Thermodynamic and structural analysis of highly stabilized BPTIs by single and double mutations

Enhancing protein conformational stability is an important aspect of protein engineering and biotechnology. However, protein stabilization is difficult to rationalize as it often results from the small cumulative and intertwined effects of multiple mutations. Here, we analyzed the mechanisms behind...

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Published in:Proteins, structure, function, and bioinformatics structure, function, and bioinformatics, 2009-12, Vol.77 (4), p.962-970
Main Authors: Islam, Mohammad Monirul, Sohya, Shihori, Noguchi, Keiichi, Kidokoro, Shun-ichi, Yohda, Masafumi, Kuroda, Yutaka
Format: Article
Language:English
Subjects:
Amino Acid Substitution
Animals
Aprotinin - chemistry
Aprotinin - genetics
Binding Sites - genetics
Calorimetry, Differential Scanning
Cattle
Crystallography, X-Ray
enthalpy stabilization
Entropy
In Vitro Techniques
Models, Molecular
Mutagenesis, Site-Directed
Protein Conformation
protein engineering
Protein Stability
protein structure
Recombinant Proteins - chemistry
Recombinant Proteins - genetics
steric clash
Thermodynamics
Water - chemistry
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Summary:Enhancing protein conformational stability is an important aspect of protein engineering and biotechnology. However, protein stabilization is difficult to rationalize as it often results from the small cumulative and intertwined effects of multiple mutations. Here, we analyzed the mechanisms behind a remarkable 13° stabilization produced by a single A14G and a double A14GA38V mutation in BPTI‐[5,55], a natively folded bovine pancreatic trypsin inhibitor variant. Differential scanning calorimetry analysis of three BPTI‐[5,55] variants (A14G, A38V, and A14GA38V) indicated that the A14G mutation stabilized the structure enthalpically, whereas the A38V stabilization was entropy driven. We also determined the structure of the A14GA38V mutant at 1.09 Å resolution, whereas the A38V variant did not crystallize, and we previously reported the A14G variant's structure (2ZJX). The overall structures of the A14G and A14GA38V variants were very similar to that of wild‐type BPTI, but small local structure perturbations around residues 14 and 38 strongly suggested potential factors contributing to the enthalpy stabilization. First, the A14G mutation displaced the local backbone structures around residues 14 and 38 by up to 0.7 Å, presumably increasing local van der Waals interactions. Next, this displacement produced steric clashes between neighboring residue's side‐chains in all but the variants containing the A14G mutation. Noteworthy, these clashes are not predicted from the wild type BPTI structure. These observations provide one of the first unambiguous analyses of how a subtle interplay between the sidechain and backbone structures can have a major effect on protein stability. Proteins 2009. © 2009 Wiley‐Liss, Inc.
ISSN:0887-3585
1097-0134
DOI:10.1002/prot.22522