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PDBsum entry 1m7t
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Electron transport
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PDB id
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1m7t
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Contents |
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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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Solution structure and dynamics of the human-Escherichia coli thioredoxin chimera: insights into thermodynamic stability.
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Authors
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B.Dangi,
A.V.Dobrodumov,
J.M.Louis,
A.M.Gronenborn.
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Ref.
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Biochemistry, 2002,
41,
9376-9388.
[DOI no: ]
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PubMed id
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Abstract
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We have determined the high-resolution solution structure of the oxidized form
of a chimeric human and Escherichia coli thioredoxin (TRX(HE)) by NMR. The
overall structure is well-defined with a rms difference for the backbone atoms
of 0.27 +/- 0.06 A. The topology of the protein is identical to those of the
human and E. coli parent proteins, consisting of a central five-stranded
beta-sheet surrounded by four alpha-helices. Analysis of the interfaces between
the two domains derived from the human and E. coli sequences reveals that the
general hydrophobic packing is unaltered and only subtle changes in the details
of side chain interactions are observed. The packing of helix alpha(4) with
helix alpha(2) across the hybrid interface is less optimal than in the parent
molecules, and electrostatic interactions between polar side chains are missing.
In particular, lysine-glutamate salt bridges between residues on helices
alpha(2) and alpha(4), which were observed in both human and E. coli proteins,
are not present in the chimeric protein. The origin of the known reduced
thermodynamic stability of TRX(HE) was probed by mutagenesis on the basis of
these structural findings. Two mutants of TRX(HE), S44D and S44E, were created,
and their thermal and chemical stabilities were examined. Improved stability
toward chaotropic agents was observed for both mutants, but no increase in the
denaturation temperature was seen compared to that of TRX(HE). In addition to
the structural analysis, the backbone dynamics of TRX(HE) were investigated by
(15)N NMR relaxation measurements. Analysis using the model free approach
reveals that the protein is fairly rigid with an average S(2) of 0.88. Increased
mobility is primarily present in two external loop regions comprising residues
72-74 and 92-94 that contain glycine and proline residues.
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