RESUMO
Recently, a novel transmembrane protein was found to be up-regulated in the auditory learning pathway of birds and mammals. The protein, FnTm2, was predicted to have an extracellular fibronectin III (Fn3) domain and a single transmembrane domain. By contrast to other studied Fn3 domains the extracellular domain of FnTm2 bears several cysteine residues, which are predicted to form disulfide bonds. The Fn3 domain of the FnTm2 protein was expressed in DH5-α Escherichia coli (E. coli) cells, purified and characterized by circular dichroism (CD). In order to identify binding partners to Fn3, the isolated protein was incubated with bird brain lysate for a pull down treatment. Of the proteins recognized, myelin basic protein (MBP) was identified as a bona fide partner; it was further characterized for binding to Fn3 in vitro via fluorescence spectroscopy and confirmed via isothermal calorimetry (ITC).
Assuntos
Proteínas Aviárias/metabolismo , Fibronectinas/química , Proteínas Recombinantes/metabolismo , Actinas/química , Actinas/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Aviárias/química , Proteínas Aviárias/genética , Química Encefálica , Calorimetria , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Tentilhões , Masculino , Dados de Sequência Molecular , Proteína Básica da Mielina/química , Proteína Básica da Mielina/metabolismo , Domínios e Motivos de Interação entre Proteínas , Redobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Espectrometria de FluorescênciaRESUMO
Some viruses use phosphatidylinositol phosphate (PIP) to mark membranes used for genome replication or virion assembly. PIP-binding motifs of cellular proteins do not exist in viral proteins. Molecular-docking simulations revealed a putative site of PIP binding to poliovirus (PV) 3C protein that was validated using nuclear magnetic resonance spectroscopy. The PIP-binding site was located on a highly dynamic α helix, which also functions in RNA binding. Broad PIP-binding activity was observed in solution using a fluorescence polarization assay or in the context of a lipid bilayer using an on-chip, fluorescence assay. All-atom molecular dynamics simulations of the 3C protein-membrane interface revealed PIP clustering and perhaps PIP-dependent conformations. PIP clustering was mediated by interaction with residues that interact with the RNA phosphodiester backbone. We conclude that 3C binding to membranes will be determined by PIP abundance. We suggest that the duality of function observed for 3C may extend to RNA-binding proteins of other viruses.