Hedgehog (Hh) precursor proteins contain an autoprocessing domain called HhC whose native function is protein cleavage and C-terminal glycine sterylation. The transformation catalyzed by HhC occurs in cis from a precursor protein and exhibits wide tolerance toward both sterol and protein substrates. Here, we repurpose HhC as a 1:1 protein−nucleic acid ligase, with the sterol serving as a molecular linker. A procedure is described for preparing HhCactive sterylated DNA, called steramers, using aqueous compatible chemistry and commercial reagents. Steramers have K M values of 7−11 μM and reaction t 1/2 values of ∼10 min. Modularity of the HhC/steramer method is demonstrated using four different proteins along with structured and unstructured sterylated nucleic acids. The resulting protein−DNA conjugates retain the native solution properties and biochemical function. Unlike self-tagging domains, HhC does not remain fused to the conjugate; rather, enzymatic activity is mechanistically coupled to conjugate release. That unique feature of HhC, coupled with efficient kinetics and substrate tolerance, may ease access and open new applications for these suprabiological chimeras.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.