Escherichia coli thioredoxin is a 108
amino acid oxidoreductase and contains a single Met residue
at position 37. The protein contains a long α-helical
stretch between residues 32 and 49. The central residue
of this helix, Pro40, has been replaced by Ser. The stabilities
of the oxidized states of two proteins, the single mutant
M37L and the double mutant M37L,P40S, have been characterized
by differential scanning calorimetry (DSC) and also by
a series of isothermal guanidine hydrochloride (GuHCl)
melts in the temperature range of 277 to 333 K. The P40S
mutation was found to stabilize the protein at all temperatures
upto 340 K though both proteins had similar Tm
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 calorimetric data was carried out
to determine the enthalpy, entropy, and heat capacity change
upon unfolding. At 298 K there was a large, stabilizing
enthalpic effect in P40S though significant enthalpy-entropy
compensation was observed and the two proteins had similar
values of ΔCp. Thus, replacement
of a Pro in the interior of an α helix can have substantial
effects on protein stability.