Enthalpy of Unfolding (ΔH)
Circular Dichroism (CD)
10 mM sodium phosphate
222 nm all but Ghel (216 nm)
see Tm assay.
Tm details: Thermally induced unfolding was monitored by CD, on a Jasco-710 dichrograph previously calibrated with d- 10-camphorsulphonic acid. Measurements were per- formed in a 0.2 cm pathlength cuvette in the temperature range 277-371 K. The temperature was increased step- wise by 0.5 K at a rate of 50 K/hour, and the ellipticity was recorded with a 1 nm bandwidth and a two second response. CD was monitored at 222 nm (GB1, Gin, Gout and GY) or 216 nm (Ghel). The sample concentrations were ~20 μM, in 10 mM sodium phosphate buffer (pH 7). For all proteins, the reversibility of the transition was tested and was found to be completely reversible. Fitting of temperature denaturation curves was performed with the program Kaleidagraph 3.1 (Synergy Software). The curves corresponding to the wild-type and mutant proteins were used to find the linear depen-dence of the mean residue ellipticity at 222 nm of the native (EN) and denatured states (EU). The ΔCp,U value used is that of Alexander et al. (1992):
E = (EN + AT + (EU + BT) exp (ΔHTm (1−T/Tm) - ΔCp,U(Tm − T+ T ln(T/Tm)) /RT)) / (1+ exp( ΔHTm(1 − T/Tm) - Cp,U(Tm − T +T ln(T/Tm))/RT
where A and B are constants that take into consideration the dependence of the native and denatured ellipticities with temperature, E is the observed ellipticity and EN, and EU are the ellipticities of the native and denatured states, respectively.