The adaptability of Escherichia coli thioredoxin to non-conservative amino acid substitutions.


Abstract

The adaptability of Escherichia coli thioredoxin to the substitution of a series of non-natural amino acids has been investigated. Different thiosulfonated alkyl groups were inserted into the hydrophobic core of the protein in position 78 via disulfide bonding with a buried cysteine residue as previously described (Wynn R, Richards FM. 1993. Unnatural amino acid packing mutants of Escherichia coli thioredoxin produced by combined mutagenesis/chemical modification techniques. Protein Sci 2:395-403). The side chains added to the cysteine included methyl, ethyl, n-propyl, n-butyl, n-pentyl, and cyclo-pentyl derivatives. The side chains appear to exploit the presence of the large cavities to incorporate these variant forms, enabling the protein to fold and have some activity. Solution structural and kinetic data suggested that these substitutions had little effect on the overall fold of the protein. Thermodynamic data revealed that the entropic effect of restricting the side chains in the folded protein has an effect on the stability. The variant forms of thioredoxin have different propensities to form dimers despite the limited structural perturbations. Molecular modeling studies allow the conformation of the side chains to be assessed. Study holds ProTherm entries: 9392, 9393, 9394, 9395, 9396, 9397, 9398, 9399, 9400, 9401, 9402, 9403, 9404, 9405, 9406, 9407, 9408, 9409, 9410, 9411, 9412 Extra Details: calorimetry; cysteine modification; differential scanning; kinetics; protein folding; thermodynamics: thioredoxin; unnatural amino acids

Submission Details

ID: kHz5G3mZ3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:38 p.m.

Version: 1

Publication Details
O'Brien R;Wynn R;Driscoll PC;Davis B;Plaxco KW;Sturtevant JM;Ladbury JE,Protein Sci. (1997) The adaptability of Escherichia coli thioredoxin to non-conservative amino acid substitutions. PMID:9194193
Additional Information

Study Summary

Number of data points 59
Proteins Thioredoxin 1 ; Thioredoxin 1
Unique complexes 1
Assays/Quantities/Protocols Experimental Assay: dCp prot_conc:31 micro M ; Experimental Assay: dHcal prot_conc:31 micro M ; Experimental Assay: Tm prot_conc:31 micro M ; Experimental Assay: dCp prot_conc:73 micro M ; Experimental Assay: dHcal prot_conc:73 micro M ; Experimental Assay: Tm prot_conc:73 micro M ; Experimental Assay: dCp prot_conc:97 micro M ; Experimental Assay: dHcal prot_conc:97 micro M ; Experimental Assay: Tm prot_conc:97 micro M ; Experimental Assay: dCp prot_conc:110 micro M ; Experimental Assay: dHcal prot_conc:110 micro M ; Experimental Assay: Tm prot_conc:110 micro M ; Experimental Assay: dCp prot_conc:232 micro M ; Experimental Assay: dHcal prot_conc:232 micro M ; Experimental Assay: Tm prot_conc:232 micro M ; Experimental Assay: dCp prot_conc:327 micro M ; Experimental Assay: dHcal prot_conc:327 micro M ; Experimental Assay: Tm prot_conc:327 micro M ; Experimental Assay: dCp prot_conc:37 micro M ; Experimental Assay: dHcal prot_conc:37 micro M ; Experimental Assay: Tm prot_conc:37 micro M ; Experimental Assay: dCp prot_conc:83 micro M ; Experimental Assay: dHcal prot_conc:83 micro M ; Experimental Assay: Tm prot_conc:83 micro M ; Experimental Assay: dCp prot_conc:226 micro M ; Experimental Assay: dHcal prot_conc:226 micro M ; Experimental Assay: Tm prot_conc:226 micro M ; Experimental Assay: dCp prot_conc:134 micro M ; Experimental Assay: dHcal prot_conc:134 micro M ; Experimental Assay: Tm prot_conc:134 micro M ; Experimental Assay: dCp prot_conc:258 micro M ; Experimental Assay: dHcal prot_conc:258 micro M ; Experimental Assay: Tm prot_conc:258 micro M ; Experimental Assay: dCp prot_conc:333 micro M ; Experimental Assay: dHcal prot_conc:333 micro M ; Experimental Assay: Tm prot_conc:333 micro M ; Experimental Assay: dCp prot_conc:467 micro M ; Experimental Assay: dHcal prot_conc:467 micro M ; Experimental Assay: Tm prot_conc:467 micro M ; Experimental Assay: dCp prot_conc:198 micro M ; Experimental Assay: dHcal prot_conc:198 micro M ; Experimental Assay: Tm prot_conc:198 micro M ; Experimental Assay: dCp prot_conc:310 micro M ; Experimental Assay: dHcal prot_conc:310 micro M ; Experimental Assay: Tm prot_conc:310 micro M ; Experimental Assay: dCp prot_conc:346 micro M ; Experimental Assay: dHcal prot_conc:346 micro M ; Experimental Assay: Tm prot_conc:346 micro M ; Experimental Assay: dHcal prot_conc:120 micro M ; Experimental Assay: Tm prot_conc:120 micro M ; Experimental Assay: dHcal prot_conc:162 micro M ; Experimental Assay: Tm prot_conc:162 micro M ; Experimental Assay: dHcal prot_conc:330 micro M ; Experimental Assay: Tm prot_conc:330 micro M
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
5IKN 2016-03-03T00:00:00+0000 4.8 Crystal Structure of the T7 Replisome in the Absence of DNA
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
1TKD 2004-06-08T00:00:00+0000 2.49 T7 DNA polymerase ternary complex with 8 oxo guanosine and dCMP at the elongation site
1TK0 2004-06-07T00:00:00+0000 2.3 T7 DNA polymerase ternary complex with 8 oxo guanosine and ddCTP at the insertion site
2EIQ 2007-03-13T00:00:00+0000 1.9 Design of Disulfide-linked Thioredoxin Dimers and Multimers Through Analysis of Crystal Contacts
2AJQ 2005-08-02T00:00:00+0000 2.6 Structure of replicative DNA polymerase provides insigts into the mechanisms for processivity, frameshifting and editing
1ZCP 2005-04-12T00:00:00+0000 2.3 Crystal Structure of a catalytic site mutant E. coli TrxA (CACA)
2EIO 2007-03-13T00:00:00+0000 2.6 Design of Disulfide-linked Thioredoxin Dimers and Multimers Through Analysis of Crystal Contacts
1X9M 2004-08-23T00:00:00+0000 2.1 T7 DNA polymerase in complex with an N-2-acetylaminofluorene-adducted DNA

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