In vitro evolutionary thermostabilization of congerin II: A limited reproduction of natural protein evolution by artificial selection pressure

Clara Shionyu-Mitsuyama, Yoshimaro Ito, Ayumu Konno, Yukiko Miwa, Tomohisa Ogawa, Koji Muramoto, Tsuyoshi Shirai

Research output: Contribution to journalArticlepeer-review

12 Citations (Scopus)

Abstract

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 KA/KS 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.

Original languageEnglish
Pages (from-to)385-397
Number of pages13
JournalJournal of Molecular Biology
Volume347
Issue number2
DOIs
Publication statusPublished - 2005 Mar 25

Keywords

  • Crystal structure
  • Galectin
  • Random mutagenesis
  • Thermostability

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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