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. 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.
Shionyu-Mitsuyama
Clara
C
Department of Computational Biology, Biomolecular Engineering Research Institute, 6-2-3 Furuedai, Suita, Osaka 565-0874, Japan.
Ito
Yoshimaro
Y
Konno
Ayumu
A
Miwa
Yukiko
Y
Ogawa
Tomohisa
T
Muramoto
Koji
K
Shirai
Tsuyoshi
T
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D018360:Crystallography, X-Ray; D004524:Eels; D019143:Evolution, Molecular; D037161:Galectins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006384:Hemagglutination; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012641:Selection, Genetic; D016415:Sequence Alignment; D013696:Temperature
D037161:Galectins; D020033:Protein Isoforms; C122080:congerin II protein, Conger eel