Limits...
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).

Show MeSH

Related in: MedlinePlus

Photocycle kinetics of pR at selected wavelengths at pH 9.5. Time traces were measured at 400, 500, and 580 nm. The 400-nm trace shows the kinetics of the M intermediate, i.e. the deprotonated Schiff base, as in Fig. 3. The 500-nm trace shows the depletion signal of pR at the earliest times, and then the time course of the N intermediate as well as return of the pR resting state. The 580-nm trace is indicative of an O-like intermediate. The conditions are 1% DHPC, 100 mM NaCl, pH9.5 at 22°C. The laser excitation is as in fig. 3.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC103662&req=5

Figure 4: Photocycle kinetics of pR at selected wavelengths at pH 9.5. Time traces were measured at 400, 500, and 580 nm. The 400-nm trace shows the kinetics of the M intermediate, i.e. the deprotonated Schiff base, as in Fig. 3. The 500-nm trace shows the depletion signal of pR at the earliest times, and then the time course of the N intermediate as well as return of the pR resting state. The 580-nm trace is indicative of an O-like intermediate. The conditions are 1% DHPC, 100 mM NaCl, pH9.5 at 22°C. The laser excitation is as in fig. 3.

Mentions: At pH 9.5 in the presence of DHPC, and observing transient changes at 500 nm (fig. 4), pR undergoes a 2-phase decay after the initial unresolved absorbance decrease. Multiexponential fits show that the first decay phase has a time constant of 4 μs, in good agreement with the 4-μs rise time of the 400 nm signal (Fig. 4). The amplitude of this decay represents about 80% of the initial absorbance depletion. The second phase of the 500 nm absorbance decay occurs with a substantially slower time constant of 0.5 s, returning the remaining 20% of initial absorbance change. The slowest decay components of the positive 400-nm signal and the negative 500-nm signal follow similar kinetics, although the amplitudes of these components differ by a factor of 3. At pH 9.5, the 580 nm trace has no significant positive values indicative of an O-like intermediate, although, in agreement with earlier measurements [1], at lower pH values a red-shifted transient is the predominant positive absorbance signal (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)

Photocycle kinetics of pR at selected wavelengths at pH 9.5. Time traces were measured at 400, 500, and 580 nm. The 400-nm trace shows the kinetics of the M intermediate, i.e. the deprotonated Schiff base, as in Fig. 3. The 500-nm trace shows the depletion signal of pR at the earliest times, and then the time course of the N intermediate as well as return of the pR resting state. The 580-nm trace is indicative of an O-like intermediate. The conditions are 1% DHPC, 100 mM NaCl, pH9.5 at 22°C. The laser excitation is as in fig. 3.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Photocycle kinetics of pR at selected wavelengths at pH 9.5. Time traces were measured at 400, 500, and 580 nm. The 400-nm trace shows the kinetics of the M intermediate, i.e. the deprotonated Schiff base, as in Fig. 3. The 500-nm trace shows the depletion signal of pR at the earliest times, and then the time course of the N intermediate as well as return of the pR resting state. The 580-nm trace is indicative of an O-like intermediate. The conditions are 1% DHPC, 100 mM NaCl, pH9.5 at 22°C. The laser excitation is as in fig. 3.
Mentions: At pH 9.5 in the presence of DHPC, and observing transient changes at 500 nm (fig. 4), pR undergoes a 2-phase decay after the initial unresolved absorbance decrease. Multiexponential fits show that the first decay phase has a time constant of 4 μs, in good agreement with the 4-μs rise time of the 400 nm signal (Fig. 4). The amplitude of this decay represents about 80% of the initial absorbance depletion. The second phase of the 500 nm absorbance decay occurs with a substantially slower time constant of 0.5 s, returning the remaining 20% of initial absorbance change. The slowest decay components of the positive 400-nm signal and the negative 500-nm signal follow similar kinetics, although the amplitudes of these components differ by a factor of 3. At pH 9.5, the 580 nm trace has no significant positive values indicative of an O-like intermediate, although, in agreement with earlier measurements [1], at lower pH values a red-shifted transient is the predominant positive absorbance signal (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