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

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 112; no. 19; pp. E2437 - E2446
• Main Authors: Lerch, Michael T., López, Carlos J., Yang, Zhongyu, Kreitman, Margaux J., Horwitz, Joseph, Hubbell, Wayne L.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 12.05.2015; National Acad Sciences
• Series: PNAS Plus
• Subjects: Bacteriophage T4 - enzymology; Biological Sciences; Circular Dichroism; Electron Spin Resonance Spectroscopy; Electrons; energy; High pressure; Hydration; Hydrogen-Ion Concentration; Hydrostatic Pressure; Immune system; Ligands; lysozyme; Magnetic Resonance Spectroscopy; Models, Molecular; Muramidase - chemistry; Muramidase - genetics; Mutagenesis, Site-Directed; mutants; Mutation; PNAS Plus; Pressure; Protein Denaturation; Protein Folding; Protein Structure, Secondary; Proteins; Solvents; Structure-Activity Relationship; Temperature; Thermodynamics; USA; DEER; conformational exchange; protein structural dynamics; EPR
• ISSN: 0027-8424; 1091-6490; 1091-6490
• DOI: 10.1073/pnas.1506505112

Significance High pressure has emerged as a powerful tool for exploring the energy landscape of proteins, but structural origins of the pressure response remain controversial. The results of this study on a cavity mutant of T4 lysozyme (L99A) provide direct evidence for a structure-relaxation mechanism wherein pressure shifts conformational equilibria toward states with alternative packing arrangements that fill cavities or voids in the core. Both structure relaxation and cavity hydration can occur in response to pressure, and which dominates is found to depend on details of the energy landscape. The results also address conflicting views regarding the pressure response of L99A that have recently been published. 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 [Lóópez 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 electron–electron 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.