Experimental verification of the 'stability profile of mutant protein' (SPMP) data using mutant human lysozymes

The stability profile of mutant protein (SPMP) (Ota,M., Kanaya,S. and Nishi kawa,K., 1995, J. Mol. Biol., 248, 733-738) estimates the changes in confor mational stability due to single amino acid substitutions using a pseudo-en ergy potential developed for evaluating structure-sequence compatibility in the structure prediction method, the 3D-1D compatibility evaluation. Nine mutant human lysozymes expected to significantly increase in stability from SPMP were constructed, in order to experimentally verify the reliability o f SPMP, The thermodynamic parameters for denaturation and crystal structure s of these mutant proteins were determined. One mutant protein was stabiliz ed as expected, compared with the wild-type protein. However, the others we re not stabilized even though the structural changes were subtle, indicatin g that SPMP overestimates the increase in stability or underestimates negat ive effects due to substitution. The stability changes in the other mutant human lysozymes previously reported were also analyzed by SPMP, The correla tion of the stability changes between the experiment and prediction depende d on the types of substitution: there were some correlations for proline mu tants and cavity-creating mutants, but no correlation for mutants related t o side-chain hydrogen bonds. The present results may indicate some addition al factors that should be considered in the calculation of SPMP, suggesting that SPMP can be refined further.