title: Introduction of Ca(2+)-binding amino-acid sequence into the T4 lysozyme.
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.
authors.affiliation: Institute of Protein Research, Academy of Sciences of Russia, Pushchino, Moscow Region.
authors.forename: V V
authors.initials: VV
authors.lastname: Leontiev
authors.forename: V N
authors.initials: VN
authors.lastname: Uversky
authors.forename: E A
authors.initials: EA
authors.lastname: Permyakov
authors.forename: A G
authors.initials: AG
authors.lastname: Murzin
mesh_terms: D000595:Amino Acid Sequence; D017122:Bacteriophage T4 / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry* / Q000235:genetics; D016254:Mutagenesis, Insertional; D011487:Protein Conformation; D011489:Protein Denaturation; D014364:Tryptophan / Q000032:analysis
chemical_list: D002135:Calcium-Binding Proteins; D014364:Tryptophan; D009113:Muramidase; D002118:Calcium