Thermodynamics of replacing an alpha-helical Pro residue in the P40S mutant of Escherichia coli thioredoxin

Escherichia coli thioredoxin is a 108 amino acid oxidoreductase and contain s a single Met residue at position 37. The protein contains a long alpha-he lical stretch between residues 32 and 49. The central residue of this helix , Pro40, has been replaced by Ser. The stabilities of the oxidized states o f two proteins, the single mutant M37L and the double mutant M37L,P40S, hav e been characterized by differential scanning calorimetry (DSC) and also by a series of isothermal guanidine hydrochloride (GuHCl) melts in the temper ature range of 277 to 333 K. The P40S mutation was found to stabilize the p rotein at all temperatures upto 340 K though both proteins had similar T-m values of about 356 K. At 298 K, the M37L,P40S mutant was found to be more stable than M37L by 1.5 kcal/mol. A combined analysis of GuHCl and calorime tric data was carried out to determine the enthalpy. entropy, and heat capa city change upon unfolding. At 298 K there was a large, stabilizing enthalp ic effect in P40S though significant enthalpy-entropy compensation was obse rved and the two proteins had similar values of Delta C-p. Thus, replacemen t of a Pro in the interior of an alpha helix can have substantial effects o n protein stability.