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. 2015 May 12;112(19):E2437-46.
doi: 10.1073/pnas.1506505112. Epub 2015 Apr 27.

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

Michael T Lerch  1 , Carlos J López  1 , Zhongyu Yang  1 , Margaux J Kreitman  1 , Joseph Horwitz  2 , Wayne L Hubbell  3
Affiliations

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

Michael T Lerch et al. Proc Natl Acad Sci U S A. .

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 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.

Keywords: DEER; EPR; conformational exchange; protein structural dynamics.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Distance mapping of the G and E conformations at atmospheric pressure and pH 5.5 with DEER spectroscopy. (A) An overlay in cylinder representation of the G (PDB ID code 3DMV) and E (PDB ID code 2LC9) (5) conformations of L99A in blue and magenta, respectively. Models of the R1 side chain are shown in stick representation; helix H is rendered in ribbon form to show the 150R1 side chain and its parent helix J. The direction of movement of helix F in the G→E structural transition is indicated by a red arrow. The dashed lines show the distances measured involving residue T109R1 with respect to an R1 reference for the G state (blue) and E state (magenta). (B) Ribbon diagram showing the interspin distances measured between the indicated reference sites in the G and E conformations. (C) DEFs, model-free fits of the DEFs (dashed yellow traces), and corresponding distance distributions for the indicated spin-labeled mutants in the WT* (black), L99A (gray), and L99A/G113A/R119P (blue) backgrounds in buffer consisting of 50 mM phosphate, 25 mM NaCl, and 20% (vol/vol) glycerol at pH 5.5. The DEFs and distance distributions after addition of benzene to mutants in the L99A/G113A/R119P background are shown in orange. The blue arrows identify distances only observed in the E conformation. The range of distances corresponding to the G and E conformations are indicated by brackets above the distributions.
Fig. 2.
Fig. 2.
CW EPR spectra of T109R1 in the indicated genetic background. Spectra were recorded in 30% (wt/wt) sucrose at pH 6.8.
Fig. 3.
Fig. 3.
High-pressure CD and CW EPR of T4L mutants. (A) High-pressure far-UV CD of L99A and (B) L99A/G113A/R119P. Both proteins contained spin labels D89R1/T109R1. CD Spectra were recorded in 5 mM MES and 2.5 mM NaCl at pH 5.5 for L99A and 10 mM MES and 25 mM NaCl at pH 6.8 for L99A/G113A/R119P at the indicated pressure. The typical protein concentration was ∼15 μM. (C) CW EPR spectra of the indicated protein at 0 and 2 kbar are shown in blue and red traces, respectively. For clarity, the low field lines in the spectra of D72R1/L99A and T109R1/L99A are amplified. Arrows identify a new component observed at 2 kbar. Spectra were recorded in 25% (wt/wt) Ficoll-70 at pH 5.5.
Fig. 4.
Fig. 4.
Effect of pressure on L99A monitored with DEER using spin pairs (A) T109R1/N140R1 and (B) D89R1/T109R1. DEFs and model-free fits (dashed yellow traces) (Left) and corresponding distance distributions (Right) are shown from 0 to 4 kbar. The DEFs and distance distributions are color-coded as indicated. The approximated distances corresponding to the G and E states are indicated with brackets above the distributions. PR DEER experiments were conducted in buffer consisting of 50 mM MES, 25 mM NaCl, and 20% (vol/vol) glycerol at pH 5.5. The red and black bars indicate the upper limit of reliable shape and distance of the distribution, respectively (Materials and Methods).
Fig. 5.
Fig. 5.
The effect of pressure on the G ↔ E equilibrium in L99A/G113A/R119P. PR DEER was used to map distance changes (A) between indicated reference sites and (B) between T109R1 and selected references to monitor the position of helix F. DEFs, model-free fits of the DEFs (dashed yellow traces), and corresponding distance distributions are shown at various pressures between 0 and 4 kbar. DEFs and distance distributions are color-coded as indicated. The black arrows identify populations in reference pairs increased by pressure. The approximate distance ranges corresponding to G and E are indicated by brackets above the distributions. PR DEER data were collected for the protein in buffer consisting of 50 mM MOPS, 25 mM NaCl, and 20% (vol/vol) glycerol at pH 6.8. The red and black bars indicate the upper limit of reliable shape and distance of the distribution, respectively (Materials and Methods). (C) Plots of ln(K/Ko) vs. pressure for the indicated mutants, and fits (red trace) using a two-state model to measure ΔVo¯ for the G → E transition.
Fig. 6.
Fig. 6.
Effect of pressure on the equilibrium between the GH, G, and E states of T4L mutants L99A (Left) and L99A/G113A/R119P (Right). Relative configurational free energies (ΔGo) are shown for 0 and 2 kbar; values for the GH and E states are relative to G. Values for ΔVo¯ and ΔGo are −36 mL/mol and −0.2 kcal/mol, respectively, for the G-to-E transition (based on PR DEER of L99A/G113A/R119P), and −75 mL/mol and 2.5 kcal/mol, respectively, for the G-to-GH transition [based on high-pressure tryptophan fluorescence of L99A (14) and the total L99A cavity volume]. Populations of each state are indicated in the local minima of the landscape. Structural models for the C domain are shown below the corresponding energy minima. Helix F is shown in red. The location of Phe114 (red spheres) in the G and E states are indicated. The empty cavity is shown in gray surface representation. A blue surface is used to represent pressure-populated hydration of the cavity in the L99A mutant. Green spheres at the Cα are used to indicate the position of G113A and R119P mutants in the E state structure (PDB ID code 2LC9) (5).
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