RESUMO
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.
Assuntos
Anquirinas/metabolismo , Anquirinas/química , Anquirinas/genética , Dicroísmo Circular , Cisteína/metabolismo , Humanos , Mutagênese Sítio-Dirigida , Ligação Proteica , Engenharia de Proteínas , Receptor ErbB-2/química , Receptor ErbB-2/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
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.
Assuntos
Marcadores de Afinidade/química , Biotina/química , Maleimidas/química , Estreptavidina/química , Concentração de Íons de Hidrogênio , Hidrólise , Modelos Moleculares , Estrutura Terciária de Proteína , TemperaturaRESUMO
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.
RESUMO
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.
Assuntos
Proteínas de Fluorescência Verde/química , Compostos de Sulfônio/química , Cisteína , Proteínas de Fluorescência Verde/genética , Modelos Moleculares , Mutação , Conformação ProteicaRESUMO
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.
