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.

Hydrophobicity of protein surfaces: Separating geometry from chemistry

“…4a indicates approximately the region of cavitation between the dimers. The size of this region is comparable to that of nonpolar protein cavities found in experiments (36,37). Fig.…”

Capturing Invisible Motions in the Transition from Ground to Rare Excited States of T4 Lysozyme L99A

“…We attribute this discrepancy to variability in methods of measuring cavities in proteins (50). The volume reduction seen in the Anton trajectory aligns with the previously described rationale for using high pressure to shift protein conformational equilibria to alternative packing of the hydrophobic core by filling void volumes (51), and has been seen experimentally in L99A for F114 or water filling the void volume (16)(17)(18)52).…”

“…Remarkably, there is a high degree of cavity plasticity, exceeding what might be expected from previous crystallographic studies (18,19), that involves residues, metastates, and conformations beyond the discrete crystallographic states observed for L99A bound to a congeneric series of benzene-related compounds (24,30). Further, the number of rotamers observed for buried L99A residues exceeds that seen in the several-hundred-microsecond simulations of other protein cores, such as ubiquitin, RNaseH, and b-lactamase (56).…”

“…L99A is a model system for studying ligand binding to buried protein cavities and protein excited states, and the conformational changes that govern these phenomena have been enigmatic despite nearly 25 years of experimental study (5,6,(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30). Experimental studies of the L99A mutant of T4 lysozyme have focused on defining the structures of and transition times between the ground state, the excited state, and ligand-bound states.…”

Role of cavities and hydration in the pressure unfolding of T4lysozyme

“…1). Crystallographic investigation found no electron density within this pocket at ambient pressure (22,23). In contrast, solution NMR and molecular-dynamics simulations suggest that the region of the protein around the hydrophobic pocket is highly dynamic, possibly to the extent that the pocket may be transiently accessible to solvent (22,(24)(25)(26)(27).…”