Dependence of diffusion coefficient (D) as a function of pH.

<p>The error bars of the measurements were obtained by using three different measurements. The histogram shows the aggregation percentage (axis on the right) as a function of pH values. The sphere and the two ellipsoids represent (in scale) the possible shapes of MalE2 (at pH 7.0, pH 4.0 and pH 10.0) compatible with the corresponding diffusion coefficients measured by FCS (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064840#pone-0064840-t001" target="_blank">Table 1</a> and Materials and Methods section).</p

Diffusion coefficients obtained by FCS measurements (Figure 2) and geometrical properties of MalE2 molecule (see Materials and Methods section for more details).

(1)<p>Errors on FCS measurements<b>.</b></p>(2)<p>Molecular radius (R_Sph calculated from the D₁ diffusion coefficient using <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064840#pone.0064840.e003" target="_blank">equation 3</a>).</p>(3)<p>Prolate ellipsoid semi-axes (a, b) and corresponding diffusion coefficient (DE) calculated using <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064840#pone.0064840.e004" target="_blank">Equation 4</a>, assuming the same spherical volume obtained at pH 7.0 (i.e. ≈ 1·10⁵ ų).</p

Fluorescence correlation spectroscopy measurements.

<p>Autocorrelation spectra and residual distribution (inset) of MalE2 at pH 7.0 (panel A), pH 4.0 (panel B) and pH 10.0 (panel C). The best fitting of the data was obtained by the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064840#pone.0064840.e001" target="_blank">Equation 1</a>.</p

Dynamic light scattering measurements.

<p>Particle size distribution as a function of pH of MalE2 at pH 2.0 (black), pH 4.0 (red), pH 7.0 (green) and pH 10.0 (blue). The measurements were performed at 25°C. The final concentration of protein was 0.1 mg/ml.</p

Analysis of RMSD of MalE2.

<p>Panel A : RMSD of MalE2 along the simulation, with respect to the starting structures at pH 4.0 (red), pH 7.0 (green) and pH 10.0 (blue). Panel B: RMS distribution of the structures in the clusters calculated along the three simulation at pH 4.0 (red), pH 7.0 (green) and pH 10.0 (blue).</p

Free energy landscape of MalE2.

<p>Projection of the trajectories of MalE2 at pH 4.0 (panel A), pH 7.0 (panel B) and pH 10.0 (panel C) in the essential plane defined by the two first eigenvectors calculated for the simulations. The probability is expressed as relative to the maximum frequency. The color scale (blue-red) defines the most probable conformation.</p

Model of the 3D structure of MalE2 in the un-liganded form.

<p>The backbone of the protein is represented as a ribbon, and segments of secondary structures are shown as cylinders (helices) and arrows (strands).</p

A Loose Domain Swapping Organization Confers a Remarkable Stability to the Dimeric Structure of the Arginine Binding Protein from <i>Thermotoga maritima</i>

<div><p>The arginine binding protein from <i>Thermatoga maritima</i> (TmArgBP), a substrate binding protein (SBP) involved in the ABC system of solute transport, presents a number of remarkable properties. These include an extraordinary stability to temperature and chemical denaturants and the tendency to form multimeric structures, an uncommon feature among SBPs involved in solute transport. Here we report a biophysical and structural characterization of the TmArgBP dimer. Our data indicate that the dimer of the protein is endowed with a remarkable stability since its full dissociation requires high temperature as well as SDS and urea at high concentrations. In order to elucidate the atomic level structural properties of this intriguing protein, we determined the crystallographic structures of the apo and the arginine-bound forms of TmArgBP using MAD and SAD methods, respectively. The comparison of the liganded and unliganded models demonstrates that TmArgBP tertiary structure undergoes a very large structural re-organization upon arginine binding. This transition follows the Venus Fly-trap mechanism, although the entity of the re-organization observed in TmArgBP is larger than that observed in homologous proteins. Intriguingly, TmArgBP dimerizes through the swapping of the C-terminal helix. This dimer is stabilized exclusively by the interactions established by the swapping helix. Therefore, the TmArgBP dimer combines a high level of stability and conformational freedom. The structure of the TmArgBP dimer represents an uncommon example of large tertiary structure variations amplified at quaternary structure level by domain swapping. Although the biological relevance of the dimer needs further assessments, molecular modelling suggests that the two TmArgBP subunits may simultaneously interact with two distinct ABC transporters. Moreover, the present protein structures provide some clues about the determinants of the extraordinary stability of the biomolecule. The availability of an accurate 3D model represents a powerful tool for the design of new TmArgBP suited for biotechnological applications.</p></div

Swapping dimer of HoloTmArgBP.

<p>(A) Cartoon representation of HoloTmArgBP domain-swapped dimer. (B) Omit (Fo-Fc) map of the hinge region contoured at 2σ.</p

Isothermal titration calorimetry experiments with (A) arginine and (B) glutamine.

<p>Top panels report raw data for the titrations at 25°C, whereas bottom panels report integrated heats of binding obtained from the raw data after subtracting the heats of dilution. The solid line (in A) represents the best curve fit to the experimental data using the ‘one set of sites’ model from MicroCal Origin.</p