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

Study Summary

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

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
4WRD 2014-10-23T00:00:00+0000 1.65 Crystal structure of Staphylcoccal nulease variant Delta+PHS V66E L125E at cryogenic temperature
2LKV 2011-10-21T00:00:00+0000 0 Staphylococcal Nuclease PHS variant
2M00 2012-10-14T00:00:00+0000 0 Solution structure of staphylococcal nuclease E43S mutant in the presence of ssDNA and Cd2+
2OXP 2007-02-20T00:00:00+0000 2.0 Crystal Structure of Staphylococcal Nuclease mutant V66D/P117G/H124L/S128A
3D4W 2008-05-15T00:00:00+0000 1.9 Crystal structure of Staphylococcal nuclease variant Delta+PHS A109R at cryogenic temperature
3D8G 2008-05-23T00:00:00+0000 1.99 Crystal structure of Staphylococcal nuclease variant Delta+PHS I72R at cryogenic temperature
3MVV 2010-05-04T00:00:00+0000 1.72 Crystal structure of Staphylococcal nuclease variant Delta+PHS F34A at cryogenic temperature
3QOJ 2011-02-10T00:00:00+0000 1.6 Cryogenic structure of Staphylococcal nuclease variant D+PHS/V23K
3QOL 2011-02-10T00:00:00+0000 1.9 Crystal structure of Staphylococcal nuclease variant D+PHS/V23E at pH 6 determined at 100 K
3R3O 2011-03-16T00:00:00+0000 1.9 Crystal structure of Staphylococcal nuclease variant Delta+PHS T62A at cryogenic temperature and with high redundancy

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
99.3 Thermonuclease Q6GIK1 NUC_STAAR
99.3 Thermonuclease Q8NXI6 NUC_STAAW
99.3 Thermonuclease Q6GB41 NUC_STAAS
99.1 Thermonuclease Q7A6P2 NUC_STAAN
99.1 Thermonuclease Q99VJ0 NUC_STAAM
99.3 Thermonuclease Q5HHM4 NUC_STAAC
100.0 Thermonuclease P00644 NUC_STAAU