title: A fluorimetric readout reporting the kinetics of nucleotide-induced human ribonucleotide reductase oligomerization.
abstract: Human ribonucleotide reductase (hRNR) is a target of nucleotide chemotherapeutics in clinical use. The nucleotide-induced oligomeric regulation of hRNR subunit α is increasingly being recognized as an innate and drug-relevant mechanism for enzyme activity modulation. In the presence of negative feedback inhibitor dATP and leukemia drug clofarabine nucleotides, hRNR-α assembles into catalytically inert hexameric complexes, whereas nucleotide effectors that govern substrate specificity typically trigger α-dimerization. Currently, both knowledge of and tools to interrogate the oligomeric assembly pathway of RNR in any species in real time are lacking. We therefore developed a fluorimetric assay that reliably reports on oligomeric state changes of α with high sensitivity. The oligomerization-directed fluorescence quenching of hRNR-α, covalently labeled with two fluorophores, allows for direct readout of hRNR dimeric and hexameric states. We applied the newly developed platform to reveal the timescales of α self-assembly, driven by the feedback regulator dATP. This information is currently unavailable, despite the pharmaceutical relevance of hRNR oligomeric regulation.
authors.affiliation: Department of Chemistry and Chemical Biology Cornell University, Ithaca, NY 14853.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Fu
authors.affiliation: Department of Chemistry and Chemical Biology Cornell University, Ithaca, NY 14853.
authors.forename: Hongyu
authors.initials: H
authors.lastname: Lin
authors.affiliation: Department of Chemistry and Chemical Biology Cornell University, Ithaca, NY 14853.
authors.forename: Somsinee
authors.initials: S
authors.lastname: Wisitpitthaya
authors.affiliation: Department of Chemistry and Chemical Biology Cornell University, Ithaca, NY 14853.
authors.forename: William A
authors.initials: WA
authors.lastname: Blessing
authors.affiliation: Department of Chemistry and Chemical Biology Cornell University, Ithaca, NY 14853.|Department of Biochemistry Weill Cornell Medical College, New York, NY, 10065.
authors.forename: Yimon
authors.initials: Y
authors.lastname: Aye
mesh_terms: D003838:Deoxyadenine Nucleotides / Q000378:metabolism / Q000494:pharmacology*; D005470:Fluorometry; D006801:Humans; D007700:Kinetics; D055503:Protein Multimerization / Q000187:drug effects*; D012264:Ribonucleotide Reductases / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D003838:Deoxyadenine Nucleotides; D012264:Ribonucleotide Reductases; C026600:2'-deoxyadenosine triphosphate
keywords: feedback inhibition; fluorescence reporter assay; human ribonucleotide reductase; oligomeric regulation; stopped-flow kinetics