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. 2012 May 1;109(18):6945-50.
doi: 10.1073/pnas.1200915109. Epub 2012 Apr 10.

Cavities determine the pressure unfolding of proteins

Julien Roche  1 , Jose A Caro, Douglas R Norberto, Philippe Barthe, Christian Roumestand, Jamie L Schlessman, Angel E Garcia, Bertrand E García-Moreno, Catherine A Royer
Affiliations

Cavities determine the pressure unfolding of proteins

Julien Roche et al. Proc Natl Acad Sci U S A. .

Abstract

It has been known for nearly 100 years that pressure unfolds proteins, yet the physical basis of this effect is not understood. Unfolding by pressure implies that the molar volume of the unfolded state of a protein is smaller than that of the folded state. This decrease in volume has been proposed to arise from differences between the density of bulk water and water associated with the protein, from pressure-dependent changes in the structure of bulk water, from the loss of internal cavities in the folded states of proteins, or from some combination of these three factors. Here, using 10 cavity-containing variants of staphylococcal nuclease, we demonstrate that pressure unfolds proteins primarily as a result of cavities that are present in the folded state and absent in the unfolded one. High-pressure NMR spectroscopy and simulations constrained by the NMR data were used to describe structural and energetic details of the folding landscape of staphylococcal nuclease that are usually inaccessible with existing experimental approaches using harsher denaturants. Besides solving a 100-year-old conundrum concerning the detailed structural origins of pressure unfolding of proteins, these studies illustrate the promise of pressure perturbation as a unique tool for examining the roles of packing, conformational fluctuations, and water penetration as determinants of solution properties of proteins, and for detecting folding intermediates and other structural details of protein-folding landscapes that are invisible to standard experimental approaches.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Structures of Δ + PHS SNase and cavity-containing variants. Left Panel, from Top to Bottom: Structure of Δ + PHS SNase (3BDC) with the Cα positions of the 10 cavity-containing variants indicated with red spheres and with the surface representation of the central cavity in purple. Cavity volume was calculated using a 1.1 Å sphere and McVol (11). Structures of the cavity mutants L125A (3NXW), I92A (3MEH) and V66A (3NQT) with the engineered cavities in green and the mutated residue in red. Right Panel: Estimation of the void density (Top) and hydration density (Bottom) for each Cα position of the Δ + PHS reference protein. See SI Materials and Methods in SI Appendix and (9) for the details concerning calculation of void and hydration density. Briefly, the 1,000 configurations resulting from a 10 ns all-atom MD simulation in explicit solvent were submitted to Monte Carlo point oversampling. All points that fell within the structure and not on an atom of solvent or the protein were counted for void density. Water density was calculated as the number of oxygen atoms of water molecules within 5 Å of each Cα carbon, normalized to the largest number found for a Cα carbon.
Fig. 2.
Fig. 2.
Pressure unfolding monitored with Trp fluorescence. (A) High-pressure fluorescence average emission wavelength profile for (Left Panel) Δ + PHS variant at 2.0 M (circle), 2.3 M (triangle) and 2.6 M (square) GuHCl; and (Right Panel) I92A cavity mutant at 0.8 M (circle), 1.0 M (triangle) and 1.2 M (square) GuHCl. (B) Folding volume change, ΔVf( = -ΔVu) values obtained from analysis of the high-pressure fluorescence unfolding profiles for Δ + PHS and 10 cavity-containing variants assuming a two-state model. The dashed line indicates the value for the Δ + PHS protein used as reference. Measurements were performed in the presence of guanidinium chloride (GuHCl) to ensure complete unfolding in the pressure range of the instrumentation used (< 3 kbar).
Fig. 3.
Fig. 3.
Pressure unfolding monitored with NMR spectroscopy. (Top): Examples of HSQC cross-peak intensity profiles for four representative residues of the Δ + PHS variant at 1.5 M GuHCl (Left), I92A at 0.85 M GuHCl (Middle), and L125A at 0.85 M GuHCl (Right). (Middle Panel): Distributions of the ΔVf( = -ΔVu) values obtained from high-pressure NMR spectroscopy experiments for Δ + PHS (Left), I92A (Middle), and L125A (Right). (Lower Panel): Structural mapping of the ΔVf values obtained from high-pressure NMR experiments for Δ + PHS (Left), I92A (Middle), and L125A (Right). Residues of the first quartile (the 25% lowest values) are in green; residues of the third quartile (25% highest values) are in red; and residues lying between the first and the third quartile (50% of the values) are in blue.
Fig. 4.
Fig. 4.
Fractional contact maps. (A–C) the Δ + PHS variant at 1.5 M GuHCl and 400 bar (A), 600 bar (B), and 800 bar (C); and (D) L125A at 0.85 M GuHCl and 700 bar. The complete set of native contacts is represented by dark dots on the bottom half of the contact maps. The probabilities of contact, calculated as the product of the cross-peak fractional intensities of the implied pair of residues, are indicated by color dots on the upper half of the contact maps. The color scale is dark blue > 90% probability of contact; light blue, 80–90%; green, 70–80%; yellow, 60–70%; orange, 50–60%; and red < 50%. The heterogeneity of contact for the probabilities is most apparent for the Δ + PHS variant at 800 bar (C). It can be seen that although contacts in the core region remain dark or light blue in color (high probability of contact), those in the C terminus and the region connecting the C terminus to the core have shifted to green, yellow, red, and orange (much lower contact probability). In C and D, the fractional contacts for 1.5 M GuHCl at 800 bar for Δ + PHS and for the L125A variant at 0.85 M and 600 bar can be compared. Even if the experiments were not performed at the same concentration of GuHCl, precluding any direct correspondence of the pressures, these two conditions were found to be appropriate for comparison in terms of fractional contacts. It can be seen that compared to the contact probabilities for Δ + PHS, which are very heterogeneous, those for the L125A variant are more homogeneous, remaining mostly dark or light blue, with only a few green residues.
Fig. 5.
Fig. 5.
Go-model calculations. (A) Pseudo free-energy profiles calculated from structure and NMR-based Go-model simulations of the Δ + PHS variant at 1 bar (blue), 400 bar (purple), 600 bar (pink), and 800 bar (red). (B) Representative conformations extracted from simulations with Δ + PHS SNase at 800 bar with (from Top to Bottom) fraction of native contacts, Q, Q = 0.55, Q = 0.83 and Q = 0.92. (C) Comparison of the pseudo free-energy profiles obtained from simulations of Δ + PHS at 800 bar (red) and L125A at 700 bar (green). Pressures shown are below the unfolding midpoint.
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Comment in

  • Proteins under pressure.
    Matthews BW. Matthews BW. Proc Natl Acad Sci U S A. 2012 May 1;109(18):6792-3. doi: 10.1073/pnas.1204795109. Epub 2012 Apr 23. Proc Natl Acad Sci U S A. 2012. PMID: 22529364 Free PMC article. No abstract available.

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