To identify interactions between amino acid positions in staphylococcal 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 the basis of their m value [m = d(ΔG)/d [GuHCl]], a parameter that has been correlated with energetically significant changes in the structure of the denatured state [Green et al. (1992) Biochemistry 31, 5717–5728]. Several mutant pairs from five of the six possible double-mutant classes were analyzed by guanidine hydrochloride denaturation to determine the extent to which changes in stability ([formula omitted]) and changes in the m value (ΔmGuHCl) reflect the sum of the effects of the individual mutants, The differences between the values for [formula omitted] and ΔmGuHCl estimated on the assumption of additivity and those obtained by experiment, i.e., ΔΔΔG and ΔΔm, were calculated for each double-mutant protein. Surprisingly, a large majority of double mutants from four of the five classes exhibited positive values of ΔΔΔG and ΔΔm; i.e., they were more stable and displayed a higher sensitivity to GuHCl than predicted on the basis of additivity. Statistical analysis of the data reveals (1) a highly significant correlation between the value of ΔΔΔG (the nonadditivity in stability) and ΔΔm (the nonadditivity in GuHCl sensitivity), (2) only weak correlations between the distance separating two mutant positions and the magnitude of ΔΔΔG and ΔΔm, (3) evidence for unique patterns of interactions between some pairs of mutations, and (4) a triad of positions remote in sequence but relatively close in the native structure that show a dramatic degree of nonadditivity. The proposal is made that, for some mutants, the dominant pathway of interaction between widely separated positions involves changes in the residual structure of the denatured state, structure which retains a cooperative character and can extend out 20 or more angstroms.
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