PATTERNS OF NONADDITIVITY BETWEEN PAIRS OF STABILITY MUTATIONS IN STAPHYLOCOCCAL NUCLEASE

To identify interactions between amino acid positions in staphylococca l nuclease that affect its stability, a collection of 71 double-mutant forms was constructed from 22 previously characterized single mutants . These single mutations were assigned to three different classes on t he basis of their m value [m = d(DELTAG)/d[GuHCl]], a parameter that h as been correlated with energetically significant changes in the struc ture of the denatured state [Green et al. (1992) Biochemistry 31,5717- 5728]. Several mutant pairs from five of the six possible double-mutan t classes were analyzed by guanidine hydrochloride denaturation to det ermine the extent to which changes in stability (DELTADELTAG(H2O)) and changes in the m value (DELTAm(GuHCl)) reflect the sum of the effects of the individual mutants. The differences between the values for DEL TADELTAG(H2O) and DELTAm(GuHCl) estimated on the assumption of additiv ity and those obtained by experiment, i.e., DELTADELTADELTAG and DELTA DELTAm, were calculated for each double-mutant protein. Surprisingly, a large majority of double mutants from four of the five classes exhib ited positive values of DELTADELTADELTAG and DELTADELTAm; i.e., they w ere more stable and displayed a higher sensitivity to GuHCl than predi cted on the basis of additivity. - Statistical analysis of the data re veals (1) a highly significant correlation between the value of DELTAD ELTADELTAG (the nonadditivity in stability) and DELTADELTAm (the nonad ditivity in GuHCl sensitivity), (2) only weak correlations between the distance separating two mutant positions and the magnitude of DELTADE LTADELTAG and DELTADELTAm, (3) evidence for unique patterns of interac tions between some pairs of mutations, and (4) a triad of positions re mote in sequence but relatively close in the native structure that sho w a dramatic degree of nonadditivity. The proposal is made that, for s ome mutants, the dominant pathway of interaction between widely separa ted positions involves changes in the residual structure of the denatu red state, structure which retains a cooperative character and can ext end out 20 or more angstroms.