Figure 1. Ball and stick diagram of the cavity residues of wild-type Il-1β. The wire contour represents positionally disordered water observed crystallographically. The triad of phenylalanine residues that circumscribe the cavity are shown in gray. Phenylalanine 101 (dark gray) is not solvent-accessible from the cavity. All other residues shown in light grey are the residues that form the cavity. Image adapted from Yu et al.²

Figure 2. Differential scanning calorimetry data of wild-type Il-1β. Profile (bold black) represents an average of three sample scans of protein. The overlaid gray line is a representative two-state model. The scatter plot reflecting the difference between the model and the data is the dotted line near the 0 C_p position. The lower broken line represents a linear fit to the native state heat capacity baseline. The dash-dot line represents a linear fit to the denatured state heat capacity baseline.

Figure 3. Heat capacity curves for tyrosine mutants. Each curve represents the average of thee different sample scans. The dotted line below each curve is for visual reference. The area between the data profile and the reference line approximates the relative enthalpy of the unfolding transition.

Figure 4. Heat capacity curves for tryptophan mutants. Each curve represents the average of thee different sample scans. The dotted line below each curve is for visual reference. The area between the data profile and the reference line approximates the relative enthalpy of the unfolding transition.

Figure 5. Local structure of F101Y around the mutation site. The stereograph ball and stick model of the mutant structure is shown superposed over the darker, smaller, wild-type structure. Large dark spheres represent water molecules. Dotted lines represent putative hydrogen bonds.

Figure 6. Local structure of F146Y around the mutation site. The stereograph ball and stick model of the mutant structure is shown superposed over the darker, smaller, wild-type structure. Large dark spheres represent water molecules. Dotted lines represent putative hydrogen bonds.

Figure 7. Local structure of F42W around the mutation site. The stereograph ball and stick model of the mutant structure is shown superposed over the darker, smaller, wild-type structure. Large dark spheres represent water molecules. Dotted lines represent putative hydrogen bonds.

Figure 8. Local structure of F101W around the mutation site. The stereograph ball and stick model of the mutant structure is shown superposed over the darker, smaller, wild-type structure. Large dark spheres represent water molecules. Dotted lines represent putative hydrogen bonds.

Figure 9. Local structure of F146W around the mutation site. The stereograph ball and stick model of the mutant structure is shown superposed over the darker, smaller, wild-type structure. Large dark spheres represent water molecules. Dotted lines represent putative hydrogen bonds.

Thermodynamic parameters obtained from DSC analyses

T_m is the transition midpoint or melting temperature. ΔH is the calorimetric enthalpy. ΔH_vH/H_Cal is ratio of the van't Hoff to calorimetric enthalpies. ΔΔG^25 °C is the difference in the free energy of unfolding extrapolated to 25 °C between wild-type Il-1β and the mutants. ΔΔG^WT is the free energy of unfolding of the mutants at the melting temperature of the wild-type protein.

Data and model statistics

WT (Yu) values were taken from Yu et al., 1999.² WT represents our wild-type data and refined model. Il-1β wild-type statistics were again obtained from Yu et al.²

Free energy differences of transfer to organic solvent

ΔΔG values are in reference to phenylalanine. Positive values indicate a more energetically favorable transfer. Values were obtained from Sharp et al.¹⁸