Role of cavities and hydration in the pressure unfolding of T4 lysozyme

Abstract

It is well known that high hydrostatic pressures can induce the unfolding of proteins. The physical underpinnings of this phenomenon have been investigated extensively but remain controversial. Changes in solvation energetics have been commonly proposed as a driving force for pressure-induced unfolding. Recently, the elimination of void volumes in the native folded state has been argued to be the principal determinant. Here we use the cavity-containing L99A mutant of T4 lysozyme to examine the pressure-induced destabilization of this multidomain protein by using solution NMR spectroscopy. The cavity-containing C-terminal domain completely unfolds at moderate pressures, whereas the N-terminal domain remains largely structured to pressures as high as 2.5 kbar. The sensitivity to pressure is suppressed by the binding of benzene to the hydrophobic cavity. These results contrast to the pseudo-WT protein, which has a residual cavity volume very similar to that of the L99A-benzene complex but shows extensive subglobal reorganizations with pressure. Encapsulation of the L99A mutant in the aqueous nanoscale core of a reverse micelle is used to examine the hydration of the hydrophobic cavity. The confined space effect of encapsulation suppresses the pressure-induced unfolding transition and allows observation of the filling of the cavity with water at elevated pressures. This indicates that hydration of the hydrophobic cavity is more energetically unfavorable than global unfolding. Overall, these observations point to a range of cooperativity and energetics within the T4 lysozyme molecule and illuminate the fact that small changes in physical parameters can significantly alter the pressure sensitivity of proteins.

Keywords: high-pressure NMR; protein folding and cooperativity; protein hydration; protein stability; reverse micelle encapsulation.

Fig. 1.

Hydration of T₄ lysozyme L99A at ambient pressure (∼1 bar). A backbone ribbon representation of L99A [Protein Data Bank (PDB) ID code 1L90 (63)] is shown with the N-terminal domain (residues 13–65) illustrated in blue, and the C-terminal domain (residues 1–12 and 66–164) is colored green. The hydrophobic pocket created by the L99A mutation is shown as orange mesh, and the three tryptophan side chains are shown as stick representations. The helices are numbered as a reference for discussion in the text. Cyan spheres are shown at the positions of amide hydrogens where an NOE to the water resonance was detected. Yellow spheres indicate the positions of amide hydrogens within NOE distance (5 Å) of the interior of the hydrophobic pocket, but outside NOE distance to the protein surface. These are the sites where detection of NOEs to the water resonance would indicate hydration of the pocket. No NOE cross-peaks from these sites to the water resonance were observed, suggesting that the pocket is not hydrated at ambient pressure.

Fig. 2.

The intensities of cross-peaks in the ¹⁵N HSQC spectra shown in Figs. S2 and S3 were fitted to a two-state pressure unfolding model to extract thermodynamic parameters of the unfolding process. The P_u is mapped to the backbone structures of (A) T₄ lysozyme L99A [PDB ID code 1L90 (63)], (B) L99A with benzene bound [PDB ID code 1L84 (34)], and (C) WT* [PDB ID code 1L63 (64)] with sites linearly color coded as indicated at bottom right. To further illustrate these data, the thickness of the backbone cartoon is scaled with the color code such that the thickest sites are those that unfold at the lowest pressures. Sites at which data could not be quantitatively analyzed are colored gray and are shown at the minimal thickness.

Fig. 3.

Hydration of T₄ lysozyme L99A at elevated pressure. (Upper) Slices of ¹⁵N-resolved NOESY-HSQC spectra of reverse micelle-encapsulated L99A at the ¹H resonance of water (4.875 ppm). Cross-peaks centered at this resonance are labeled according to the amide hydrogen resonance of the residue from which they arise. Unlabeled cross-peaks are those centered at planes above and below the water resonance. Planes are shown at 1 bar (red), 1 kbar (purple), and 2 kbar (blue). At 1 kbar, an NOE is seen from the amide hydrogen of residue 101 to the water resonance. This cross-peak increases in intensity at 2 kbar and is joined by the appearance of an NOE cross-peak between the amide hydrogen of residue 100 and the water resonance. (Lower) The structure of L99A is shown with the ribbon and pocket illustrated as in Fig. 1. Sites where a cross-peak to the water resonance is observed at 1 bar are again shown in cyan. Approximately half of the protein–water cross-peaks at these sites are broadened into the noise as pressure is increased as a result of slower macromolecular tumbling caused by increased viscosity of the alkane solvent. Residues 100 and 101 are highlighted in magenta. These are the only sites where new protein–water cross-peaks are observed as pressure is increased, providing conclusive evidence of water entry into the hydrophobic pocket under solution conditions.