Introduction of Ca2+-binding Amino-acid Sequence Into the T4 Lysozyme
Calcium Ion Binding, Ef-hand Motif, Site-directed Mutagenesis, T4 Lysozyme, T4
Digital Object Identifier (DOI)
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 Ca2+-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 Ca2+-induced destabilization of the protein.
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Citation / Publisher Attribution
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, v. 1162, issue 1-2, p. 84-88
Scholar Commons Citation
Leontiev, Vladimir V.; Uversky, Vladimir N.; Permyakova, Maria E.; and Murzin, N. V., "Introduction of Ca2+-binding Amino-acid Sequence Into the T4 Lysozyme" (1993). Molecular Medicine Faculty Publications. 624.