Cooperative water filling of a nonpolar protein cavity observed by high-pressure crystallography and simulation

Abstract

Formation of a water-expelling nonpolar core is the paradigm of protein folding and stability. Although experiment largely confirms this picture, water buried in "hydrophobic" cavities is required for the function of some proteins. Hydration of the protein core has also been suggested as the mechanism of pressure-induced unfolding. We therefore are led to ask whether even the most nonpolar protein core is truly hydrophobic (i.e., water-repelling). To answer this question we probed the hydration of an approximately 160-A(3), highly hydrophobic cavity created by mutation in T4 lysozyme by using high-pressure crystallography and molecular dynamics simulation. We show that application of modest pressure causes approximately four water molecules to enter the cavity while the protein itself remains essentially unchanged. The highly cooperative filling is primarily due to a small change in bulk water activity, which implies that changing solvent conditions or, equivalently, cavity polarity can dramatically affect interior hydration of proteins and thereby influence both protein activity and folding.

Fig. 1.

Electron density in the main cavity of T4 lysozyme mutant L99A at high pressure. Helix E is shown behind a cut-away view of the ≈160-ų cavity. (A) Experimental density at 100 MPa (yellow), 150 MPa (cyan), and 200 MPa (magenta) is contoured at 0.1 electrons per ų. (B) Experimental electron density at 150 MPa (cyan) compared with simulation density at 200 MPa (magenta), contoured at 0.1 electrons per ų, viewed as described above. The distribution of atoms at 100 MPa (using the occupancies of N = 1, 2, 3, 4, 5 at 200 MPa) is shown in yellow for comparison. [The figures were made with pymol (http://pymol.sourceforge.net).]

Fig. 2.

Average number of water molecules inside the cavity as a function of pressure. Solid circles show the results from x-ray crystallography, dividing the integrated electron density by 10 electrons per water molecule. The open squares show the average occupancies calculated from the MD simulations. The solid line is the result of perturbation theory about the ≈200-MPa simulation reference state. Shifting the pressure by 53 MPa (or ≈0.4 k_BT) produces the dashed line.

Fig. 3.

Probability distribution (logarithmic scale) of the number N of water molecules in the cavity from computer simulations. Symbols show results from MD simulations at 0.1, 100, and 200 MPa. Lines are the results of perturbation theory using the 200-MPa simulations as a reference point. Error bars indicate statistical uncertainties corresponding to one estimated standard deviation.