RESUMO
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.
Assuntos
Bacteriófago T4/enzimologia , Proteínas de Ligação ao Cálcio/química , Muramidase/química , Sequência de Aminoácidos , Bacteriófago T4/genética , Sequência de Bases , Sítios de Ligação , Cálcio , Proteínas de Ligação ao Cálcio/genética , Estabilidade Enzimática , Escherichia coli/genética , Dados de Sequência Molecular , Muramidase/genética , Mutagênese Insercional , Conformação Proteica , Desnaturação Proteica , Triptofano/análiseRESUMO
Dihydrofolate reductase mutants with amino acid replacements in the active center (Thr35-->Asp mutant, Arg57-->His mutant and the mutant with triple replacement Thr35-->Asp, Asn37-->Ser, Arg57-->His) were obtained by site-directed mutagenesis. The stabilization effect of trimethoprim and NADP.H on the protein tertiary structure in vitro has been investigated. In the case of mutants with a 'weak' tertiary structure (Thr35-->Asp35 and the triple mutant) the separate addition of ligands does not affect their stability. The simultaneous addition of these ligands to Thr35-->Asp35 and the triple mutant leads to the large increase in their stability. A distinct correlation was found between the in vitro studied stability of the mutant proteins to the urea- or heat-induced denaturation and the level of proteolytic degradation of these mutants previously observed in vivo.
Assuntos
Estabilidade Enzimática/genética , Escherichia coli/enzimologia , Tetra-Hidrofolato Desidrogenase/química , Sistema Livre de Células , Escherichia coli/efeitos dos fármacos , Temperatura Alta , Mutagênese , Desnaturação Proteica , Estrutura Terciária de Proteína , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/metabolismo , Trimetoprima/farmacologia , Tripsina/farmacologiaRESUMO
The triple amino acid replacement (Asp10-->His, Asn101-->Asp, Arg148-->Ser) in T4 phage lysozyme was carried out by site-directed mutagenesis. At acid pH (2.7) the mutant is in a conformational state with the properties of the molten globule: (i) the mutant protein molecule is essentially compact; (ii) its CD spectrum in the near UV region is drastically reduced in intensity as compared with the wild type protein spectrum; (iii) the CD spectrum in the far UV region indicates the presence of pronounced secondary structure in the mutant; (iv) unlike the wild type protein the mutant protein can bind the hydrophobic fluorescent probe, ANS.
