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Introduction of Ca(2+)-binding amino-acid sequence into the T4 lysozymeстатья
- Авторы: Leontiev Vladimir V., Uversky Vladimir N., Permyakov Evgeny A., Murzin Alexey G.
- Журнал: Biochimica et Biophysica Acta - Proteins and Proteomics
- Том: 1162
- Номер: 1-2
- Год издания: 1993
- Издательство: Elsevier BV
- Местоположение издательства: Netherlands
- Первая страница: 84
- Последняя страница: 88
- DOI: 10.1016/0167-4838(93)90131-a
- Аннотация: The 51-62 loop of T4 phage lysozyme was altered by site-directed mutagenesis to obtain maximal homology with the typical EF-hand motif. A Ca(2+)-binding site was designed and created by replacing both Gly-51 and Asn-53 with aspartic acid. The mutant T4 lysozyme (G51D/N53D) was expressed in Escherichia coli. The activity of the G51D/N53D-mutant was about 60% of that of the wild-type protein. This mutant can bind Ca2+ ions specifically, while the effective dissociation constant was essentially greater than that of the EF-hand proteins. Stability of the G51D/N53D-mutant apo-form to urea- or temperature-induced denaturation was the same as that of the wild-type protein. In the presence of Ca2+ ions in solution the stability of the mutant T4 phage lysozyme was less than that of the wild-type protein. It is suggested that the binding of Ca2+ by the mutant is accompanied by the considerable conformational changes in the 'corrected' loop, which can lead to the Ca(2+)-induced destabilization of the protein.
- Добавил в систему: Уверский Владимир Николаевич