In vitro evolutionary thermostabilization of congerin II: a limited reproduction of natural protein evolution by artificial selection pressure
| Autor: | Tsuyoshi Shirai, Ayumu Konno, Koji Muramoto, Yoshimaro Ito, Tomohisa Ogawa, Clara Shionyu-Mitsuyama, Yukiko Miwa |
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| Rok vydání: | 2004 |
| Předmět: |
Models
Molecular Galectins Mutant Molecular Sequence Data Protein Data Bank (RCSB PDB) Biology medicine.disease_cause Crystallography X-Ray Evolution Molecular Structural Biology medicine Animals Protein Isoforms Amino Acid Sequence Selection Genetic Molecular Biology Gene Galectin Thermostability Mutation Eels Base Sequence Hemagglutination Mutagenesis Temperature Protein engineering Protein Structure Tertiary Biochemistry Sequence Alignment |
| Zdroj: | Journal of molecular biology. 347(2) |
| ISSN: | 0022-2836 |
| Popis: | The thermostability of the conger eel galectin, congerin II, was improved by in vitro evolutionary protein engineering. Two rounds of random PCR mutagenesis and selection experiments increased the congerin II thermostability to a level comparative to its naturally thermostable isoform, congerin I. The crystal structures of the most thermostable double mutant, Y16S/T88I, and the related single mutants, Y16S and T88I, were determined at 2.0 angstroms, 1.8 angstroms, and 1.6 angstroms resolution, respectively. The exclusion of two interior water molecules by the Thr88Ile mutation, and the relief of adjacent conformational stress by the Tyr16Ser mutation were the major contributions to the thermostability. These features in the congerin II mutants are similar to those observed in congerin I. The natural evolution of congerin genes, with the K(A)/K(S) ratio of 2.6, was accelerated under natural selection pressures. The thermostabilizing selection pressure artificially applied to congerin II mimicked the implied natural pressure on congerin I. The results showed that the artificial pressure made congerin II partially reproduce the natural evolution of congerin I. |
| Databáze: | OpenAIRE |
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