The stability of WT and mutant proteins was determined using urea-induced equilibrium denaturation followed by fluorimetry using a Cary Eclipse spectrometer (Varian); the excitation wavelength was 280 nm, the emission wavelength was 360 nm and the concentration of protein was ≤ 1–2 μM. The stability of the WT protein was also determined by CD using an Applied Photophysics Chirascan circular dichroism spectropolarimeter; data were collected at 222 nm and the concentration of protein was ≤ 5 μM. All experiments were performed at 25 °C in 50 mM sodium phosphate, pH 7.0, 150 mM NaCl, 5 mM DTT. The data were fit to a two-state transition as described.60,61