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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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Flash-induced absorptionchanges of GLR at pH 8.5 in 10 mM KClat several characteristic wavelengths, 410, 510, 550, and 590 nm.The concentration of NaCl is <1 μM. The traces were globallyfit with six kinetic components with time constants shown at the top.
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fig2: Flash-induced absorptionchanges of GLR at pH 8.5 in 10 mM KClat several characteristic wavelengths, 410, 510, 550, and 590 nm.The concentration of NaCl is <1 μM. The traces were globallyfit with six kinetic components with time constants shown at the top.

Mentions: At lowconcentrations of Na+ (≤1 μM), theturnover of the GLR photocycle is very slow (Figure 2): the absorption changes produced by flash illumination relaxfully after ∼8 s. The first detected intermediate is a red-shiftedphotoproduct, which in analogy with BR and other retinal proteinscan be called K. Its relaxation back to the initial state involvesat least six or seven distinctive transitions. In <6 μs,a fraction of K is replaced by a blue-shifted, M-like intermediate(see the increase in absorbance at 410 nm and the simultaneous decreasein absorbance at 550 and 590 nm). The absorption maximum of this stateis at or below 400 nm, which suggests that the retinal Schiff baseis deprotonated. It accumulates in only small amounts, as in othereubacterial rhodopsins.31,38,43 In the subsequent transition, with a time constant of ∼60μs, an intermediate absorbing at ∼470 nm is formed (X470). This state and M may be in equilibrium. In KR2, an equilibriumbetween L and M had been suggested,17 butwe find that the intermediate in question arises after M, rather thanbefore as L would. In the following steps, these two blue-shiftedintermediates decay to a red-shifted species that we tentatively identifyas an N-like state [see the increase in absorbance at 550 and 590nm with time constants at 2.7 and 274 ms, the latter coincident withthe major component in M decay (see the 410 nm trace)]. The last twotransitions involve a strongly red-shifted intermediate (O-like) thatarises in 1 s and decays with a time constant of 2.6 s (Figure 2, 590 nm trace), which determines the rate of recoveryof the initial state (Figure 2, 510 nm trace).As shown below, this intermediate contains a reisomerized but distortedall-trans-retinal, as O of BR. The formation of theM-like intermediate and its decay to the N intermediate slow in D2O by ∼2.5- and ∼8-fold, respectively, consistentwith deprotonation and reprotonation of the Schiff base during thesesteps, and the movement of protons limiting the rates.


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)

Flash-induced absorptionchanges of GLR at pH 8.5 in 10 mM KClat several characteristic wavelengths, 410, 510, 550, and 590 nm.The concentration of NaCl is <1 μM. The traces were globallyfit with six kinetic components with time constants shown at the top.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Flash-induced absorptionchanges of GLR at pH 8.5 in 10 mM KClat several characteristic wavelengths, 410, 510, 550, and 590 nm.The concentration of NaCl is <1 μM. The traces were globallyfit with six kinetic components with time constants shown at the top.
Mentions: At lowconcentrations of Na+ (≤1 μM), theturnover of the GLR photocycle is very slow (Figure 2): the absorption changes produced by flash illumination relaxfully after ∼8 s. The first detected intermediate is a red-shiftedphotoproduct, which in analogy with BR and other retinal proteinscan be called K. Its relaxation back to the initial state involvesat least six or seven distinctive transitions. In <6 μs,a fraction of K is replaced by a blue-shifted, M-like intermediate(see the increase in absorbance at 410 nm and the simultaneous decreasein absorbance at 550 and 590 nm). The absorption maximum of this stateis at or below 400 nm, which suggests that the retinal Schiff baseis deprotonated. It accumulates in only small amounts, as in othereubacterial rhodopsins.31,38,43 In the subsequent transition, with a time constant of ∼60μs, an intermediate absorbing at ∼470 nm is formed (X470). This state and M may be in equilibrium. In KR2, an equilibriumbetween L and M had been suggested,17 butwe find that the intermediate in question arises after M, rather thanbefore as L would. In the following steps, these two blue-shiftedintermediates decay to a red-shifted species that we tentatively identifyas an N-like state [see the increase in absorbance at 550 and 590nm with time constants at 2.7 and 274 ms, the latter coincident withthe major component in M decay (see the 410 nm trace)]. The last twotransitions involve a strongly red-shifted intermediate (O-like) thatarises in 1 s and decays with a time constant of 2.6 s (Figure 2, 590 nm trace), which determines the rate of recoveryof the initial state (Figure 2, 510 nm trace).As shown below, this intermediate contains a reisomerized but distortedall-trans-retinal, as O of BR. The formation of theM-like intermediate and its decay to the N intermediate slow in D2O by ∼2.5- and ∼8-fold, respectively, consistentwith deprotonation and reprotonation of the Schiff base during thesesteps, and the movement of protons limiting the rates.

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