Influence of (GlcNAc)_n and muropeptides on the 290-nm CD denaturation profile of Cpl-7.

<p>Denaturation parameters were determined in 20 mM Pi buffer pH 8.0. <i>T_m</i>, Δ<i>H</i> and <i>f</i> are defined as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046654#pone-0046654-t002" target="_blank">Table 2</a>; CM and CWBM superscripts indicate the module involved in the transition.</p>1<p> and values in the absence of ligands for this batch of protein were 44.0°C and 60°C, respectively.</p

Dependence on pH of thermal transitions under the DSC envelope of Cpl-7.

<p>Dependence on pH of thermal transitions under the DSC envelope of Cpl-7.</p

Dependence on pH of CM-Cpl-7 thermal denaturation parameters.

<p><i>T_mi</i> and Δ<i>H_i</i> are the half transition temperature and the enthalpy change for transition <i>i</i>, respectively and <i>f_i</i> is the relative contributions of transition <i>i</i> to the total ellipticity change at a given wavelength. Incertitude in fitted parameters are ≈0.2–1% for <i>T_mi</i>, 3–15% for Δ<i>H_i</i> and ±(0.1–0.2) for <i>f_i</i>.</p

Influence of pH on Cpl-7 DSC denaturation profiles.

<p>Panels (<b>A</b>) to (<b>D</b>) show the thermograms registered at 20°C h⁻¹ in 20 mM Pi buffer at the pH values indicated in the curve labels. Dotted lines depict the results of endotherm deconvolution in independent two-state transitions (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046654#pone-0046654-t003" target="_blank">Table 3</a>) and grey traces are the theoretical envelopes.</p

Influence of pH on Cpl-7 thermal stability.

<p>Panels (<b>A</b>) to (<b>D</b>) depict full-length protein denaturation profiles at 195 nm (circles), 203 nm (triangles), 208 nm (diamonds) and 290 nm (hexagons) at different pHs. Continuous lines are the theoretical curves calculated as indicated in the text using the fitting parameters shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046654#pone-0046654-t002" target="_blank">Table 2</a>. Measurements were carried out in 20 mM Pi buffer (pH 6.0, 6.5 and 8.0) and 25 mM sodium borate buffer (pH 8.5) at a scan rate of 20°C h⁻¹ (195, 203 and 208 nm) and 40°C h⁻¹ (290 nm).</p

Dependence of Cpl-7 CD spectra with temperature at neutral pH.

<p>(<b>A</b>) and (<b>B</b>) show the spectroscopic changes induced by the temperature increase in the far- and near-UV regions, respectively. Isodichroic points in the far-UV region are marked as “a” and “b”, and the T arrow indicates the temperature increase (20, 30, 40, 45, 50, 55, 60, 65, 70, 85 and 95°C). The near-UV spectra correspond to 20°C (black), 50°C (green), 55°C (blue), 65°C (red) and 95°C (magenta). (<b>C</b>) compares the thermal denaturation profiles of Cpl-7 at 195 nm (circles), 203 nm (triangles), 208 nm (diamonds) and 290 nm (hexagons). Measurements were performed in 20 mM Pi buffer, pH 7.0, at heating rates of 20°C h⁻¹ (solid symbols) and 40°C h⁻¹ (open symbols).</p

Denaturation parameters of Cpl-7 endolysin derived from CD thermal profiles at different pH values.

<p><i>T_mi</i>, Δ<i>H_i</i> and <i>f_i</i> are defined as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046654#pone-0046654-t001" target="_blank">Table 1</a>. The uncertainty in the fitting parameters was 0.2–1% in <i>T_mi</i>, 3–15% in Δ<i>H</i>_i, and ±(0.1–0.2) in <i>f_i</i>. ND, not determined.</p

CD spectra of the full-length Cpl-7 endolysin and its isolated catalytic (CM) module.

<p>(<b>A</b>) and (<b>B</b>) show the far- and near-UV CD spectra, respectively, of the full-length protein at 20°C (black circles), 55°C (triangles), 95°C (white circles). Spectra obtained under renaturing conditions (20°C) with protein samples heated up to 90°C (grey circles) and 55°C (grey squares) are also depicted. The insets compare the spectra of CPL-7 (black circles) and the isolated CM (white squares) with the ellipticities expressed per mole of molecule.</p

Influence of Cpl-7 potential ligands in the CM and CWBM structural stabilities.

<p>(<b>A</b>) shows the CD denaturation profiles of Cpl-7 at 290 nm in the absence (grey circles) and in the presence of 10 mM (GlcNAc)₆ or 10 mM MurNAc-</p><p>l</p>-Ala-<p>d</p>-isoGln (white triangles and grey diamonds, respectively). (<b>B</b>) depicts the denaturation profiles in the absence and in the presence of 17.3 mM GlcNAc-MurNAc-<p>l</p>-Ala-<p>d</p>-isoGln (grey and black circles, respectively) at the same wavelength and pH.<p></p

DNA Binding Induces a Nanomechanical Switch in the RRM1 Domain of TDP-43

Understanding the molecular mechanisms governing protein–nucleic acid interactions is fundamental to many nuclear processes. However, how nucleic acid binding affects the conformation and dynamics of the substrate protein remains poorly understood. Here we use a combination of single molecule force spectroscopy AFM and biochemical assays to show that the binding of TG-rich ssDNA triggers a mechanical switch in the RRM1 domain of TDP-43, toggling between an entropic spring devoid of mechanical stability and a shock absorber bound-form that resists unfolding forces of ∼40 pN. The fraction of mechanically resistant proteins correlates with an increasing length of the TG_<i>n</i> oligonucleotide, demonstrating that protein mechanical stability is a direct reporter of nucleic acid binding. Steered molecular dynamics simulations on related RNA oligonucleotides reveal that the increased mechanical stability fingerprinting the holo-form is likely to stem from a unique scenario whereby the nucleic acid acts as a “mechanical staple” that protects RRM1 from mechanical unfolding. Our approach highlights nucleic acid binding as an effective strategy to control protein nanomechanics