RESUMEN
In this report we present a method to identify functional artificial lantipeptides. In vitro translation coupled with an enzyme-free protocol for posttranslational modification allows preparation of more than 10(11) different lanthionine containing peptides. This diversity can be searched for functional molecules using mRNA-lantipeptide display. We validated this approach by isolating binders toward Sortase A, a transamidase which is required for virulence of Staphylococcus aureus. The interaction of selected lantipeptides with Sortase A is highly dependent on the presence of a (2S,6R)-lanthionine in the peptide and an active conformation of the protein.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Péptidos/genética , Péptidos/metabolismo , Ingeniería de Proteínas/métodos , Secuencia de Aminoácidos , Aminoaciltransferasas/metabolismo , Proteínas Bacterianas/química , Cisteína Endopeptidasas/metabolismo , Biblioteca de Genes , Péptidos/químicaRESUMEN
A series of 16 synthetic scramblase candidates were prepared from a tris(aminoethyl)amine (TREN) scaffold and evaluated for ability to facilitate translocation of fluorescent phospholipid probes across vesicle membranes and endogenous phosphatidylserine across the plasma membrane of nucleated cells. More than half of the compounds were found to greatly accelerate phospholipid translocation in vesicles. However, they were generally unable to induce large increases in the amount of phosphatidylserine on the surface of nucleated mammalian cells, which contrasts with previous results using erythrocytes. Fluorescence microscopy showed that the synthetic scramblases are rapidly trafficked out of the cell plasma membrane and into the membranes of internal organelles. Future molecular designs of synthetic scramblases should focus on structures that are more amphiphilic, a structural feature that is expected to increase plasma membrane residence time.
Asunto(s)
Fosfolípidos/metabolismo , Transporte Biológico , Membrana Celular , Células , Eritrocitos , Colorantes Fluorescentes , Humanos , Membranas Intracelulares , Células Jurkat , Liposomas , Microscopía Fluorescente , Imitación Molecular , Fosfatidilserinas/metabolismo , Proteínas de Transferencia de FosfolípidosRESUMEN
We have developed a synthesis of phosphoarginine containing peptides using a bis(2,2,2-trichloroethyl) protected phosphoarginine derivative as building block. Binding studies and computer modelling demonstrate the ability of the SH2 domain from Src kinase to recognize a phosphoarginine-containing peptide in a phosphoryl group-dependent manner.