Leontiev V V, Uversky V N, Permyakov E A, Murzin A G
Institute of Protein Research, Academy of Sciences of Russia, Pushchino, Moscow Region.
Biochim Biophys Acta. 1993 Mar 5;1162(1-2):84-8. doi: 10.1016/0167-4838(93)90131-a.
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.
通过定点诱变改变T4噬菌体溶菌酶的51-62环，以使其与典型的EF-手基序具有最大同源性。通过用天冬氨酸取代甘氨酸-51和天冬酰胺-53来设计并创建一个钙离子结合位点。突变型T4溶菌酶（G51D/N53D）在大肠杆菌中表达。G51D/N53D突变体的活性约为野生型蛋白活性的60%。该突变体能特异性结合钙离子，但其有效解离常数基本上大于EF-手蛋白的解离常数。G51D/N53D突变体脱辅基形式对尿素或温度诱导变性的稳定性与野生型蛋白相同。在溶液中存在钙离子的情况下，突变型T4噬菌体溶菌酶的稳定性低于野生型蛋白。有人提出，突变体对钙离子的结合伴随着“校正”环中相当大的构象变化，这可能导致钙离子诱导的蛋白质不稳定。
