Hydrogen exchange kinetics were measured on the native states of wild type staphylococcal nuclease and four mutants with values of mGuHCl (defined as dDeltaG/d[guanidine hydrochloride]) ranging from 0.8 to 1.4 of the wild type value. Residues within the five-strand beta-barrel of wild type and E75A and D77A, two mutants with reduced values of m GuHCl, were significantly more protected from exchange than expected on the basis of global stability as measured by fluorescence. In contrast, mutants V23A and M26G with elevated values of mGuHCl approach a flat profile of more or less constant protection independent of position in the structure. Differences in exchange protection between the C-terminus and the beta-barrel region correlate with mGuHCl, suggesting that a residual barrel-like structure becomes more highly populated in the denatured states of m- mutants and less populated in m+ mutants. Variations in the population of such a molten globule-like structure would account for the large changes in solvent accessible surface area of the denatured state thought to underlie m value effects. Study holds ProTherm entries: 7024, 7025, 7026, 7027, 7028, 7029, 7030, 7031, 7032, 7033, 7034, 7035, 7036, 7037, 7038 Extra Details: hydrogen exchange kinetic; five-strand beta-barrel;,global stability; solvent accessible surface area; m value
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:32 p.m.
|Number of data points||30|
|Proteins||Thermonuclease ; Thermonuclease|
|Assays/Quantities/Protocols||Experimental Assay: m pH:5.2, temp:25.0 C, buffers:Sodium acetate: 50 mM ; Experimental Assay: dG_H2O temp:25.0 C, buffers:Sodium acetate: 50 mM, pH:5.2 ; Experimental Assay: m pH:5.6, temp:25.0 C, buffers:Sodium acetate: 50 mM ; Experimental Assay: dG_H2O pH:5.6, buffers:Sodium acetate: 50 mM, temp:25.0 C ; Experimental Assay: m buffers:Sodium phosphate: 25 mM, temp:20.0 C, pH:7.0 ; Experimental Assay: dG_H2O buffers:Sodium phosphate: 25 mM, temp:20.0 C, pH:7.0|
|Libraries||Mutations for sequence ATSTKKLHKEPATLIKAIDGDTVKLMYKGQPMTFRLLLVDTPETKHPKKGVEKYGPEASAFTKKMVENAKKIEVEFDKGQRTDKYGRGLAYIYADGKMVNEALVRQGLAKVAYVYKPNNTHEQHLRKSEAQAKKEKLNIWSEDNADSGQ|