Distances of Cl ion from particular residues, Glu/Gln194, Glu/Gln204 and Arg82, during 100 ns MD simulations for each of nine bR molecules.

<p>Colors indicate distance ranges: light gray < = 0.5 nm (binding area), dark gray < = 0.8 nm (proximity area), and red > 0.8 nm (escape).</p

X ray structure of extracellular side of ground state bacteriorhodopsin, Protein Data Bank entry 1C3W [12,50].

<p>Added and optimized are hydrogen atoms, presenting the continuous hydrogen bonding network, connecting proton release group with protein active site. Arg82 connects with Glu194 through water molecules W403 and W404 (green), while the side chains of Glu194 and Glu204 form a direct hydrogen bond. In addition, Arg82 connects to Asp85 through Trp 402, Asp212, Tyr57 and Trp407.</p

Light minus dark (LA–DA) absorption difference spectra of 2Glu and 3Glu at pH 6.0, room temperature.

<p>(A) LA–DA difference spectra of 2Glu in 150 mM KCl (the dotted line corresponds to 0.02 M KCl). (B) LA–DA difference spectra of 3Glu in 150 mM KCl. (C) LA–DA difference spectra of 2Glu (green line) and 3Glu (red line) in 1.5 M KCl. For comparison the difference spectrum of WT (black line) is also plotted. The difference spectra of 4Glu (not shown) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162952#pone.0162952.ref026" target="_blank">26</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162952#pone.0162952.ref035" target="_blank">35</a>], recorded in the presence of low and high concentrations of KCl are identical to those of 3Glu.</p

Identification of Specific Effect of Chloride on the Spectral Properties and Structural Stability of Multiple Extracellular Glutamic Acid Mutants of Bacteriorhodopsin

<div><p>In the present work we combine spectroscopic, DSC and computational approaches to examine the multiple extracellular Glu mutants E204Q/E194Q, E204Q/E194Q/E9Q and E204Q/E194Q/E9Q/E74Q of bacteriorhodopsin by varying solvent ionic strength and composition. Absorption spectroscopy data reveal that the absorption maxima of multiple EC Glu mutants can be tuned by the chloride concentration in the solution. Visible Circular dichroism spectra imply that the specific binding of Cl^- can modulate weakened exciton chromophore coupling and reestablish wild type-like bilobe spectral features of the mutants. The DSC data display reappearance of the reversible thermal transition, higher T_m of denaturation and an increase in the enthalpy of unfolding of the mutants in 1 M KCl solutions. Molecular dynamics simulations indicate high affinity binding of Cl^- to Arg82 and to Gln204 and Gln194 residues in the mutants. Analysis of the experimental data suggests that simultaneous elimination of the negatively charged side chain of Glu194 and Glu204 is the major cause for mutants’ alterations. Specific Cl^- binding efficiently coordinates distorted hydrogen bonding interactions of the EC region and reconstitutes the conformation and structure stability of mutated bR in WT-like fashion.</p></div

Calorimetric parameters from thermal unfolding of WT, 2Glu and 3Glu, measured in water and 1 M KCl, at pH 7.0 by DSC.

<p>Calorimetric parameters from thermal unfolding of WT, 2Glu and 3Glu, measured in water and 1 M KCl, at pH 7.0 by DSC.</p

Positions of chloride ion close to the proton release group in MD simulations.

<p>Starting points and summary positions for two different locations are shown in columns. Starting positions are exemplified in WT structure (top row). For subsequent rows: in green summary locations of Cl^- ion (from 9 monomers of bR) in WT, 2Glu and 3Glu during 100 ns MD simulation.</p

Circular dichroism (CD) experiments in the visible spectral region.

<p>(A) Vis-CD spectra of WT (■), E194Q () and 2Glu () in water, pH 7.0. The spectra of E9Q, E74Q and E204Q single mutants (not shown) are identical with those of E194Q. The CD spectra of WT and of all single mutants reveal the well-known biphasic bands (535/600 nm) of the retinal chromophore, while the multiple mutants show only one band (at about 468 nm). (B) Representative Vis–CD spectra of 3Glu in the presence of increasing (by 0.2 M) concentrations of KCl (pH 6.0). Arrows indicate the direction of spectral changes with KCl increases. Upon addition of KCl, the monophasic spectrum is converted to a biphasic, indicative for conformational changes in the retinal binding pocket.</p

Effects of the ionic strength and the composition of the media on the UV/ VIS absorption spectra of multiple EC Glu mutants.

<p>(A) Representative absorption spectra of 3Glu mutant in KCl concentrations ranging from 0 to 2 M, pH 6.0, 25°C. The arrow indicates the direction of absorption maximum changes with increasing salt concentration. The isosbestic point at 517 nm indicates that a transition between only two spectral species, purple and red, is taking place. (B) Difference spectra of 2Glu and 3Glu, calculated by subtraction of an absorption spectrum, recorded in 1 M KCl minus a spectrum in water, pH 6.0, 25°C. The difference spectrum of 4 Glu (not shown) shows similar spectral features [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162952#pone.0162952.ref035" target="_blank">35</a>]. (C) Absorption spectra of 3Glu in 1 M KI, 1 M LaCl₃ and 1 M Na₂SO₄ at pH 6.0, 25°C. (D) Plots of the absorption maxima (λ_max) of dark-adapted samples of 2Glu and 3Glu vs KCl concentration. Dotted lines represent the best nonlinear curve fits using the Hill1 equation (OriginLab software) yielding the apparent dissociation constants.</p

Thermal unfolding experiments.

<p>DSC data.Differential scanning calorimetric traces for WT (left panel) and for 2Glu (right panel) recorded in water (black) and in 1 M KCl (colored line) at pH 7.0. a) First scan up to 65°C; b) Second scan of the same sample after cooling down the sample. Scans were taken at scanning rate of 1 K·min^-1. The DSC data were corrected for the instrumental and chemical base lines and analyzed as explained in Materials and Methods.</p

Thermal stability of inter-trimer chromophore interactions CD data.

<p>(A) Temperature-dependent Visible–CD spectra of WT in water, pH 6.5. (B) Representative spectra of 3Glu in 1 M KCl, pH 6.5. (C) Representative spectra of 3Glu in water, pH 6.5. (D) Plots of the ratio of the ellipticity at any temperature to that at 20°C, measured at 530 nm for WT(), 3Glu () and 2Glu () in 1 M KCl and at 460 nm for 3Glu () in water, pH 6.5. The dotted lines represent the best fit of the experimental data, applying non-linear fit with single sigmoid Boltzmann function for 2Glu and 3Glu. The curve for WT shows bi-phase temperature dependence and was fitted to a sum of two sigmoid functions, applying the Boltzmann equation.</p