RESUMEN
Thiol-reactive reagents designed for the chemical modification of proteins cannot, in general, be used directly for the modification of intracellular targets because the presence of millimolar concentrations of glutathione inside cells effectively outcompetes reaction with target thiols. Here we report an equilibrium, entropic strategy for achieving target selectivity using a cyanoacrylate-based thiol-reactive cross-linker (BCNA) with two reactive sites. This compound exhibits â³200-fold selectivity for reaction with target peptides and proteins containing appropriately spaced pairs of thiols, versus reaction with mono-thiols. Photo-isomerization of the azobenzene moiety of the cross-linker can be used to affect the conformation of the target peptide or protein. This approach suggests a general strategy for the chemical modification of intracellular peptide and protein targets.
Asunto(s)
Compuestos Azo , Proteínas , Reactivos de Enlaces Cruzados/química , Compuestos Azo/química , Péptidos/química , Compuestos de Sulfhidrilo/químicaRESUMEN
The photo-control of protein activity can often be achieved via the photo-control of protein structure. Intramolecular cross-linkers that change length upon photoisomerization provide a means to photo-control protein structure by linking to pairs of Cys residues in a protein sequence. In this protocol, we describe general methods for introducing intramolecular cross-linkers, both UV light switchable and red-light switchable, under either denaturing or native conditions.
Asunto(s)
Compuestos Azo/química , Reactivos de Enlaces Cruzados/química , Cisteína/química , Proteínas/química , Secuencia de Aminoácidos , Compuestos Azo/síntesis química , Técnicas de Química Sintética/métodos , Cromatografía Líquida de Alta Presión/métodos , Reactivos de Enlaces Cruzados/síntesis química , Cisteína/síntesis química , Luz , Modelos Moleculares , Procesos Fotoquímicos , Conformación Proteica , Pliegue de Proteína , Proteínas/síntesis química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Espectrofotometría Ultravioleta/métodos , Rayos UltravioletaRESUMEN
Photo-controlled affinity reagents seek to provide modular spatiotemporal control of bioactivity by conferring photo-switchability of function on an affinity reagent scaffold. Here we used Rosetta-based computational methods to screen for sites on the Fynomer affinity reagent structure for attachment of photoswitchable cross-linkers. Both established UV-based cross-linkers (azobenzene-iodoacetamide (IAC)) and an azonium-based efficient red light switchable cross-linker, piperazino-tetra-ortho-methoxy azobenzene (PIP), were then tested experimentally. Several sites compatible with Fynomer function were identified, including sites showing rapid (<10s) red light (633 nm) modulation of function. While a range of overall target binding affinities were observed, the degree of photo-switchability of Fynomer function was generally small (<2-fold). Computational models suggest that local flexibility limits the degree of switching seen in these designs.