The pH-denaturation of HPr^bs.

<p>Changes in ellipticity at 222 nm (left side, blank circles) and in ANS-binding at 480 nm (right side, blue filled circles) upon pH variation are shown. Experiments were carried out at 25°C with protein concentration of 15 μM in both techniques.</p

Spectroscopic features of HPr^bs at physiological pH.

<p>(A) Far-UV CD spectrum of HPr^bs at physiological pH; protein concentration was 15 μM. (B) 1D-¹H-NMR spectrum of HPr^bs in the amide (left side) and methyl (right side) regions. Protein concentration was 50 μM. Experiments were carried at 25°C.</p

Thermodynamic parameters for the binding reactions as determined by ITC^a_.

a<p>All titrations were conducted in Mes and Mops buffers 10 mM (pH 7.0) at 25°C. ^b<i>K</i>_D is the dissociation constant; ^c<i>K</i>_D, for HPr^bs dilution, is the self-dissociation constant. Typical relative errors are 15% for <i>K</i>_D, 5% for Δ<i>G</i>⁰ and 10% for Δ<i>H</i>⁰, −TΔ<i>S</i> and <i>n</i>_H. Experiments were performed in duplicate. ^dΔ<i>H</i>⁰ is the buffer independent enthalpy for the binding reaction; its value was determined by conducting experiments in Mes and Mops buffers, 10 mM (pH 7.0) at 25°C, and by using Eq 5. ^eΔ<i>G</i>⁰ is the binding free energy at 25°C, determined as Δ<i>G</i>⁰  =  RT ln <i>K</i>_D. ^f−TΔ<i>S</i>⁰ is the value of the entropic contribution of the binding reaction at 25°C, determined as −<i>T</i>Δ<i>S</i>⁰  =  Δ<i>G</i>⁰ – Δ<i>H</i>⁰. ^g<i>n</i>_H is the number of exchanged protons, determined by conducting experiments in Mes and Mops buffers, 10 mM (pH 7.0) at 25°C, by using the Eq. 5.</p

Aggregation propensity of two HPr species as predicted by Zyggregator.

<p>The plot shows the intrinsic aggregation propensity of HPr^bs (red, blank circles) and HPr^sc (blue, filled circles). Because of the way the scores are normalized, aggregation-prone regions have a score larger than one. A positive value of the score indicates an aggregation-prone region (especially when the value is larger than 1). The rectangles at the bottom indicate the α-helical regions, and the double-headed arrows the presence of β-strands.</p

Thermal and chemical denaturations of HPr^bs.

<p>(A) Thermal denaturations followed by the changes in ellipticity at 222 nm (left side, blank circles) and in the intrinsic fluorescence emission at 305 nm, after excitation at 278 nm (right side, filled blue circles). (B) Urea-denaturations followed by the changes in ellipticity at 222 nm (left side, blank circles) and in the intrinsic fluorescence emission at 305 nm (similar results were obtained at 315 and 330 nm), after excitation at 278 nm (right side, blue filled circles).</p

Determination of the affinity constants of the complexes by ITC.

<p>The fitting curves were obtained assuming a 1∶1 model. Binding curve for the titration between: (A) wild-type EIN^sc:HPr^bs; (B) EINH186D:HPr^bs; (C) EINbsite:HPr^bs; (D) EINosite:HPr^bs; (E) (EINbsite+EINosite):HPr^bs (ligands were at equimolar concentrations); and, (F) HPr^bs dilution. All the titrations were carried out at 25°C in Mes buffer, 10 mM (pH 7.0).</p

Molecular modelling of the EIN^sc:HPr^bs complex.

<p>The EIN^sc is shown in skin representation, color code: green, hydrophobic; red, hydrogen bond acceptor; blue, hydrogen bond donor. The HPr^bs protein is shown as a magenta ribbon, and key residues are explicitly displayed. Numbers without the prime symbol correspond to EIN^sc numbering, and numbers with the prime symbol are those from HPr^bs. The figure was obtained with ICM <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069307#pone.0069307-ICM1" target="_blank">[36]</a>.</p

Hydrodynamic features of HPr^bs.

<p>(A) The CONTIN analysis of the DLS experiment where the protein size distribution is shown as intensity. (B) Example of the AUC absorbance scans at 11 times of centrifugation, from 3447 s (top, in black) to 60556 s (bottom, in green), with the same time interval between scans. It is important to note the initial, baseline absorbance of only 0.08 units. On the second panel, the residuals of SEDFIT fitting for each time are shown; it is important to note that the residuals are large but random, without any anomalous systematic trend. (C) SEC profile, as monitored by the absorbance at 280 nm, of the main peak of HPr^bs in a Superdex 75 HR 10/30. Other peaks were observed at larger elution volumes (larger than 19 ml, the bed volume of the column, as it can be observed on the right side of the panel) probably due to protein-column interactions. The arrows indicate the position of the protein standards used in column calibration, from left to right: bovine serum albumin (9.31 ml), ovoalbumin (10.29 ml), chymotrypsinogen (12.33 ml) and ribonuclease A (13.14 ml). (D) Cross-linking experiments: Left lane: The BioRad marker with the indicated molecular weights. Right lane: the cross-linked HPr^bs.</p

Examples for the determination of radial magnification errors.

<p>(A) Radial intensity profile measured in scans of the precision mask. Blue lines are experimental scans, and shaded areas indicate the regions expected to be illuminated on the basis of the known mask geometry. In this example, the increasing difference between the edges corresponds to a calculated radial magnification error of -3.1%. (B—D) Examples for differences between the experimentally measured positions of the light/dark transitions (blue circles, arbitrarily aligned for absolute mask position) and the known edge distances of the mask. The solid lines indicate the linear or polynomial fit. (B) Approximately linear magnification error with a slope corresponding to an error of -0.04%. Also indicated as thin lines are the confidence intervals of the linear regression. (C) A bimodal shift pattern of left and right edges, likely resulting from out-of-focus location of the mask, with radial magnification error of -1.7%. (D) A non-linear distortion leading to a radial magnification error of -0.53% in the <i>s</i>-values from the analysis of back-transformed data. The thin grey lines in C and D indicate the best linear fit through all data points.</p

Examples of transient changes in the console temperature reading during the SV experiment, as saved in the scan file data.

<p>For comparison, the maximum adiabatic cooling of -0.3°C would be expected after approximately 300 sec, recovering to the equilibrium temperature after approximately 1,200 s (see Fig 3 in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126420#pone.0126420.ref033" target="_blank">33</a>]).</p