Evaluating the effects of a single amino acid substitution on both the native and denatured states of a protein.


Abstract

For proteins that contain a disulfide bond, stability is linked thermodynamically to thiol-disulfide exchange. We use this relationship to obtain unfolding free energies for both the reduced and oxidized forms of Escherichia coli thioredoxin from measurements of the effective concentrations of protein thiols. We then evaluate the effect of an amino acid substitution on disulfide bond formation in both the native and denatured states of the protein. Although the Pro-34----Ser substitution in thioredoxin results in a decrease of the effective concentration of protein thiols in the native state, the effective concentration increases in the denatured state. The net effect of the amino acid substitution is to increase the stability of reduced thioredoxin by approximately 2.4 kcal/mol, whereas the stability of the oxidized protein remains the same. By assuming a two-state unfolding equilibrium and a mutation free energy of -7.7 kcal/mol for the Pro-34----Ser substitution in the reduced, urea-unfolded state (based on estimates of solvation and entropic changes), we obtained relative free energies for the native and denatured states of the mutant and wild-type proteins, in both the reduced and oxidized forms. Study holds ProTherm entries: 10994, 10995, 10996, 10997 Extra Details: additive : EDTA(1 mM),reduced thiol-disulphide exchange; amino acid substitution

Submission Details

ID: uEzTz3L9

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:41 p.m.

Version: 1

Publication Details
Lin TY;Kim PS,Proc. Natl. Acad. Sci. U.S.A. (1991) Evaluating the effects of a single amino acid substitution on both the native and denatured states of a protein. PMID:1961723
Additional Information

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
3DXB 2008-07-24T00:00:00+0000 2.2 Structure of the UHM domain of Puf60 fused to thioredoxin
5E4W 2015-10-07T00:00:00+0000 2.8 Crystal structure of cpSRP43 chromodomains 2 and 3 in complex with the Alb3 tail
5IKN 2016-03-03T00:00:00+0000 4.8 Crystal Structure of the T7 Replisome in the Absence of DNA
1F6M 2000-06-22T00:00:00+0000 2.95 CRYSTAL STRUCTURE OF A COMPLEX BETWEEN THIOREDOXIN REDUCTASE, THIOREDOXIN, AND THE NADP+ ANALOG, AADP+
1KEB 2001-11-15T00:00:00+0000 1.8 Crystal Structure of Double Mutant M37L,P40S E.coli Thioredoxin
1M7T 2002-07-22T00:00:00+0000 0 Solution Structure and Dynamics of the Human-Escherichia coli Thioredoxin Chimera: Insights into Thermodynamic Stability
1OAZ 2003-01-21T00:00:00+0000 2.78 IgE Fv SPE7 complexed with a recombinant thioredoxin
1SKR 2004-03-05T00:00:00+0000 2.4 T7 DNA Polymerase Complexed To DNA Primer/Template and ddATP
1SKS 2004-03-05T00:00:00+0000 2.3 Binary 3' complex of T7 DNA polymerase with a DNA primer/template containing a cis-syn thymine dimer on the template
1SKW 2004-03-05T00:00:00+0000 2.3 Binary 3' complex of T7 DNA polymerase with a DNA primer/template containing a disordered cis-syn thymine dimer on the template

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Thioredoxin 1 P0AA30 THIO_SHIFL
100.0 Thioredoxin 1 P0AA28 THIO_SALTY
100.0 Thioredoxin 1 P0AA29 THIO_SALTI
100.0 Thioredoxin 1 P0AA25 THIO_ECOLI
100.0 Thioredoxin 1 P0AA26 THIO_ECOL6
100.0 Thioredoxin 1 P0AA27 THIO_ECO57