein, corresponding to spectrum B of fig. 1. Lane E contains the hydroxylapatite-purified pR wild type protein, corresponding to spectrum C in fig. 1.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: SDS-PAGE of pR (wild type and pR-triple cysteine mutant). Lane A contains bacteriorhodopsin (bR). Lanes B and F contain BioRad protein molecular weight markers including labeled bands at 21.5 (trypsin inhibitor), 31 (bovine carbonic anhydrase), and 45 (ovalbumin) kDa. Lane C is of the pR triple cysteine mutant (TCM). Lane D contains the Phenylsepharose™-purified pR wild type protein, corresponding to spectrum B of fig. 1. Lane E contains the hydroxylapatite-purified pR wild type protein, corresponding to spectrum C in fig. 1.
Mentions: Relative to protein standards, the apparent molecular weight of bR is 25,000 while the apparent molecular weights of pR-wt and pR-TCM are 36,000 and 31,000, respectively (fig. 2, lanes E and C, respectively). SDS-PAGE (fig. 2) also confirms the estimates of purity level based on the assumed ε280/ε546 ratio identical with that of detergent solubilized bR. Interestingly, the pR appears to be a doublet band whose relative concentrations remain almost unchanged during purification. This doublet is also present in the less-purified sample of pR-TCM, with both bands shifted down by approximately the same amount (fig. 2, lane C).

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