화학공학소재연구정보센터
Journal of the American Chemical Society, Vol.133, No.44, 17855-17868, 2011
Nonenzymatic Assembly of Natural Polyubiquitin Chains of Any Linkage Composition and Isotopic Labeling Scheme
Polymeric chains made Of a small protein ubiquitin act as molecular signals regulating a variety of cellular processes controlling essentially all aspects of eukaryotic biology. Uncovering the mechanisms that allow differently linked polyubiquitin chains to serve as distinct molecular signals requires the ability to make these chains with the native connectivity, defined length, linkage composition, and in sufficient quantities. This, however, has been a major impediment in the ubiquitin field. Here; we present a robust, efficient, and widely accessible method for controlled iterative nonenzymatic assembly of polyubiquitin chains using recombinant ubiquitin monomers as the primary building blocks. This method uses silver-mediated:condensation reaction between the C-terminal thioester of one ubiquitin and the E-amine of a specific lysine ()tithe other ubiquitin. We augment the nonenzymatic approaches developed recently by using removable orthogonal amine protecting groups, Alloc and Boc. The use of bacterially expressed ubiquitins allows cost effective isotopic enrichment, Of any individual :monomer in the Chain. We demonstrate that our method yield's completely natural polyubiquitin chains (free of mutations and linked through native isopeptide bonds) Of essentially any desired length, linkage composition; and isotopic labeling scheme, and in milligram quantities. Specifically, we successfully made Lys11-linked di-, tri-, and tetra-ubiquitins, Lys33-linked diubiquitin, and a mixed-linkage Lys33, Lys33,Lys11-linked triubiquitin. We also demonstrate the ability to obtain, by high-resolution NMR, residue-specific information on ubiquitin units at any desired position in such chains. This method opens up essentially endless possibilities for rigorous structural and functional studies of polyubiquitin signals.