Staphylococcal nuclease, at low pH and in the presence of high salt concentrations, has previously been proposed to exist in a partially folded or molten globule form called the "A-state" (Fink et al., 1993, Protein Sci 2:1155-1160). We have found that the A-state of nuclease at pH 2.1 in the presence of moderate to high salt concentrations and at low temperature exists in a substantially folded form structurally more similar to a native state. The A-state has the far-UV circular dichroism spectra characteristic of the native protein, which indicates that it has a large degree of secondary structure. Upon heating, the A-state denatures with a sigmoidal change in far-UV ellipticity and an observable peak in a differential scanning calorimeter trace, indicating that it is thermodynamically distinct from the denatured state. Three different mutations in a residue normally buried in the protein's core stabilize or destabilize the A-state in the same way as they affect the denaturation of the native state. The A-state must, therefore, contain at least some tertiary packing of side chains. Unlike the native state, which shows cold denaturation at low temperatures, the A-state is most stable at temperatures below 0 degrees C. Study holds ProTherm entries: 2778, 2779, 2780, 2781, 2782 Extra Details: additive : EDTA(1 mM), denatured state; molten globule; protein denaturation;,structural intermediate; unfolding
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:19 p.m.
|Number of data points||20|
|Proteins||Thermonuclease ; Thermonuclease|
|Assays/Quantities/Protocols||Experimental Assay: dCp pH:4.1, buffers:glycine-HCl: 20 mM ; Experimental Assay: Tm pH:4.1, buffers:glycine-HCl: 20 mM ; Experimental Assay: dHvH pH:4.1, buffers:glycine-HCl: 20 mM ; Experimental Assay: dCp buffers:Sodium phosphate: 20 mM, pH:7.0 ; Experimental Assay: Tm buffers:Sodium phosphate: 20 mM, pH:7.0 ; Experimental Assay: dHvH buffers:Sodium phosphate: 20 mM, pH:7.0 ; Derived Quantity: dTm pH:4.1, buffers:glycine-HCl: 20 mM ; Derived Quantity: dTm buffers:Sodium phosphate: 20 mM, pH:7.0|
|Libraries||Mutations for sequence ATSTKKLHKEPATLIKAIDGDTVKLMYKGQPMTFRLLLVDTPETKHPKKGVEKYGPEASAFTKKMVENAKKIEVEFDKGQRTDKYGRGLAYIYADGKMVNEALVRQGLAKVAYVYKPNNTHEQHLRKSEAQAKKEKLNIWSEDNADSGQ|