Combined NMR-observation of cold denaturation in supercooled water and heat denaturation enables accurate measurement of ΔCp of protein unfolding

Cold and heat denaturation of the double mutant Arg 3 -> Glu/Leu 66 -> Glu of cold shock protein Csp of Bacillus caldolyticus was monitored using 1D H-1 NMR spectroscopy in the temperature range from -12 degrees C in supercooled water up to +70 degrees C. The fraction of unfolded protein, f(u), was determined as a function of the temperature. The data characterizing the unfolding transitions could be consistently interpreted in the framework of two-state models: cold and heat denaturation temperatures were determined to be -11 degrees C and 39 degrees C, respectively. A joint fit to both cold and heat transition data enabled the accurate spectroscopic determination of the heat capacity difference between native and denatured state, Delta C-p of unfolding. The approach described in this letter, or a variant thereof, is generally applicable and promises to be of value for routine studies of protein folding.