Patterns of nonadditivity between pairs of stability mutations in staphylococcal nuclease.
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(delta G)/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 (delta delta GH2O) and changes in the m value (delta mGuHCl) reflect the sum of the effects of the individual mutants. The differences between the values for delta delta GH2O and delta mGuHCl estimated on the assumption of additivity and those obtained by experiment, i.e., delta delta delta G and delta delta m, were calculated for each double-mutant protein. Surprisingly, a large majority of double mutants from four of the five classes exhibited positive values of delta delta delta G and delta delta m; i.e., they were more stable and displayed a higher sensitivity to GuHCl than predicted on the basis of additivity.(ABSTRACT TRUNCATED AT 250 WORDS)
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Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
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D000595:Amino Acid Sequence; D004274:DNA, Recombinant / Q000378:metabolism; D004795:Enzyme Stability; D019791:Guanidine; D006146:Guanidines; D008836:Micrococcal Nuclease / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008961:Models, Structural; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D004274:DNA, Recombinant; D006146:Guanidines; D011994:Recombinant Proteins; D008836:Micrococcal Nuclease; D019791:Guanidine