In Vitro Evolutionary Thermostabilization of Congerin II: A Limited Reproduction of Natural Protein Evolution by Artificial Selection Pressure

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. T...

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Bibliographic Details
Published in:Journal of molecular biology 2005-03, Vol.347 (2), p.385-397
Main Authors: Shionyu-Mitsuyama, Clara, Ito, Yoshimaro, Konno, Ayumu, Miwa, Yukiko, Ogawa, Tomohisa, Muramoto, Koji, Shirai, Tsuyoshi
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Base Sequence
crystal structure
Crystallography, X-Ray
Eels
Evolution, Molecular
galectin
Galectins - chemistry
Galectins - genetics
Galectins - metabolism
Hemagglutination
Models, Molecular
Molecular Sequence Data
Mutation
Protein Isoforms - chemistry
Protein Isoforms - genetics
Protein Isoforms - metabolism
Protein Structure, Tertiary
random mutagenesis
Selection, Genetic
Sequence Alignment
Temperature
thermostability
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Summary: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 Å, 1.8 Å, and 1.6 Å 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.
ISSN:0022-2836
1089-8638
DOI:10.1016/j.jmb.2005.01.027