RESUMO
Three different forms of dihydrofolate reductase (DHFR) from Escherichia coli with amino acid replacements Thr35----Asp, Asn37----Ser and Arg57----His, and one form containing all three of these changes were obtained by oligonucleotide-directed mutagenesis. These amino acids are on the surface of the protein and two of them (Thr35 and Arg57) are invariant for known sequences of DHFR. Conversion of Asn37----Ser has no effect on the functional activity or the protein level in the cells. The Thr35----Asp replacement leads to a sharp decrease in the protein level, while the addition of a DHFR inhibitor, trimethoprim (Tmp), to the growth medium increases the level of DHFR in the cells. There is a very small quantity of DHFR with all three amino acid changes. The addition of Tmp to the growth medium also leads to an increase in the mutant protein levels. The mutant with the Arg57----His replacement renders the cells sensitive to Tmp, but the level of DHFR is the same as for the wild-type protein. It is suggested that the invariant Thr35 is important for the stable conformation of DHFR whereas Arg57 is essential for protein activity. Various structural and functional aspects of these results are discussed.
Assuntos
Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Tetra-Hidrofolato Desidrogenase/genética , Sequência de Aminoácidos , Sequência de Bases , Eletroforese em Gel de Poliacrilamida , Escherichia coli/enzimologia , Escherichia coli/crescimento & desenvolvimento , Antagonistas do Ácido Fólico , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica , Engenharia de Proteínas , Mapeamento por Restrição , Tetra-Hidrofolato Desidrogenase/metabolismo , Trimetoprima/farmacologiaRESUMO
Three circularly permuted variants of Escherichia coli dihydrofolate reductase genes were constructed. Linkers coding tri- and pentapeptides were used to connect the natural 5'- and 3'-terminal ends. Only one variant of circularly permuted protein with tripeptide linker and the cleavage of the peptide bond between 107 and 108 amino acid residues was produced in a good yield. The expressed protein was insoluble in the cells, but at pH 8.0 and higher the isolated protein was soluble. Enzymatic assay and physical studies have shown that permuted dihydrofolate reductase has a destabilized tertiary structure. Only the addition of the natural substrates or inhibitors lead to the protein with the native-like structure and functional activity.
Assuntos
Escherichia coli/enzimologia , Estrutura Terciária de Proteína , Tetra-Hidrofolato Desidrogenase/química , Sequência de Aminoácidos , Sequência de Bases , Dicroísmo Circular , Estabilidade Enzimática , Escherichia coli/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Desnaturação Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Espectrometria de Fluorescência , Tetra-Hidrofolato Desidrogenase/efeitos dos fármacos , Tetra-Hidrofolato Desidrogenase/genética , Ureia/farmacologiaRESUMO
The structure of a chromatin binding domain from mouse chromatin modifier protein 1 (MoMOD1) was determined using nuclear magnetic resonance (NMR) spectroscopy. The protein consists of an N-terminal three-stranded anti-parallel beta-sheet which folds against a C-terminal alpha-helix. The structure reveals an unexpected homology to two archaebacterial DNA binding proteins which are also involved in chromatin structure. Structural comparisons suggest that chromo domains, of which more than 40 are now known, act as protein interaction motifs and that the MoMOD1 protein acts as an adaptor mediating interactions between different proteins.
Assuntos
Proteínas Arqueais , Proteínas de Transporte/química , Cromatina/química , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Cromatina/metabolismo , Cromatografia Líquida de Alta Pressão , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/metabolismo , Clonagem Molecular , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Espectroscopia de Ressonância Magnética , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , SoluçõesRESUMO
The heterochromatin protein 1 (HP1) family of proteins is involved in gene silencing via the formation of heterochromatic structures. They are composed of two related domains: an N-terminal chromo domain and a C-terminal shadow chromo domain. Present results suggest that chromo domains may function as protein interaction motifs, bringing together different proteins in multi-protein complexes and locating them in heterochromatin. We have previously determined the structure of the chromo domain from the mouse HP1beta protein, MOD1. We show here that, in contrast to the chromo domain, the shadow chromo domain is a homodimer. The intact HP1beta protein is also dimeric, where the interaction is mediated by the shadow chromo domain, with the chromo domains moving independently of each other at the end of flexible linkers. Mapping studies, with fragments of the CAF1 and TIF1beta proteins, show that an intact, dimeric, shadow chromo domain structure is required for complex formation.