RESUMO
Proteins represent powerful biomacromolecules due to their unique functionality and broad utility both in the cell and in non-biological applications. The genetic encoding of non-canonical amino acids (ncAAs) facilitates functional diversification of these already powerful proteins. Specifically, ncAAs have been demonstrated to provide unique functional handles to bioorthogonally introduce novel functionality via conjugation reactions. Herein we examine the ability of a single ncAA to serve as a handle to generate multivalent bioconjugates to introduce two or more additional components to a protein, yielding a multivalent conjugate. To accomplish this aim, p-bromopropargyloxyphenyalanine (pBrPrF) was genetically encoded into both superfolder green fluorescent protein (sfGFP) and ubiquitin model proteins to serve as a conjugation handle. A sequential bioconjugation sequence involving a copper-assisted cycloaddition reaction coupled with a subsequent Sonogashira cross-coupling was then optimized. The linkage of two additional molecules to the model protein via these reactions yielded the desired multivalent bioconjugate. This domino approach using a single ncAA has a plethora of applications in both therapeutics and diagnostics as multiple unique moieties can be introduced into proteins in a highly controlled fashion.
Assuntos
Aminoácidos , Aminoácidos/química , Proteínas de Fluorescência Verde/químicaRESUMO
Protein methyltransferases are vital to the epigenetic modification of gene expression. Thus, obtaining a better understanding of and control over the regulation of these crucial proteins has significant implications for the study and treatment of numerous diseases. One ideal mechanism of protein regulation is the specific installation of a photolabile-protecting group through the use of photocaged non-canonical amino acids. Consequently, PRMT1 was caged at a key tyrosine residue with a nitrobenzyl-protected Schultz amino acid to modulate protein function. Subsequent irradiation with UV light removes the caging group and restores normal methyltransferase activity, facilitating the spatial and temporal control of PRMT1 activity. Ultimately, this caged PRMT1 affords the ability to better understand the protein's mechanism of action and potentially regulate the epigenetic impacts of this vital protein.