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Light-driven Na(+) pump from Gillisia limnaea: a high-affinity Na(+) binding site is formed transiently in the photocycle.

Balashov SP, Imasheva ES, Dioumaev AK, Wang JM, Jung KH, Lanyi JK - Biochemistry (2014)

Bottom Line: However, very low concentrations of Na(+) cause profound differences in the decay and rise time of photocycle intermediates, consistent with a switch from a "Na(+)-independent" to a "Na(+)-dependent" photocycle (or photocycle branch) at ∼60 μM Na(+).A greater concentration of Na(+) is needed for switching the reaction path at lower pH.Binding of Na(+) to the mutant shifts the chromophore maximum to the red like that of H(+), which occurs in the photocycle of the wild type.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, University of California , Irvine, California 92697, United States.

ABSTRACT
A group of microbial retinal proteins most closely related to the proton pump xanthorhodopsin has a novel sequence motif and a novel function. Instead of, or in addition to, proton transport, they perform light-driven sodium ion transport, as reported for one representative of this group (KR2) from Krokinobacter. In this paper, we examine a similar protein, GLR from Gillisia limnaea, expressed in Escherichia coli, which shares some properties with KR2 but transports only Na(+). The absorption spectrum of GLR is insensitive to Na(+) at concentrations of ≤3 M. However, very low concentrations of Na(+) cause profound differences in the decay and rise time of photocycle intermediates, consistent with a switch from a "Na(+)-independent" to a "Na(+)-dependent" photocycle (or photocycle branch) at ∼60 μM Na(+). The rates of photocycle steps in the latter, but not the former, are linearly dependent on Na(+) concentration. This suggests that a high-affinity Na(+) binding site is created transiently after photoexcitation, and entry of Na(+) from the bulk to this site redirects the course of events in the remainder of the cycle. A greater concentration of Na(+) is needed for switching the reaction path at lower pH. The data suggest therefore competition between H(+) and Na(+) to determine the two alternative pathways. The idea that a Na(+) binding site can be created at the Schiff base counterion is supported by the finding that upon perturbation of this region in the D251E mutant, Na(+) binds without photoexcitation. Binding of Na(+) to the mutant shifts the chromophore maximum to the red like that of H(+), which occurs in the photocycle of the wild type.

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Related in: MedlinePlus

Light minusdark FTIR spectra of photostationary states at 270K: spectrum 1, in the absenceof NaCl (<1 μM); spectrum 2, in 150 mM NaCl (pH8.6). Theethylenic stretch, the fingerprint, and the HOOP regions of spectrain the infrared and the corresponding spectra in the visible rangeof the same samples (not shown) indicate that the intermediate trappedis like the O state of bacteriorhodopsin. The Na+ dependenceof the C=O stretch region is discussed in the text.
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fig6: Light minusdark FTIR spectra of photostationary states at 270K: spectrum 1, in the absenceof NaCl (<1 μM); spectrum 2, in 150 mM NaCl (pH8.6). Theethylenic stretch, the fingerprint, and the HOOP regions of spectrain the infrared and the corresponding spectra in the visible rangeof the same samples (not shown) indicate that the intermediate trappedis like the O state of bacteriorhodopsin. The Na+ dependenceof the C=O stretch region is discussed in the text.

Mentions: FTIR spectrameasured under photostationary conditions at 270 K (Figures 6) revealed that the phototrapped state(s) are O-like.The ethylenic stretch frequency at 1523 cm–1 indicatesthat this state is red-shifted by ∼76 nm (from the ethylenicto λmax correlation), consistent with the stronglyred-shifted O state observed in the visible range. The HOOP band at955 cm–1 and the pattern in the fingerprint region(1150–1270 cm–1) indicate that the chromophoreis in a distorted all-trans conformation like theO state of bacteriorhodopsin. Shifts in the amide I (1630–1680cm–1) and amide II (at 1552 cm–1) bands, comparable to those in the N state of BR,50,51 indicate strong conformational perturbation of the protein.


Light-driven Na(+) pump from Gillisia limnaea: a high-affinity Na(+) binding site is formed transiently in the photocycle.

Balashov SP, Imasheva ES, Dioumaev AK, Wang JM, Jung KH, Lanyi JK - Biochemistry (2014)

Light minusdark FTIR spectra of photostationary states at 270K: spectrum 1, in the absenceof NaCl (<1 μM); spectrum 2, in 150 mM NaCl (pH8.6). Theethylenic stretch, the fingerprint, and the HOOP regions of spectrain the infrared and the corresponding spectra in the visible rangeof the same samples (not shown) indicate that the intermediate trappedis like the O state of bacteriorhodopsin. The Na+ dependenceof the C=O stretch region is discussed in the text.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263435&req=5

fig6: Light minusdark FTIR spectra of photostationary states at 270K: spectrum 1, in the absenceof NaCl (<1 μM); spectrum 2, in 150 mM NaCl (pH8.6). Theethylenic stretch, the fingerprint, and the HOOP regions of spectrain the infrared and the corresponding spectra in the visible rangeof the same samples (not shown) indicate that the intermediate trappedis like the O state of bacteriorhodopsin. The Na+ dependenceof the C=O stretch region is discussed in the text.
Mentions: FTIR spectrameasured under photostationary conditions at 270 K (Figures 6) revealed that the phototrapped state(s) are O-like.The ethylenic stretch frequency at 1523 cm–1 indicatesthat this state is red-shifted by ∼76 nm (from the ethylenicto λmax correlation), consistent with the stronglyred-shifted O state observed in the visible range. The HOOP band at955 cm–1 and the pattern in the fingerprint region(1150–1270 cm–1) indicate that the chromophoreis in a distorted all-trans conformation like theO state of bacteriorhodopsin. Shifts in the amide I (1630–1680cm–1) and amide II (at 1552 cm–1) bands, comparable to those in the N state of BR,50,51 indicate strong conformational perturbation of the protein.

Bottom Line: However, very low concentrations of Na(+) cause profound differences in the decay and rise time of photocycle intermediates, consistent with a switch from a "Na(+)-independent" to a "Na(+)-dependent" photocycle (or photocycle branch) at ∼60 μM Na(+).A greater concentration of Na(+) is needed for switching the reaction path at lower pH.Binding of Na(+) to the mutant shifts the chromophore maximum to the red like that of H(+), which occurs in the photocycle of the wild type.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, University of California , Irvine, California 92697, United States.

ABSTRACT
A group of microbial retinal proteins most closely related to the proton pump xanthorhodopsin has a novel sequence motif and a novel function. Instead of, or in addition to, proton transport, they perform light-driven sodium ion transport, as reported for one representative of this group (KR2) from Krokinobacter. In this paper, we examine a similar protein, GLR from Gillisia limnaea, expressed in Escherichia coli, which shares some properties with KR2 but transports only Na(+). The absorption spectrum of GLR is insensitive to Na(+) at concentrations of ≤3 M. However, very low concentrations of Na(+) cause profound differences in the decay and rise time of photocycle intermediates, consistent with a switch from a "Na(+)-independent" to a "Na(+)-dependent" photocycle (or photocycle branch) at ∼60 μM Na(+). The rates of photocycle steps in the latter, but not the former, are linearly dependent on Na(+) concentration. This suggests that a high-affinity Na(+) binding site is created transiently after photoexcitation, and entry of Na(+) from the bulk to this site redirects the course of events in the remainder of the cycle. A greater concentration of Na(+) is needed for switching the reaction path at lower pH. The data suggest therefore competition between H(+) and Na(+) to determine the two alternative pathways. The idea that a Na(+) binding site can be created at the Schiff base counterion is supported by the finding that upon perturbation of this region in the D251E mutant, Na(+) binds without photoexcitation. Binding of Na(+) to the mutant shifts the chromophore maximum to the red like that of H(+), which occurs in the photocycle of the wild type.

Show MeSH
Related in: MedlinePlus