THERMODYNAMIC EFFECTS OF MUTATIONS ON THE DENATURATION OF T4 LYSOZYME

We investigated the folding of substantially destabilized mutant forms of T4 lysozyme using differential scanning calorimetry and circular d ichroism measurements. Three mutations in an alpha-helix in the protei n's N-terminal region, the alanine insertion mutations S44[A] and K48[ A], and the substitution A42K had previously been observed to result i n unexpectedly low apparent enthalpy changes of melting, compared to a pseudo-wild-type reference protein. The pseudo-wild-type reference pr otein thermally unfolds in an essentially two-state manner. However, w e found that the unfolding of the three mutant proteins has a reduced cooperativity, which partially explains their lower apparent enthalpy changes. A three-state unfolding model including a discrete intermedia te is necessary to describe the melting of the mutant proteins. The re duction in cooperativity must be considered for accurate calculation o f the energy changes of folding, Unfolding in two stages reflects the underlying two-subdomain structure of the lysozyme protein family.