Intrinsic Fluorescence of OBP.

<p>Intrinsic fluorescence emission spectra of native dimer OBP in the absence (dashed trace) and in the presence of 1.5 M GdnHCl (solid line) at 0.1 MPa.</p

Successive pressurization cycles of OBP binding protein in the presence of substrate.

<p>A) The protein fluorescence intensity at 338 nm is shown for increasing (filled symbols) and decreasing (empty symbols) pressures in a first (circles) and a second (squares) pressurization cycle. B) Pressure-jump induced relaxation kinetics. The p-jumps were from 185 to 350 MPa. The upper and lower traces reflect the kinetics in the first (close circles) and second pressurization cycle (open circle), respectively. C) Pressure-dependence of <i>k</i>_obs in the first and second pressurization cycles. The rate constants were determined by fitting the kinetic traces of 40 MPa downward pressure jumps to mono-exponential decays for the first (open circle) and the second (open square) cycles. D) Pressure dependence of the individual rate constants k_f (filled symbols) and k_u (empty symbols) of relaxation kinetics induced by downward pressure-jumps in the first (circles) and second (diamonds) pressurization cycles.</p

DSSP analysis made on the representative structures for each cluster of the three MD simulations.

a<p>DSSP codes H+G+I.</p>b<p>DSSP code E.</p>c<p>DSSP codes B+S+T.</p>d<p>structures not identified by DSSP code.</p

Analysis of the distances between the center of mass of the two subunits during the simulation time.

<p>Variation of the distances of the center of mass of the two subunits of bOBP during the time, for simulations at 0.1 MPa (black), 250 MPa (blue) and 600 MPa (orange).</p

Effect of the binding of the natural substrate 1-octen-3-ol on the OBP stability under pressure.

<p>Spectral transition curves were obtained by plotting the fluorescence center of spectral mass (panel A) and the maximum intensity of protein fluorescence emission (panel B) as a function of pressure. OBP proteins (dimer: circle, monomer: triangle) in 1.5 M GdnHCl, 20 mM Tris buffer pH 7.4 were incubated in the absence (•,▾) or in the presence (○,▿) of 1-octen-3-ol for 1 hour at 25°C before application of pressure. The best fit was made according to a two-step transition and the corresponding values are reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050489#pone-0050489-t001" target="_blank">Table 1</a>.</p

Structural stability of dimer OBP after pressure treatment analyzed by size exclusion chromatography.

<p>Chromatograms of the dimer protein OBP in the absence of substrate before (solid line) and after (dashed line) pressure treatment up to 600 MPa and 250 MPa. The dash-dot-dot line represents the relative contribution of the monomer state of OBP assuming a Gaussian profile of elution for each oligomeric state of the protein. (<i>Inset</i>) The quantification of the monomer species was evaluated using bovine serum albumin (67 kDa), ovalbumin (36.9 kDa), chymotrypsinogen (23 kDa) and ribonuclease A (13.7 kDa) as standard proteins (filled circles). From the calculated partition coefficient K_AV, molecular masses of 34.8 kDa and 18.97 kDa were determined for the initial dimer form (open square) and the pressure-induced dissociated form (open diamond), respectively.</p

Protein fluorescence characterization in the presence of ANS.

<p>Panel A: ANS fluorescence emission spectra of dimer (full line) and monomer (dashed line) bOBP in the absence of substrate at 0.1 MPa. In the absence of proteins, the ANS probe exhibits only low fluorescence (dotted line). Panel B: ANS fluorescence intensity at 450 nm of the dimer OBP protein as a function of pressure in the absence (filled symbols) or in the presence (empty symbols) of 1 mM 1-octen-3-ol, respectively.</p

Thermodynamic parameters calculated from pressure unfolding curves of OBP at 25°C in the presence of 1.5 M GdnHCl.

<p>Thermodynamic parameters calculated from pressure unfolding curves of OBP at 25°C in the presence of 1.5 M GdnHCl.</p