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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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UV/visible absorption spectra of pR in octylglucoside solution (1–3%) at three stages of purification. All three spectra were measured in the presence of octylglucoside at pH 8, and are normalized to the 280-nm protein peak. Spectrum A, the OG extract of cholate-washed E. coli membranes; spectrum B, pooled 546-nm absorbing fractions from Phenylsepharose column; spectrum C, same material after hydroxylapatite column.
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Figure 1: UV/visible absorption spectra of pR in octylglucoside solution (1–3%) at three stages of purification. All three spectra were measured in the presence of octylglucoside at pH 8, and are normalized to the 280-nm protein peak. Spectrum A, the OG extract of cholate-washed E. coli membranes; spectrum B, pooled 546-nm absorbing fractions from Phenylsepharose column; spectrum C, same material after hydroxylapatite column.

Mentions: PR was obtained in 85% purity, assuming that values of ε280 and ε546 for pR are the same as for bR solubilized in DMPC/cholate/SDS mixtures at pH 8 (ε280= 7.85 × 104 cm-1 M-1 and ε551 = 4.8 × 104 cm-1 M-1) [5]. This assumption is actually expected to underestimate the purity of pR produced, by up to ~20%, since the pR we expressed has 10 tryptophan and 14 tyrosine residues, as compared to 8 tryptophans and 11 tyrosines in bR from H. salinarum. The absorbance of contaminant proteins was assumed to be 1.1 for a 1 mg/mL solution. By using these assumptions, the relative concentrations of pR and other proteins can be determined from the absorbance spectra of the various fractions (fig. 1). The resulting purity values correlate well with those Coomasie-stained SDS-PAGE gels (see below). The OG extract of cholate-washed membrane pellets starts out at a pR content of 7% total protein (w/w). The Phenylsepharose column increases the purity level to 24%, with approximately 5% loss. The final purification step by hydroxylapatite column chromatography produces pR with ~85% purity and a further loss of ~60%, i.e. the overall yield of the two column procedure was ~30%.


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)

UV/visible absorption spectra of pR in octylglucoside solution (1–3%) at three stages of purification. All three spectra were measured in the presence of octylglucoside at pH 8, and are normalized to the 280-nm protein peak. Spectrum A, the OG extract of cholate-washed E. coli membranes; spectrum B, pooled 546-nm absorbing fractions from Phenylsepharose column; spectrum C, same material after hydroxylapatite column.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: UV/visible absorption spectra of pR in octylglucoside solution (1–3%) at three stages of purification. All three spectra were measured in the presence of octylglucoside at pH 8, and are normalized to the 280-nm protein peak. Spectrum A, the OG extract of cholate-washed E. coli membranes; spectrum B, pooled 546-nm absorbing fractions from Phenylsepharose column; spectrum C, same material after hydroxylapatite column.
Mentions: PR was obtained in 85% purity, assuming that values of ε280 and ε546 for pR are the same as for bR solubilized in DMPC/cholate/SDS mixtures at pH 8 (ε280= 7.85 × 104 cm-1 M-1 and ε551 = 4.8 × 104 cm-1 M-1) [5]. This assumption is actually expected to underestimate the purity of pR produced, by up to ~20%, since the pR we expressed has 10 tryptophan and 14 tyrosine residues, as compared to 8 tryptophans and 11 tyrosines in bR from H. salinarum. The absorbance of contaminant proteins was assumed to be 1.1 for a 1 mg/mL solution. By using these assumptions, the relative concentrations of pR and other proteins can be determined from the absorbance spectra of the various fractions (fig. 1). The resulting purity values correlate well with those Coomasie-stained SDS-PAGE gels (see below). The OG extract of cholate-washed membrane pellets starts out at a pR content of 7% total protein (w/w). The Phenylsepharose column increases the purity level to 24%, with approximately 5% loss. The final purification step by hydroxylapatite column chromatography produces pR with ~85% purity and a further loss of ~60%, i.e. the overall yield of the two column procedure was ~30%.

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