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

Abstract

Application of hydrostatic pressure shifts protein conformational equilibria in a direction to reduce the volume of the system. A current view is that the volume reduction is dominated by elimination of voids or cavities in the protein interior via cavity hydration, although an alternative mechanism wherein cavities are filled with protein side chains resulting from a structure relaxation has been suggested [López CJ, Yang Z, Altenbach C, Hubbell WL (2013) Proc Natl Acad Sci USA 110(46):E4306-E4315]. In the present study, mechanisms for elimination of cavities under high pressure are investigated in the L99A cavity mutant of T4 lysozyme and derivatives thereof using site-directed spin labeling, pressure-resolved double electronelectron resonance, and high-pressure circular dichroism spectroscopy. In the L99A mutant, the ground state is in equilibrium with an excited state of only ∼3% of the population in which the cavity is filled by a protein side chain [Bouvignies et al. (2011) Nature 477(7362):111-114]. The results of the present study show that in L99A the native ground state is the dominant conformation to pressures of 3 kbar, with cavity hydration apparently taking place in the range of 2-3 kbar. However, in the presence of additional mutations that lower the free energy of the excited state, pressure strongly populates the excited state, thereby eliminating the cavity with a native side chain rather than solvent. Thus, both cavity hydration and structure relaxation are mechanisms for cavity elimination under pressure, and which is dominant is determined by details of the energy landscape.DEER | EPR | conformational exchange | protein structural dynamics P roteins in solution exist in conformational equilibria that cannot be appreciated from structures observed in crystal lattices (1-5). The members of a folded conformational ensemble may have distinct functions and hence are of interest in elucidating mechanisms of protein action (5-7). The free-energy differences between the conformations can range from zero to a few kilocalories per mole; the higher free-energy states are referred to as "invisible" or "excited" (E) states owing to their low equilibrium populations. The structural transition between the native ground state (G) and the E state may involve rigid body motion of the peptide backbone (5, 8) or local unfolding (9).For a complete understanding of molecular mechanisms underlying function, characterization of functionally relevant conformational substates is required. However, in the case of E states, low populations and short lifetimes present a challenge for biophysical characterization. The elegant high-resolution NMR studies from Akasaka (10, 11) and coworkers suggest that application of hydrostatic pressure on the order of a few kilobars may solve this problem by reversibly populating functional E states, making them amenable for study by spectroscopic methods. For example, high-pressure NMR has been used to identify and characterize E states crucial to ligand binding in ubiquitin (12) and d...

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.…”

Predicting Binding Free Energies: Frontiers and Benchmarks

“…Pressure may also cause the protein to populate an excited state (73, 61) (but see (129)) which is already present to a very limited extent at equilibrium (11). Still, as noted below, these issues do not seem to dramatically impact our ability to calculate binding free energies at standard temperature and pressure, probably in large part because these are effects which come into play only at high pressures (96, 66, 73), though as we discuss below, some ligands do induce a protein conformational change which affects the same helix as the proposed excited state (74). It seems likely that the conformational hetereogeneity observed experimentally will make lysozyme even more of a valuable benchmark system, as test cases here can range from simple to challenging, depending on the ligand and the pressure.…”

“…These binding sites do exhibit some surprising experimental complexities which make them interesting ongoing topics of study, such as the fact that the L99A site is empty of water when ligands are not bound (96, 66, 22), yet the protein can undergo pressure-induced filling (22, 66) or denaturation (96), which can be inhibited by binding of ligand (96, 66). Pressure may also cause the protein to populate an excited state (73, 61) (but see (129)) which is already present to a very limited extent at equilibrium (11).…”