RESUMO
Seventeen mutants with one, two or three amino acids substitutions of human protein p14.5, homologue to well-known tumor antigen from goat liver UK114 and a member of proteins YER057c/YIL051c/YjgF family, have been used for structure-functional relation studies and ligand binding analysis using cross-linking by triacryloyl-hexahydro-s-triazine (TAT), size exclusion chromatography, free fatty acid and 8-anilino-1-naphthalenesulfonic acid (ANS) binding assays. Amino acids having the most significant impact on the ligand binding activity have been determined: R107, N93, Y21 and F89. Arginine 107 has been identified as the most accessible amino acid in the cleft. Trimeric structure of protein p14.5 has been confirmed as being essential for stoichiometric small ligand binding activity and oligomeric structure of p14. Ligand binding activity may be related with the biological functions of these proteins, which still are not understood well.
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Choque Térmico/química , Proteínas Mitocondriais/química , Ribonucleases/química , Proteínas de Saccharomyces cerevisiae/química , Naftalenossulfonato de Anilina/farmacologia , Sítios de Ligação , Bioquímica/métodos , Cromatografia , Clonagem Molecular , Reagentes de Ligações Cruzadas/farmacologia , DNA Complementar/metabolismo , Eletroforese em Gel de Poliacrilamida , Ácidos Graxos/química , Corantes Fluorescentes/farmacologia , Humanos , Cinética , Ligantes , Modelos Moleculares , Conformação Molecular , Mutagênese Sítio-Dirigida , Mutação , Ligação Proteica , Relação Estrutura-Atividade , Triazinas/farmacologiaRESUMO
Proteins UK114 and p14.5 are both members of the putative family of small proteins YER057c/YIL051c/YjgF. The biological role of these proteins is not understood very well, and in addition, their oligomeric structure in solution remains controversial. We therefore investigated the oligomeric structure of UK114 and p14.5 using a number of methods. Both proteins have exhibited a homotrimeric structure in solution. Indeed the trimeric structure of the two proteins appeared to be so similar that when protein subunits derived from different species were mixed, stable heterotrimeric complexes (monomer ratio of 1:2 and 2:1 of UK114 and p14.5, respectively) could be formed in vitro. Furthermore, the trimeric structure of both UK114 and p14.5 proved essential for the stoichiometric hydrophobic ligand, such as fatty acid binding activity of the two proteins.
Assuntos
Proteínas de Choque Térmico/química , Proteínas de Neoplasias/química , Naftalenossulfonato de Anilina/química , Animais , Sítios de Ligação , Cromatografia Líquida , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Ácidos Graxos/metabolismo , Proteínas de Choque Térmico/genética , Humanos , Cinética , Ligantes , Proteínas de Neoplasias/genética , Ligação Proteica , Proteínas Recombinantes de Fusão/química , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
The tumour-associated antigen UK114, isolated from goat liver, belongs to the YER057c/YIL051c/YjgF protein family, which has members in both the prokaryotes and eukaryotes. The crystal structure of a mammalian representative, goat UK114, was determined, revealing a trimeric arrangement in the crystal. It was confirmed by ultracentrifugation that UK114 is a trimer in solution. These results are in agreement with the published structures of homologues from unicellular organisms, but contrast with those reported for the rat homologue of UK114, for which a dimeric quaternary structure was proposed.
