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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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pH dependence of transient absorptionchanges of GLR in the presenceof 100 mM NaCl at selected wavelengths: (A) 410 nm and (B) 590 and510 nm. Numerals 1–4 correspond to pH 8.3, 6.1, 5.1, and 3.5,respectively.
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fig7: pH dependence of transient absorptionchanges of GLR in the presenceof 100 mM NaCl at selected wavelengths: (A) 410 nm and (B) 590 and510 nm. Numerals 1–4 correspond to pH 8.3, 6.1, 5.1, and 3.5,respectively.

Mentions: Between pH 7.0 and 8.5,no significant pH dependence in the photocycle reactions is seen inthe presence of 100 mM NaCl. However, at lower pH, the amplitudesof transient changes at all three characteristic wavelengths thatreflect M rise and decay (410 nm), N/O rise and decay (590 nm), anddepletion of the initial state (510 nm) decrease (Figure 7). This decrease correlates with a decrease in thefraction of initial GLR with a deprotonated counterion, as followsfrom the titration curve in Figure 8B. At pH6.1 and especially pH 5.1, M decay is slowed in the same way as whenthe concentration of Na+ is lowered and the long-livedO intermediate from the “Na+-independent”cycle appears, providing evidence of the competition of H+ and Na+ in diverting the photocycle into the “Na+-independent” and “Na+-dependent”pathways, respectively. At pH 3.5, where the counterion is expectedto be nearly fully protonated (see below), both M and O are absent,as in bacteriorhodopsin at acidic pH.


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)

pH dependence of transient absorptionchanges of GLR in the presenceof 100 mM NaCl at selected wavelengths: (A) 410 nm and (B) 590 and510 nm. Numerals 1–4 correspond to pH 8.3, 6.1, 5.1, and 3.5,respectively.
© Copyright Policy
Related In: Results  -  Collection

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

fig7: pH dependence of transient absorptionchanges of GLR in the presenceof 100 mM NaCl at selected wavelengths: (A) 410 nm and (B) 590 and510 nm. Numerals 1–4 correspond to pH 8.3, 6.1, 5.1, and 3.5,respectively.
Mentions: Between pH 7.0 and 8.5,no significant pH dependence in the photocycle reactions is seen inthe presence of 100 mM NaCl. However, at lower pH, the amplitudesof transient changes at all three characteristic wavelengths thatreflect M rise and decay (410 nm), N/O rise and decay (590 nm), anddepletion of the initial state (510 nm) decrease (Figure 7). This decrease correlates with a decrease in thefraction of initial GLR with a deprotonated counterion, as followsfrom the titration curve in Figure 8B. At pH6.1 and especially pH 5.1, M decay is slowed in the same way as whenthe concentration of Na+ is lowered and the long-livedO intermediate from the “Na+-independent”cycle appears, providing evidence of the competition of H+ and Na+ in diverting the photocycle into the “Na+-independent” and “Na+-dependent”pathways, respectively. At pH 3.5, where the counterion is expectedto be nearly fully protonated (see below), both M and O are absent,as in bacteriorhodopsin at acidic pH.

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