Structural rigidity of a large cavity-containing protein revealed by high-pressure crystallography

Abstract

Steric constraints, charged interactions and many other forces important to protein structure and function can be explored by mutagenic experiments. Research of this kind has led to a wealth of knowledge about what stabilizes proteins in their folded states. To gain a more complete picture requires that we perturb these structures in a continuous manner, something mutagenesis cannot achieve. With high pressure crystallographic methods it is now possible to explore the detailed properties of proteins while continuously varying thermodynamic parameters. Here, we detail the structural response of the cavity-containing mutant L99A of T4 lysozyme, as well as its pseudo wild-type (WT*) counterpart, to hydrostatic pressure. Surprisingly, the cavity has almost no effect on the pressure response: virtually the same changes are observed in WT* as in L99A under pressure. The cavity is most rigid, while other regions deform substantially. This implies that while some residues may increase the thermodynamic stability of a protein, they may also be structurally irrelevant. As recently shown, the cavity fills with water at pressures above 100 MPa while retaining its overall size. The resultant picture of the protein is one in which conformationally fluctuating side groups provide a liquid-like environment, but which also contribute to the rigidity of the peptide backbone.

Figure 1

Displacement of L99A α-carbon atoms from the ambient structure in Å, at 100, 150, and 200 MPa, as obtained by aligning residues in the C terminal domain. Bars indicate positions of α-helices.

Figure 2

Displacement of the N-terminal domain. The arrow labelled “P” indicates the direction of pressure-induced displacement of the N-terminal domain. Red lines indicate the three principal axes of inertia of the ambient pressure L99A structure. The ambient pressure N-terminal domain is shown in dark blue, and the 200 MPa displacements are magnified by 5 and shown in orange. The remainder of the protein is shown in light blue, with the cavity slightly below and to the right of the beta-sheet in the N-terminal domain as viewed in this figure.

Figure 3

Displacements in helix C. The view is along helix E, with the N-terminal domain shown here behind helix C. The ambient pressure structure is shown in a cartoon view colored blue. Helices C and D are shown with their 2 kbar displacements magnified 5 times in orange. Arrow indicates position of Leu66, on the back side of the C-helix in this view. The cavity is shown in light blue for reference. While residues before Leu66 displace very little, those beyond Leu66 have progressively larger displacements under pressure, clearly visible as a “kink” in the helix near Leu66.

Figure 4

Displacements of the C, D and H helices. This view is opposite that in Figure 3; colors are as in Figure 3. Helices C and D are shown at the top of this figure, labelled by their respective letters. The arrow labelled “H” indicates the C-terminal end of helix H, which displaces slightly towards the cavity (shown in light blue.)

Figure 5

Comparison of WT* and L99A response to pressure. Bars and letters at bottom indicate positions of helices. Solid trace: displacements in L99A at 200 MPa; dashed trace: displacements in WT* at 200 MPa.