∆H pH2.5


Enthalpy of Unfolding (ΔH)


Differential Scanning Calorimetry (DSC)


The DSC experiments were preformed on a VP-DSC (MicroCal, Northampton, MA) instrument at a scan rate of 90°/h. The protein concentration in the DSC experiments varied between 1.5 and 3.5 mg/mL in 20 mM glycine or sodium acetate buffers. Temperature-induced unfolding of all the studied ubiquitin variants was routinely checked for reversibility by recording the second scan, and found to be better than 90%. Calorimetric profiles were analyzed according to a 2-state transition model using the nonlinear regression routine (NLREG) and in-house written scripts.1