Coupling between local structure and global stability of a protein: mutants of staphylococcal nuclease.


Abstract

Staphylococcal nuclease exists in solution as a mixture of two folded (N and N') and two unfolded (U and U*) forms. Earlier workers [Evans et al. (1989) Biochemistry 28, 362] have proposed that the N'/N and U/U* structural differences involve cis/trans isomerization about the Lys116-Pro117 peptide bond with N and U cis and N' and U* trans. The present results show that residue changes throughout the nuclease structure have large effects on the distribution of the N and N'forms. The N'/N ratios at 313 K for nuclease H124L (N'/N = 0.07) and nuclease G79S (N'/N = 12) differ by 2 orders of magnitude. Thermodynamic parameters for equilibria linking the two folded and two unfolded substates were evaluated for seven mutants of nuclease which were found by kinetic assays to have similar enzymatic activities but by NMR spectroscopy to have a wide dispersion of thermal stabilities. Our results indicate that mutational perturbations of the N'/N equilibrium in folded nuclease (delta G for the N in equilibrium N' reaction) are strongly coupled to changes in the stability of the N form (delta G for the N in equilibrium U reaction), but much less so to the stability of the N' form (delta G for the N' in equilibrium U* reaction). Study holds ProTherm entries: 3894, 3895, 3896, 3897, 3898, 3899, 3900, 3901, 11886, 11887, 11888, 11889, 11890, 11891, 11892, 11893 Extra Details: cis/trans isomerization; kinetic assays; enzymatic activities;,NMR spectroscopy

Submission Details

ID: TwajCaq94

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:23 p.m.

Version: 1

Publication Details
Alexandrescu AT;Hinck AP;Markley JL,Biochemistry (1990) Coupling between local structure and global stability of a protein: mutants of staphylococcal nuclease. PMID:2372535
Additional Information

Study Summary

Number of data points 48
Proteins Thermonuclease ; Thermonuclease
Unique complexes 8
Assays/Quantities/Protocols Experimental Assay: dCp pH:5.2 ; Experimental Assay: dHvH temp:40.0 C, pH:5.2 ; Experimental Assay: dG pH:5.2 ; Experimental Assay: dCp pH:5.42 ; Experimental Assay: dG pH:5.42 ; Experimental Assay: dCp pH:5.35 ; Experimental Assay: dG pH:5.35 ; Experimental Assay: dCp pH:5.21 ; Experimental Assay: dG pH:5.21 ; Experimental Assay: dCp pH:5.38 ; Experimental Assay: dG pH:5.38 ; Experimental Assay: dCp pH:5.28 ; Experimental Assay: dG pH:5.28 ; Experimental Assay: dCp pH:5.31 ; Experimental Assay: dG pH:5.31 ; Experimental Assay: Tm pH:5.2 ; Experimental Assay: dHvH pH:5.2 ; Experimental Assay: Tm pH:5.42 ; Experimental Assay: dHvH pH:5.42 ; Experimental Assay: Tm pH:5.35 ; Experimental Assay: dHvH pH:5.35 ; Experimental Assay: Tm pH:5.21 ; Experimental Assay: dHvH pH:5.21 ; Experimental Assay: Tm pH:5.38 ; Experimental Assay: dHvH pH:5.38 ; Experimental Assay: Tm pH:5.28 ; Experimental Assay: dHvH pH:5.28 ; Experimental Assay: Tm pH:5.31 ; Experimental Assay: dHvH pH:5.31 ; Derived Quantity: ddG pH:5.2 ; Derived Quantity: ddG pH:5.42 ; Derived Quantity: ddG pH:5.35 ; Derived Quantity: ddG pH:5.21 ; Derived Quantity: ddG pH:5.38 ; Derived Quantity: ddG pH:5.28 ; Derived Quantity: dTm pH:5.2 ; Derived Quantity: dTm pH:5.42 ; Derived Quantity: dTm pH:5.35 ; Derived Quantity: dTm pH:5.21 ; Derived Quantity: dTm pH:5.38 ; Derived Quantity: dTm pH:5.28 ; Derived Quantity: dTm pH:5.31
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