RESUMEN
Designed ankyrin repeat proteins (DARPins) are valuable tools in both biochemistry and medicine. Herein we describe a rapid, simple method for the dual modification of DARPins by introduction of cysteine mutations at specific positions that results in a vast difference in their thiol nucleophilicity, allowing for clean sequential modification.
Asunto(s)
Ancirinas/metabolismo , Ancirinas/química , Ancirinas/genética , Dicroismo Circular , Cisteína/metabolismo , Humanos , Mutagénesis Sitio-Dirigida , Unión Proteica , Ingeniería de Proteínas , Receptor ErbB-2/química , Receptor ErbB-2/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMEN
Reversible protein biotinylation is readily affected via conjugation with a bromomaleimide-based reagent followed by reductive cleavage. The intermediate biotinylated protein constructs are stable at physiological temperature and pH 8.0. Quantitative reversibility is elegantly delivered under mild conditions of using a stoichiometric amount of a bis-thiol, thus providing an approach that will be of general interest in chemical biology and proteomics.
Asunto(s)
Marcadores de Afinidad/química , Biotina/química , Maleimidas/química , Estreptavidina/química , Concentración de Iones de Hidrógeno , Hidrólisis , Modelos Moleculares , Estructura Terciaria de Proteína , TemperaturaRESUMEN
Local protein microenvironment is used to control the outcome of reaction between cysteine residues and 2,5-dibromohexanediamide. The differential reactivity is exploited to introduce two orthogonal reactive handles onto the surface of a double cysteine mutant of superfolder green fluorescent protein in a regioselective manner. Subsequent elaboration with commonly used thiol and alkyne containing reagents affects site-selective protein dual labelling.
RESUMEN
Smooth converter: Bioconjugation of superfolder GFP involving the formation of an unusually stable, and unprecedented, cyclic sulfonium species is described. This sulfonium can undergo smooth reaction with a range of nucleophiles to give sulfur-, selenium- and azide-modified GFP derivatives in high conversions.
Asunto(s)
Proteínas Fluorescentes Verdes/química , Compuestos de Sulfonio/química , Cisteína , Proteínas Fluorescentes Verdes/genética , Modelos Moleculares , Mutación , Conformación ProteicaRESUMEN
Bromomaleimides react rapidly and selectively with cysteine to afford thiomaleimides which can be cleaved with a phosphine to regenerate the cysteine or treated with a base to afford dehydroalanine.
