Fig. 6.
Wider applications for denaturant titrations. (A) Urea denaturation of the P60A R16 α-spectrin domain was probed between 295.7 and 318.2 K using fluorescence emission spectroscopy. The resultant titrations were fitted globally to a series of independent 2-state transitions with a shared mD–N value (see Section 2.7). This strategy allowed determination of [Denaturant]50% values at each temperature. (B) The parameters determined in (A) allowed the direct determination of ΔGD−N0 values at each temperature (black circles). The temperature dependence of ΔGD−N0 fitted well to a simple polynomial equation (dashed blue line) and a Gibbs–Helmholtz formalism (solid black line, see Section 2.7) [35]. These fits were essentially identical and defined a Tm value (blue square) close to that obtained in thermal denaturation experiments probed by far-UV CD spectroscopy (red plus sign, inset) and differential scanning calorimetry (black cross, Fig. S5). (C) Assaying protein–peptide interactions using chemical denaturation. The PDZ1 F95W domain was titrated into urea in the absence (black circles) or presence of either LQRRRETQV (triangles) or LQRRRETQ-Abu (squares) peptides. The stability of PDZ1 F95W was increased by peptide binding, with larger stability increases seen at 40 μM peptide (red and blue respectively) compared to 4 μM peptide (pink and cyan respectively). Inset: the relative affinity of the interactions was confirmed by thermophoresis measurements, where the LQRRRETQV peptide bound PDZ1 F95W more tightly (red triangles, Kd ~ 0.9 ± 0.6 μM) than the LQRRRETQ-Abu peptide (blue squares, Kd ~ 13 ± 1 μM).