PMID:8448199
| Citation |
Leontiev, VV, Uversky, VN, Permyakov, EA and Murzin, AG (1993) Introduction of Ca(2+)-binding amino-acid sequence into the T4 lysozyme. Biochim. Biophys. Acta 1162:84-8 |
|---|---|
| Abstract |
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. |
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| Keywords |
Amino Acid Sequence; Bacteriophage T4/enzymology; Bacteriophage T4/genetics; Base Sequence; Binding Sites; Calcium; Calcium-Binding Proteins/chemistry; Calcium-Binding Proteins/genetics; Enzyme Stability; Escherichia coli/genetics; Molecular Sequence Data; Muramidase/chemistry; Muramidase/genetics; Mutagenesis, Insertional; Protein Conformation; Protein Denaturation; Tryptophan/analysis |
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