Heat and cold denaturation of yeast frataxin: The effect of pressure

Abstract

Yfh1 is a yeast protein with the peculiar characteristic to undergo, in the absence of salt, cold denaturation at temperatures above the water freezing point. This feature makes the protein particularly interesting for studies aiming at understanding the rules that determine protein fold stability. Here, we present the phase diagram of Yfh1 unfolding as a function of pressure (0.1-500 MPa) and temperature 278-313 K (5-40°C) both in the absence and in the presence of stabilizers using Trp fluorescence as a monitor. The protein showed a remarkable sensitivity to pressure: at 293 K, pressures around 10 MPa are sufficient to cause 50% of unfolding. Higher pressures were required for the unfolding of the protein in the presence of stabilizers. The phase diagram on the pressure-temperature plane together with a critical comparison between our results and those found in the literature allowed us to draw conclusions on the mechanism of the unfolding process under different environmental conditions.

Figure 1

The structure of yeast frataxin (PDB: 3oeq). Tryptophan residues 131 and 149 are orange and pink, respectively. Notice that in the crystal cell the structure is a trimer, whereas the protein is monomeric in solution in the absence of iron (57). We also demonstrated that in solution the N-terminus of the protein is unstructured and flexible (58). To see the figure in color, go online.

Figure 2

Fluorescence spectrum of yeast frataxin at 293 K in 20 mM Hepes (pH 7) (solid line), plus 50 mM CaCl₂ (dash line), 1 M trehalose (short-dash line) or 8 M urea (dash-dot line). λ_ex was 289 nm. The protein concentration was 9 μM.

Figure 3

Change in center of spectral mass (cm) of frataxin fluorescence spectrum as a function of pressure at various temperatures: 278 K (squares), 293 K (circles), and 313 K (triangles). (Panel A) in the absence of salt; (panel B) in the presence of 50 mM CaCl₂; (panel C) in the presence of 1 M trehalose. Solid lines are the best fit of the experimental data to Eq. 5. Experimental conditions are as in Fig. 2.

Figure 4

Free energy changes ($\Delta G_T^0$) at atmospheric pressure (0.1 MPa) as a function of temperature, as determined by pressure unfolding experiments. Frataxin in 20 mM Hepes (pH 7) (squares), in the presence of 50 mM CaCl₂ (circles), and in the presence of 1 M trehalose (triangles). Solid lines are the best fit of the experimental data by the Eq. 7. Experimental conditions are as in Fig. 2. Error bars represent the standard deviation of three independent experiments.

Figure 5

Change in partial molar volume (ΔV) upon unfolding at different temperatures for frataxin at low ionic strength (squares), in the presence of 50 mM CaCl₂ (circles) and in the presence of 1 M trehalose (triangles). T₀ = 293 K was assumed. Solid lines are the best fit to Eq. 4 of the experimental data. Error bars represent the standard deviation of three independent experiments.

Figure 6

Pressure versus temperature stability diagram of Yfh1 as obtained by tryptophan fluorescence measurements. No salt frataxin (squares), in the presence of 50 mM CaCl₂ (circles), and in the presence of 1 M trehalose (triangles). Solid lines are the best fit of the experimental data to the following equation: $\Delta G=\Delta G^0+\Delta \kappa /2{(p-p_m)}^2+\Delta \alpha \left(p-p_0\right)\left(T-T_0\right)-\Delta Cp[T(lnT/T_0-1)+T_0]+\Delta V^0\left(p-p_0\right)-\Delta S_0\left(T-T_0\right)$, where Δκ is the difference in isothermal compressibility between unfolded and native state. Δ$G_{293}^0$, Δ$V_{293}^0$, ΔCp, and Δα values estimated at 293 K (assumed as reference temperature, T₀) were used as input parameters. Δκ and Δ$S_{293}^0$ were estimated by the fitting procedure. Error bars represent the standard deviation of three independent experiments.