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

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
Sequence Assay Result Units