C₁₂TPP enhances protonophorous effect of 2 nM FCCP (panel A) and 10 µM DNP (panel B) in bilayer lipid membrane (BLM).

<p>Diffusion potential was recorded upon the addition of KOH in one compartment to create ΔpH = 1. Incubation mixture, 10 mM Tris, 10 mM MES, 10 mM KCl, pH 7; C₁₂TPP, 0.1 µM. Control, a record without C₁₂TPP and uncouplers. Plus sign of the potential in the compartment of high pH.</p

Comparison of SkQ1 and C₁₂TPP effects on the uncoupling activity of DNP and FCCP.

<p>Effects of 2 µM SkQ1, 2 µM C₁₂TPP or 200 µM TPP on the dependence of mitochondrial membrane potential on the concentration of DNP (panel A) or FCCP (panel B). The experiments were conducted in a way shown in Fig. 6. Shown are Mean±S.E. of 4–6 experiments.</p

C₁₂TPP augments the FCCP- and DNP-induced decrease of mitochondrial membrane potential (ΔΨ) in intact yeast cells.

<p>Mitochondrial membrane potential in yeast was estimated by staining cells with the ΔΨ probe JC-1. <i>S.cerevisiae</i> cells were incubated with C₁₂TPP and/or FCCP (A), C₁₂TPP and/or DNP (B) and then loaded with 2 µM JC-1. (C) Levels of JC-1 fluorescence in yeast cells (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061902#s2" target="_blank">Materials and methods</a>). Data are presented as averages with standard errors. Samples were compared by Wilcoxon signed ranked unpaired test. Results of at least three experiments performed on separate days (number of cells, from 30 to 170). Bar, 5 µm.</p

Chemical structures of C₁₂TPP, SkQ1, SkQR4, C₁₂R4, FCCP, CCCP, DNP, and carboxyfluorescein.

<p>Chemical structures of C₁₂TPP, SkQ1, SkQR4, C₁₂R4, FCCP, CCCP, DNP, and carboxyfluorescein.</p

C₁₂TPP enlarges the FCCP- and DNP-induced increase in respiration rates of intact yeast cells.

<p>The ordinate represents the ratio of the oxygen consumption rates after (V) and before (V₀) the addition of an uncoupler. (A) FCCP-mediated stimulation of yeast respiration in the absence (solid line) and in the presence (dotted line) of 1 µM C₁₂TPP. (B) DNP stimulation of yeast respiration in the absence (solid line) and in the presence of 0.5 µM (dotted line) or 1 µM (dot and dash line) C₁₂TPP. In the absence of the anionic uncoupler, C₁₂TPP did not increase the rate of oxygen consumption at concentrations below 2 µM (insert). Shown are Mean±S.E. of 4 experiments.</p

C₁₂R4 (rhodamine B dodecyl ester) strongly increases CCCP- (panel A) or DNP-mediated (panel B) electric current through bilayer lipid membrane (BLM).

<p>Where indicated, 0.1 µM CCCP or 1 µM DNP were added to both compartments prior to the measurements; incubation mixture, 0.1 M KCl, 10 mM Tris, 10 mM MES, pH 7.0; at t = 1.5 s, 100 mV voltage was applied to the BLM. Concentration of C₁₂R4 was 0.1 µM.</p

CCCP increases the SkQ-induced efflux of carboxyfluorescein from liposomes.

<p>Carboxyfluorescein (CF) efflux from DPhPC (diphytanoylphosphatidylcholine) liposomes (50 µg/ml), induced by 2.5 µM SkQ1 (panel A) or by 0.5 µM SkQR1 (panel B) was measured with or without CCCP. The efflux was accompanied by an increase in CF fluorescence due to dilution and a relief of CF self-quenching. In panel B, the CF efflux was measured 400 s after the addition of SkQR1. Incubation mixture, 10 mM Tris, 10 mM MES, 100 mM KCl, pH 7.</p

SkQ1 (10-(6-plastoquinonyl)decyl triphenylphosphonium) affects absorption spectra of CCCP in the presence of liposomes.

<p>Incubation mixture: 4 µM CCCP, 1 mM Tris, 1 mM MES, pH = 7.4, containing DPhPC (diphytanoylphosphatidylcholine) liposomes (20 µg/ml) in the absence (curve 1) and in the presence of 1 µM, 2 µM, 4 µM and 9 µM SkQ1 (panel A, curves 2–5, respectively) or in the presence of 1 µM, 2 µM, 4 µM and 9 µM SkQR4 (panel B, curves 2–5, respectively). Insert to panel B shows the dependence of λ_max on the concentration of the SkQ derivatives.</p

SkQ1 and C₁₂TPP stimulate the uncoupling action of FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone) in rat liver mitochondria.

<p>Mitochondrial potential was assayed by measuring the absorbance of safranin O. 10 nM FCCP was added at 4, 5, 6, 7, 8, and 9 min (curves 2–4). 0.2% ethanol or ethanol solutions leading to 2 µM C₁₂TPP (curves 1 and 4) or 2 µM SkQ1 (curve 3) were added at 2 min. Oligomycin (1 µg/ml) was added at 1 min. For other conditions, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061902#s2" target="_blank">Materials and methods</a>.</p

Structural and Dynamic “Portraits” of Recombinant and Native Cytotoxin I from Naja oxiana: How Close Are They?

Today, recombinant proteins are quite widely used in biomedical and biotechnological applications. At the same time, the question about their full equivalence to the native analogues remains unanswered. To gain additional insight into this problem, intimate atomistic details of a relatively simple protein, small and structurally rigid recombinant cardiotoxin I (CTI) from cobra <i>Naja oxiana</i> venom, were characterized using nuclear magnetic resonance (NMR) spectroscopy and atomistic molecular dynamics (MD) simulations in water. Compared to the natural protein, it contains an additional Met residue at the N-terminus. In this work, the NMR-derived spatial structure of uniformly ¹³C- and ¹⁵N-labeled CTI and its dynamic behavior were investigated and subjected to comparative analysis with the corresponding data for the native toxin. The differences were found in dihedral angles of only a single residue, adjacent to the N-terminal methionine. Microsecond-long MD traces of the toxins reveal an increased flexibility in the residues spatially close to the N-Met. As the detected structural and dynamic changes of the two CTI models do not result in substantial differences in their cytotoxicities, we assume that the recombinant protein can be used for many purposes as a reasonable surrogate of the native one. In addition, we discuss general features of the spatial organization of cytotoxins, implied by the results of the current combined NMR and MD study