Concentration dependence of the inflex point of the CSM curves.

<p>The data consist of two independent experimental series (blue and red circles). Linear-regression line of the slope of 76.3 MPa and intercept of 45 MPa is shown which is used to determine the constants <i>K</i>_{<i>d</i>,<i>atm</i>} = 0.92 <i>μM</i> and Δ<i>V</i>_<i>r</i> = 32.5 <i>ml mol</i>⁻¹. Regression lines of the individual series are not shown as they are almost identical; their slopes and intercepts are 77.3 MPa and 43 MPa, respectively (blue), and 74.7 MPa and 47 MPa, respectively (red), which corresponds with <i>K</i>_{<i>d</i>,<i>atm</i>} = 0.95 <i>μM</i>, Δ<i>V</i>_<i>r</i> = 32.1 <i>ml mol</i>⁻¹ (red) and <i>K</i>_{<i>d</i>,<i>atm</i>} = 0.88 <i>μM</i>, Δ<i>V</i>_<i>r</i> = 33.2 <i>ml mol</i>⁻¹ (blue). The error bars of the individual measurements are calculated from the errors of the regression parameters of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119099#pone.0119099.e028" target="_blank">Eq. 19</a> using <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119099#pone.0119099.e029" target="_blank">Eq. 20</a> and the error-transition law. [<i>M</i>₀] is considered to be a dimensionless quantity related to the units of μM ([<i>M</i>₀] → [<i>M</i>₀]/<i>μM</i>).</p

Initial rate of substrate cleavage as a function of pressure related to the value for 10 MPa.

<p>Different symbols denote three experimental series. The solid curve shows the relative rate for the ideal case of pressure dependent <i>K</i>_<i>d</i> but pressure independent <i>K</i>_<i>m</i> and <i>k</i>_<i>cat</i>.</p

Model of the HIV-1 PR backbone in front (left) and side (right) view (structure taken from RSCB protein data bank, PDB ID: 1OHR).

<p>The residues of fluorescent amino acids, Trp6, Trp42 and Tyr59, are indicated. Trp6 is located close to the dimerization interface, therefore its spectroscopic properties are probably influenced by dimer dissociation. On the contrary, Trp42 and Tyr59 are located at the most distant site from the dimerization interface, therefore their fluorescence is probably unaffected by dimer dissociation, provided that the monomer keeps its conformation. Trp42 and Tyr59 can eventually partially influence this effect due to the change of their mutual configuration because the conformation of this part of the molecule undergoes partial changes with growing pressure, as was shown by the molecular-dynamics simulation [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119099#pone.0119099.ref032" target="_blank">32</a>]. All the fluorophores can contribute to the spectral changes induced by unfolding of monomers or protein aggregation when they undergo more extensive changes of their environment.</p

Centre of spectral mass of the emission spectra as a function of pressure for different HIV-1 protease concentrations.

<p>The curves are shifted up or down by artificial additive constants for the sake of lucidity. Solid lines express the fitting curves, empty triangles indicate the inflex points. Dashed lines represent the 95% confidence bands of the regression curves. The high pressure regions of the curves for 12 and 25 μM dimer concentration are excluded from fitting as they are influenced by protein aggregation. The smaller circles with black bordering line indicate the CSM values for the subsequent release of pressure from the highest to the lowest value.</p

Reversibility assay of HIV-1 PR of 5 μM concentration (expressed as dimer).

<p>For every enzyme sample three CSM values were determined: at 10 MPa before pressurizing, at the selected target pressure from the interval of 100 to 350 MPa after 50 min incubation and again at 10 MPa after releasing the pressure. The experiment indicates good reversibility of CSM up to 300 MPa, above this value the reversibility is perturbed by protein aggregation.</p

Time development of the tryptophan-fluorescence CSM.

<p>A. Time series for different pressures, each started with a new enzyme sample, concentration of 10 μM dimer. The individual curves are shifted up or down by artificially chosen constant for the sake of better orientation in the graph. B. Time series measured with the same sample of 2 μM concentration, pressure setting is facilitated by pressure jumps. The CSM scale is genuine for all the series.</p

Fluorescence-intensity indicated transition for 5 μM dimer.

<p>A. Time dependence of the intensity for different pressures. Each series is plotted together with the fitting single-exponential decay curve. The same sample was used for the whole set of measurements, pressure setting was facilitated by pressure jumps. The numbers indicate pressure in MPa. B. Equilibrium values of fluorescence for the same experimental series determined from the limit of the fitting curves for time tending to infinity. Inflex point is indicated by the open triangle.</p

Individual rates of folding and unfolding of the protease monomers and the observed relaxation rate for the concentration of 5 μM dimer.

<p>Both rates are fitted by linear functions. The crossing point roughly corresponds with the inflex point of the unfolding transition. Rate constants are considered as dimensionless quantities related to the units of h⁻¹.</p

Equilibrium properties of monomer unfolding indicated by intensity of the tryptophan fluorescence spectrum.

<p>Equilibrium properties of monomer unfolding indicated by intensity of the tryptophan fluorescence spectrum.</p

Equilibrium values of CSM of ANS emission spectrum for 10 μM dimer fitted by a model function.

<p>Values for the reverse course of pressure indicate irreversibility of the transition.</p