Thermodynamics of replacing an alpha-helical Pro residue in the P40S mutant of Escherichia coli thioredoxin.


Abstract

Escherichia coli thioredoxin is a 108 amino acid oxidoreductase and contains a single Met residue at position 37. The protein contains a long alpha-helical stretch between residues 32 and 49. The central residue of this helix, Pro40, has been replaced by Ser. The stabilities of the oxidized states of two proteins, the single mutant M37L and the double mutant M37L,P40S, have been characterized by differential scanning calorimetry (DSC) and also by a series of isothermal guanidine hydrochloride (GuHCl) melts in the temperature range of 277 to 333 K. The P40S mutation was found to stabilize the protein at all temperatures upto 340 K though both proteins had similar Tm values of about 356 K. At 298 K, the M37L,P40S mutant was found to be more stable than M37L by 1.5 kcal/mol. A combined analysis of GuHCl and calorimetric data was carried out to determine the enthalpy, entropy, and heat capacity change upon unfolding. At 298 K there was a large, stabilizing enthalpic effect in P40S though significant enthalpy-entropy compensation was observed and the two proteins had similar values of deltaCp. Thus, replacement of a Pro in the interior of an alpha helix can have substantial effects on protein stability. Study holds ProTherm entries: 6793, 6794, 6795, 6796, 6797, 6798, 6799, 6800, 6801, 6802, 6803, 6804, 6805, 6806, 6807, 6808, 6809, 6810, 6811, 6812, 6813, 6814, 6815, 6816, 6817 Extra Details: proline mutant; stability; thioredoxin

Submission Details

ID: aUfPYWFR

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:32 p.m.

Version: 1

Publication Details
Chakrabarti A;Srivastava S;Swaminathan CP;Surolia A;Varadarajan R,Protein Sci. (1999) Thermodynamics of replacing an alpha-helical Pro residue in the P40S mutant of Escherichia coli thioredoxin. PMID:10595549
Additional Information

Study Summary

Number of data points 75
Proteins Thioredoxin 1 ; Thioredoxin 1
Unique complexes 2
Assays/Quantities/Protocols Experimental Assay: dCp ; Experimental Assay: dHcal ; Experimental Assay: Tm ; Experimental Assay: Cm temp:281 K ; Experimental Assay: m temp:281 K ; Experimental Assay: dG_H2O temp:281 K ; Experimental Assay: Cm temp:279 K ; Experimental Assay: m temp:279 K ; Experimental Assay: dG_H2O temp:279 K ; Experimental Assay: Cm temp:277 K ; Experimental Assay: m temp:277 K ; Experimental Assay: dG_H2O temp:277 K ; Experimental Assay: Cm temp:338 K ; Experimental Assay: m temp:338 K ; Experimental Assay: dG_H2O temp:338 K ; Experimental Assay: Cm temp:333 K ; Experimental Assay: m temp:333 K ; Experimental Assay: dG_H2O temp:333 K ; Experimental Assay: Cm temp:328 K ; Experimental Assay: m temp:328 K ; Experimental Assay: dG_H2O temp:328 K ; Experimental Assay: Cm temp:323 K ; Experimental Assay: m temp:323 K ; Experimental Assay: dG_H2O temp:323 K ; Experimental Assay: Cm temp:318 K ; Experimental Assay: m temp:318 K ; Experimental Assay: dG_H2O temp:318 K ; Experimental Assay: Cm temp:313 K ; Experimental Assay: m temp:313 K ; Experimental Assay: dG_H2O temp:313 K ; Experimental Assay: Cm temp:310 K ; Experimental Assay: m temp:310 K ; Experimental Assay: dG_H2O temp:310 K ; Experimental Assay: Cm temp:303 K ; Experimental Assay: m temp:303 K ; Experimental Assay: dG_H2O temp:303 K ; Experimental Assay: Cm temp:298 K ; Experimental Assay: m temp:298 K ; Experimental Assay: dG_H2O temp:298 K ; Experimental Assay: Cm temp:293 K ; Experimental Assay: m temp:293 K ; Experimental Assay: dG_H2O temp:293 K ; Experimental Assay: Cm temp:288 K ; Experimental Assay: m temp:288 K ; Experimental Assay: dG_H2O temp:288 K ; Experimental Assay: Cm temp:283 K ; Experimental Assay: m temp:283 K ; Experimental Assay: dG_H2O temp:283 K ; Experimental Assay: Cm temp:278 K ; Experimental Assay: m temp:278 K ; Experimental Assay: dG_H2O temp:278 K
Libraries Mutations for sequence SDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLA

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