The informational structure of horse cytochrome <i>c</i> and its mutant forms: Result of the analyzis of the amino acid sequence using the ANIS method.

<p>(A) The hierarchically organized highest rank ELIS (continuous lines) and the fragments of the bipartite graph that cannot be revealed using the ANIS method (dashed line). X axis is the size of the smoothing interval a/2 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178280#pone.0178280.ref021" target="_blank">21</a>], Y axis is the number N of amino acid in the primary structure of horse cytochrome <i>c</i>; (B), (C), (D) The hierarchically organized highest rank ELIS in mutant forms T78S/K79P, I81Y/A83Y/G84N, T78N/K79Y/M80I/I81M/F82N, respectively; (E) The spatial structure of horse cytochrome <i>c</i> (1HRC.PDB). The highest rank ELIS in the spatial structure of cytochrome c are shown. His18 and Met 80 residues coordinated to the Fe atom are indicated. The ADD- site (P76-A83) with the abnormally low density of first rank ELIS is shown by the arrows.</p

The intensity ratios of selected peaks in the SERS spectra of oxidized wild-type cytochrome <i>c</i> and its mutants.

<p>Data are shown as mean values ± SE. *p<0.05 compared to wild-type cytochrome <i>c</i> (the non-parametric Kruskal-Wallis test with Dunn's multiple comparison test).</p

The structure of the oligonucleotides (direct primers) used to introduce mutations into a sequence of horse cytochrome <i>c</i>.

<p>The structure of the oligonucleotides (direct primers) used to introduce mutations into a sequence of horse cytochrome <i>c</i>.</p

Resonance Raman study of reduced cytochromes.

<p>The RRS spectra of the studied cytochromes in the reduced state: WT—wild type; M1—T78N/K79Y/M80I/I81M/F82N, M2—T78S/K79P, M3—I81Y/A83Y/G84N. For clearer presentation, the spectra are shifted in vertical position. X axis is a frequency shift, cm^-1 and Y axis is RRS intensity, a.u.</p

The intensity ratios of selected peaks in the RRS spectra of the reduced wild-type cytochrome <i>c</i> and its mutants.

<p>Data are shown as mean values ± SE. *p<0.05 compared to wild-type cytochrome <i>c</i> (non-parametric Kruskal-Wallis test with Dunn's multiple comparison test).</p

SERS study of oxidized cytochromes.

<p><b>(A)</b> The SERS spectra of the studied cytochromes in the oxidized state: WT—wild type; M1—T78N/K79Y/M80I/I81M/F82N, M2—T78S/K79P, M3—I81Y/A83Y/G84N. For clearer presentation, the spectra are shifted in vertical position. Numbers above peaks indicate positions of their maxima used in the analysis. X axis is a frequency shift, cm^-1; Y axis is SERS intensity, a.u. (B) Structural formula of heme <i>c</i> with numeration of C atoms.</p

Comparison of the kinetic parameters of the succinate:cytochrome <i>c</i> reductase and cytochrome <i>c</i> oxidase reactions in rat liver mitoplasts in the presence of the externally added WT or mutant cytochromes <i>c</i>.

<p>The ratios of the maximum reaction rates in the presence of mutant forms of cytochrome <i>c</i> to the reaction rate in the presence of a WT protein <i>A</i>max(mut)/<i>A</i>max(wt) are also calculated.</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