Effect of rSLURP-1 on the growth of Het-1A cells.

<p>(A). Amino acid sequence alignment of human SLURP-1, SLURP-2, ws-Lynx1, and non-conventional toxin WTX from <i>N</i>. <i>kaouthia</i>. Cysteine residues are labeled in gray, and the disulfide linkages are shown; additional <i>N</i>-terminal Met residues are underlined. (B). Influence of rSLURP-1 (diamonds) and ws-Lynx1 (squares) on cell growth. Each point is mean ± S.E. of six independent experiments. The Hill equation (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149733#pone.0149733.e001" target="_blank">Eq 1</a>) was fitted to rSLURP-1 data (% of control) for each of the six experiments independently. After averaging the following values for EC₅₀, nH and A1 were obtained 4.3 ± 0.6 nM, 1.4 ± 0.2 and 59.5 ± 1.3% (mean ± S.E., n = 6). (C). Effects of rSLURP-1 (1 μM), atropine (1 μM), Mec (10 μM), α-Bgtx (1 μM), polyclonal antibodies against α7 (IgG-α7, 1 μg per 50 μl), and their co-application. Each bar is mean ± S.E. of 4–6 independent experiments. ** and *** indicate significant (p<0.01 and p<0.001, t-test) differences.</p

Inhibition of ACh-evoked current at α7-nAChR expressed in <i>X</i>. <i>laevis</i> oocytes by rSLURP-1.

<p>(A). Electrophysiological recordings of α7 nAChR inhibition by 13 μM rSLURP-1. Currents were obtained in response to 20 seconds 100 μM ACh pulses (horizontal bars). Inhibition of current amplitude was observed after 5 min pre-incubation with 13 μM rSLURP-1. Vertical bar represents current scale (500 nA). (B). Dose-response curve of 1 mM acetylcholine-evoked current by rSLURP-1. Each point is mean ± S.E. of independent measurements on three oocytes. The Hill equation (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149733#pone.0149733.e001" target="_blank">Eq 1</a>) was fitted to normalized data (% of control) for each of the three experiments independently. After averaging the following values for IC₅₀, nH and A1 were obtained 1.1 ± 0.5 μM, 1.4 ± 0.3 and 31 ± 3% (mean ± S.E., n = 3). (C). Dependence of 5 μM rSLURP-1 effect at α7-nAChR on ACh concentration. Data of rSLURP-1 induced inhibition is shown by filled squares, 5 μM ws-Lynx1 effect on 1 mM ACh is shown by circle. Asterisks indicate significant (p<0.05, t-test) difference from receptor inhibition by rSLURP-1 at 1 μM ACh (6 on plot). (D). Dose-response curves of acetylcholine-evoked current amplitudes in the absence (dotted line, empty circles) and presence (solid line, filled circles) of 1 μM rSLURP-1. The calculated parameters EC₅₀ and nH were 232 ± 25 μM and 1.9 ± 0.3 in absence of rSLURP-1, and 214 ± 60 μM and 2.2 ± 0.3 in presence of rSLURP-1 (mean ± S.E., n = 3).</p

Competition of rSLURP-1 with ¹²⁵I-α-Bgtx for binding to Ls-AChBP, membrane-bound nAChR from <i>Torpedo californica</i>, and α7-nAChR in the GH₄C₁ cell line.

<p>(A). Each point is mean ± S.E. of three independent experiments. The Hill equation (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149733#pone.0149733.e001" target="_blank">Eq 1</a>, with A1 = 0%) was fitted to normalized Ls-AChBP and <i>T</i>. <i>californica</i> data (% of control binding) for each of the three experiments independently. After averaging the following values for IC₅₀ and nH were obtained 4.8 ± 0.9 μM and 0.92 ± 0.17 for Ls-AChBP, and 54 ± 15 μM and 1.3 ± 0.3 for <i>T</i>. <i>californica</i> nAChR (mean ± S.E., n = 3). Data for <i>T</i>. <i>californica</i> were taken from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149733#pone.0149733.ref023" target="_blank">23</a>]. (B). Displacement of ¹²⁵I-α-Bgtx by unlabeled Bgtx and rSLURP-1 from α7-nAChR in the GH₄C₁ cells. (C). Affinity purification of α7-nAChR subunit was performed using magnetic beads covalently coupled with rSLURP-1, rSLURP-2, and α-Bgtx or non-coupled beads (control) on GH₄C₁ cells overexpressing α7-nAChR (n = 2). The 40 μl samples of GH₄C₁ cells with final concentration of α-Bgtx-binding sites of 0.4 nM were used. The blots were analyzed by densitometry using ImageJ software (<a href="http://imagej.nih.gov/ij/" target="_blank">http://imagej.nih.gov/ij/</a>).</p

rSLURP-1 binds α7 nAChR subunits in the human brain.

<p>Affinity purification was performed using magnetic beads covalently coupled with rSLURP-1 or non-coupled beads (Ctrl) on human temporal cortical homogenates (n = 2). Samples were submitted to gel electrophoresis and Western blotting along with samples of the homogenate used for affinity purification (Input) and the remaining homogenate after affinity purification (Output) followed by detection of nAChR subunits.</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