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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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Propertiesof the D251E mutant in the initial state. (A) Shiftof the absorption spectrum from an increase in pH at low salt concentrations(1–3 mM KCl): (1) pH 5.6, (2) pH 9.0, and (3) 10.6. (B) DifferentpH dependence of the absorption maximum of the D251E mutant (curves1–3) and the WT (curves 4 and 5): (1) 100 mM KCl, (2) 100 mMNaCl, (3) 10 mM NaCl, (4) 100 mM NaCl, and (5) 100 mM KCl. (C) Absorptionchanges produced by (1) binding of H+ with a decrease inpH from 10.4 to 9.7 in 3 mM KCl, (2) addition of 10 mM NaCl to 3 mMKCl at pH 9.7, and (3) subsequent addition of 20 mM KCl (note thatthe latter change is the opposite of the others). (D) Red shift ofthe absorption maximum of the D251E mutant produced by the additionof NaCl at pH 10.3 (in the presence of 3 mM KCl). Such a shift doesnot occur in the WT under the same conditions.
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fig9: Propertiesof the D251E mutant in the initial state. (A) Shiftof the absorption spectrum from an increase in pH at low salt concentrations(1–3 mM KCl): (1) pH 5.6, (2) pH 9.0, and (3) 10.6. (B) DifferentpH dependence of the absorption maximum of the D251E mutant (curves1–3) and the WT (curves 4 and 5): (1) 100 mM KCl, (2) 100 mMNaCl, (3) 10 mM NaCl, (4) 100 mM NaCl, and (5) 100 mM KCl. (C) Absorptionchanges produced by (1) binding of H+ with a decrease inpH from 10.4 to 9.7 in 3 mM KCl, (2) addition of 10 mM NaCl to 3 mMKCl at pH 9.7, and (3) subsequent addition of 20 mM KCl (note thatthe latter change is the opposite of the others). (D) Red shift ofthe absorption maximum of the D251E mutant produced by the additionof NaCl at pH 10.3 (in the presence of 3 mM KCl). Such a shift doesnot occur in the WT under the same conditions.

Mentions: This mutant at neutral pH exhibitsa red-shifted maximum compared to that of the wild type, 562 nm (Figure 9A, spectrum 1), which implies that the counterionis protonated. Indeed, at higher pH, the absorption band undergoesa 28 nm blue shift to 534 nm (with the titration largely completeat pH 10.6), indicating deprotonation of the counterion (Figure 9A, spectrum 3). In 100 mM KCl, the pKa of this transition is 8.8 (Figure 9B, curve 1). In contrast, in 100 mM NaCl, the blue shift at highpH is much smaller, only 7 nm (Figure 9B, curve2). Titration in 10 mM NaCl is accompanied by a larger shift (Figure 9B, curve 3), but smaller than in 100 mM KCl. Inthe wild type, the pH dependence was similar in KCl and NaCl exceptfor a small (∼2 nm) blue shift in 100 mM KCl (Figures 8B and 9B). The differenteffects of KCl and NaCl are further illustrated by the absorptionchanges in panels C and D of Figure 9. Additionof 10 mM NaCl to a sample with a deprotonated counterion at pH 10.3and a low ionic strength (3 mM KCl) causes absorption changes virtuallyidentical to those that are caused by an increase in proton concentration(Figure 9C). Via the addition of more NaCl,the blue shift caused by the increase in pH from 7 to 10.3 can belargely reversed by Na+ as shown in Figure 9D. The apparent binding constant for Na+ at thispH is ∼2 mM. It appears that Na+ ions can substitutefor protons and bind to the counterion in the initial state of theD251E mutant, causing the red shift of the absorption spectrum.


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)

Propertiesof the D251E mutant in the initial state. (A) Shiftof the absorption spectrum from an increase in pH at low salt concentrations(1–3 mM KCl): (1) pH 5.6, (2) pH 9.0, and (3) 10.6. (B) DifferentpH dependence of the absorption maximum of the D251E mutant (curves1–3) and the WT (curves 4 and 5): (1) 100 mM KCl, (2) 100 mMNaCl, (3) 10 mM NaCl, (4) 100 mM NaCl, and (5) 100 mM KCl. (C) Absorptionchanges produced by (1) binding of H+ with a decrease inpH from 10.4 to 9.7 in 3 mM KCl, (2) addition of 10 mM NaCl to 3 mMKCl at pH 9.7, and (3) subsequent addition of 20 mM KCl (note thatthe latter change is the opposite of the others). (D) Red shift ofthe absorption maximum of the D251E mutant produced by the additionof NaCl at pH 10.3 (in the presence of 3 mM KCl). Such a shift doesnot occur in the WT under the same conditions.
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Related In: Results  -  Collection

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fig9: Propertiesof the D251E mutant in the initial state. (A) Shiftof the absorption spectrum from an increase in pH at low salt concentrations(1–3 mM KCl): (1) pH 5.6, (2) pH 9.0, and (3) 10.6. (B) DifferentpH dependence of the absorption maximum of the D251E mutant (curves1–3) and the WT (curves 4 and 5): (1) 100 mM KCl, (2) 100 mMNaCl, (3) 10 mM NaCl, (4) 100 mM NaCl, and (5) 100 mM KCl. (C) Absorptionchanges produced by (1) binding of H+ with a decrease inpH from 10.4 to 9.7 in 3 mM KCl, (2) addition of 10 mM NaCl to 3 mMKCl at pH 9.7, and (3) subsequent addition of 20 mM KCl (note thatthe latter change is the opposite of the others). (D) Red shift ofthe absorption maximum of the D251E mutant produced by the additionof NaCl at pH 10.3 (in the presence of 3 mM KCl). Such a shift doesnot occur in the WT under the same conditions.
Mentions: This mutant at neutral pH exhibitsa red-shifted maximum compared to that of the wild type, 562 nm (Figure 9A, spectrum 1), which implies that the counterionis protonated. Indeed, at higher pH, the absorption band undergoesa 28 nm blue shift to 534 nm (with the titration largely completeat pH 10.6), indicating deprotonation of the counterion (Figure 9A, spectrum 3). In 100 mM KCl, the pKa of this transition is 8.8 (Figure 9B, curve 1). In contrast, in 100 mM NaCl, the blue shift at highpH is much smaller, only 7 nm (Figure 9B, curve2). Titration in 10 mM NaCl is accompanied by a larger shift (Figure 9B, curve 3), but smaller than in 100 mM KCl. Inthe wild type, the pH dependence was similar in KCl and NaCl exceptfor a small (∼2 nm) blue shift in 100 mM KCl (Figures 8B and 9B). The differenteffects of KCl and NaCl are further illustrated by the absorptionchanges in panels C and D of Figure 9. Additionof 10 mM NaCl to a sample with a deprotonated counterion at pH 10.3and a low ionic strength (3 mM KCl) causes absorption changes virtuallyidentical to those that are caused by an increase in proton concentration(Figure 9C). Via the addition of more NaCl,the blue shift caused by the increase in pH from 7 to 10.3 can belargely reversed by Na+ as shown in Figure 9D. The apparent binding constant for Na+ at thispH is ∼2 mM. It appears that Na+ ions can substitutefor protons and bind to the counterion in the initial state of theD251E mutant, causing the red shift of the absorption spectrum.

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