Using microcalorimetry, we found an equilibrium intermediate state during the denaturation of the wild-type and five mutant staphylococcal nuclease proteins: V66L, V66W, G88V, D77A, and E75V. The presence of two distinct heat absorption peaks allowed direct measurement of the enthalpy differences between the native, intermediate, and denatured states. Conditions of low pH and high NaCl concentration facilitated observation of the intermediate, or I-state. We propose to consider the nuclease protein as composed of two subdomains, divided along the active-site cleft. The structure of the I-state apparently consists mainly of the folded beta-barrel subdomain, as does that of a nuclease fragment protein [Shortle, D., & Abeygunawardana, C. (1993) Structure 1, 121-134]. The cooperativity of folding of the subdomains is maintained by electrostatic bonds across the active-site cleft. Removal of these bonds by the mutation D77A or E75V results in decooperation of the protein's structure and a three-state mechanism of denaturation at pH 7.0. The origins of differences in the enthalpy change of denaturation and in the m value of guanidinium chloride-induced denaturation with mutant nucleases are discussed in terms of this three-state mechanism. Study holds ProTherm entries: 2934, 2935, 2936, 2937, 2938, 2939, 2940, 2941, 2942, 2943, 2944 Extra Details: additive : EDTA(1 mM),measurements were made in the presence of 0.1M NaCl intermediate state; three-state mechanism; thermodynamic;,enthalpy change; electrostatic; active-site cleft; structure
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:20 p.m.
|Number of data points||28|
|Proteins||Thermonuclease ; Thermonuclease|
|Assays/Quantities/Protocols||Experimental Assay: dTm ; Experimental Assay: dHcal ; Experimental Assay: Tm|
|Libraries||Mutations for sequence ATSTKKLHKEPATLIKAIDGDTVKLMYKGQPMTFRLLLVDTPETKHPKKGVEKYGPEASAFTKKMVENAKKIEVEFDKGQRTDKYGRGLAYIYADGKMVNEALVRQGLAKVAYVYKPNNTHEQHLRKSEAQAKKEKLNIWSEDNADSGQ|
|Percent Identity||Matching Chains||Protein||Accession||Entry Name|