Stability of phage T4 lysozymes. II. Unfolding with guanidinium chloride

The denaturation by guanidinium chloride of three phage lysozymes (wild type and two mutants) was investigated. The study of solvent denaturation permitted the investigation of the relative stabilities of the proteins at neutral pH, in contrast to thermal denaturation studies reported earlier which could only be performed in acid pH. The results were interpreted assuming that the free energy of solution of proteins is a linear function of denaturant concentration. Using standard thermodynamic formulas this permits the calculation of the stabilities of the three proteins in the absence of guanidinium chloride. The single point mutation Trp 138 leads to Tyr leads to relatively large changes in stability and the interaction of the protein with guanidinium chloride. The changes associated with the subsequent double mutation, Trp 126 leads to Tyr, Trp 158 leads to Tyr, are much smaller indicating a relatively smooth adjustment of the protein structure to the changed side chains. Models of the structural effects of point mutations are discussed. It is found that the mutation at position 138 does not fit a model in which the effect of a substitution is to introduce an energetic strain in the structure. It does fit a model in which there is a partial unravelling of the structure as a result of the mutation. However, there are no changes in the backbone circular dichroism spectra associated with the mutation. The two observations are not necessarily in conflict. Further physical studies are required for the resolution of the problem.