ENHANCEMENT OF PROTEIN STABILITY BY THE COMBINATION OF POINT MUTATIONS IN T4 LYSOZYME IS ADDITIVE

A number of mutations have been shown previously to stabilize T4 lysoz yme, By combining up to seven such mutations in the same protein, the melting temperature was incrementally increased by up to 8.3 degrees C at pH 5.4 (Delta Delta G = 3.6 kcal/mol), This shows that it is possi ble to engineer a protein of enhanced thermostability by combining a s eries of rationally designed point mutations, It is also shown that th is stabilization is achieved with only minor, localized changes in the structure of the protein, This is consistent with the observation tha t the change in stability of each of the multiple mutants is, in each case, additive, i.e. equal to the sum of the stability changes associa ted with the constituent single mutants, One of the seven substitution s, Asn116 --> Asp, changes a residue that participates in substrate bi nding; not surprisingly, it causes a significant loss in activity, Ign oring this mutation, there is a gradual reduction in activity as succe ssively more mutations are combined.