Exploring the temperature-pressure phase diagram of staphylococcal nuclease.


Abstract

The temperature dependence of the pressure-induced equilibrium unfolding of staphylococcal nuclease (Snase) was determined by fluorescence of the single tryptophan residue, FTIR absorption for the amide I' and tyrosine O-H bands, and small-angle X-ray scattering (SAXS). The results from these three techniques were similar, although the stability as measured by fluorescence was slightly lower than that measured by FTIR and SAXS. The resulting phase diagram exhibits the well-known curvature for heat and cold denaturation of proteins, due to the large decrease in heat capacity upon folding. The volume change for unfolding became less negative with increasing temperatures, consistent with a larger thermal expansivity for the unfolded state than for the folded state. Fluorescence-detected pressure-jump kinetics measurements revealed that the curvature in the phase diagram is due primarily to the rate constant for folding, indicating a loss in heat capacity for the transition state relative to the unfolded state. The similar temperature dependence of the equilibrium and activation volume changes for folding indicates that the thermal expansivities of the folded and transition states are similar. This, along with the fact that the activation volume for folding is positive over the temperature range examined, the nonlinear dependence of the folding rate constant upon temperature implicates significant dehydration in the rate-limiting step for folding of Snase. Study holds ProTherm entries: 16347, 16348, 16349, 16350, 16351, 16352, 16353, 16354, 16355, 16356, 16357, 16358, 16359, 16360, 16361 Extra Details: staphylococcal nuclease, curvature, pressure-induced equilibrium

Submission Details

ID: UwYE6ars

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:47 p.m.

Version: 1

Publication Details
Panick G;Vidugiris GJ;Malessa R;Rapp G;Winter R;Royer CA,Biochemistry (1999) Exploring the temperature-pressure phase diagram of staphylococcal nuclease. PMID:10194332
Additional Information

Number of data points 15
Proteins Thermonuclease ; Thermonuclease
Unique complexes 1
Assays/Quantities/Protocols Experimental Assay: dG temp:45.0 C, prot_conc:50 mg/mL, buffers:Bis-tris: 50 mM ; Experimental Assay: dG temp:40.0 C, prot_conc:50 mg/mL, buffers:Bis-tris: 50 mM ; Experimental Assay: dG temp:36.0 C, prot_conc:50 mg/mL, buffers:Bis-tris: 50 mM ; Experimental Assay: dG prot_conc:50 mg/mL, temp:25.0 C, buffers:Bis-tris: 50 mM ; Experimental Assay: dG temp:20.0 C, prot_conc:50 mg/mL, buffers:Bis-tris: 50 mM ; Experimental Assay: dG temp:15.0 C, prot_conc:50 mg/mL, buffers:Bis-tris: 50 mM ; Experimental Assay: dG prot_conc:50 mg/mL, temp:10.0 C, buffers:Bis-tris: 50 mM ; Experimental Assay: dG prot_conc:50 mg/mL, buffers:Bis-tris: 50 mM, temp:5.0 C ; Experimental Assay: dG temp:0.5 C, prot_conc:50 mg/mL, buffers:Bis-tris: 50 mM ; Experimental Assay: dG prot_conc:50 mg/mL, buffers:Bis-tris: 50 mM, temp:-4.5 C ; Experimental Assay: dG prot_conc:15 micro M, temp:40.0 C, buffers:Bis-tris: 10 mM ; Experimental Assay: dG prot_conc:15 micro M, buffers:Bis-tris: 10 mM, temp:30.0 C ; Experimental Assay: dG prot_conc:15 micro M, temp:21.0 C, buffers:Bis-tris: 10 mM ; Experimental Assay: dG prot_conc:15 micro M, buffers:Bis-tris: 10 mM, temp:10.0 C ; Experimental Assay: dG prot_conc:15 micro M, buffers:Bis-tris: 10 mM, temp:2.0 C
Libraries Mutations for sequence ATSTKKLHKEPATLIKAIDGDTVKLMYKGQPMTFRLLLVDTPETKHPKKGVEKYGPEASAFTKKMVENAKKIEVEFDKGQRTDKYGRGLAYIYADGKMVNEALVRQGLAKVAYVYKPNNTHEQHLRKSEAQAKKEKLNIWSEDNADSGQ
Sequence Assay Result Units