Role of cavities and hydration in the pressure unfolding of T₄lysozyme

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 T 4 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 T 4 lysozyme molecule and illuminate the fact that small changes in physical parameters can significantly alter the pressure sensitivity of proteins.protein stability | protein folding and cooperativity | protein hydration | high-pressure NMR | reverse micelle encapsulation

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

“…Finally, the structural details presented here are consistent with a growing body of data implicating activated volumes and mobile defects of internal cavities in protein conformational change (16,17,51). It is possible that mobile defects may not be unique to protein transitions related to excited states, and that fluctuations in small, buried volumes could contribute to conformational change in many protein mechanisms.…”

“…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).…”

“…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.…”

Structure-relaxation mechanism for the response of T4 lysozyme cavity mutants to hydrostatic pressure

“…The pressure dependence of the L99A mutant was recently studied with NMR methods by Nucci et al (27) and independently by Maeno et al (31). In the study of Maeno et al (31), the disappearance of cross-peaks in a 1 H-13 C heteronuclear singlequantum coherence (HSQC) NMR experiment at pressures up to 3 kbar was interpreted to reflect an increase in population of the E conformation with Phe114 occupying the cavity, consistent with a structure-relaxation mechanism rather than cavity hydration or unfolding.…”

“…The current view is that the volume reduction that accompanies pressure-modulated transitions in proteins, including formation of the denatured state, is dominated by the elimination of voids or cavities in the protein's interior (14, 21-24) via hydration, although other factors contribute (15,(25)(26)(27)(28). In the equilibrium between two folded conformations, G ↔ E for example, an alternative "structure-relaxation" mechanism may play a role in the pressure response.…”