ORGN 138 |
| Ribonucleotide reductases catalyze the conversion of nucleotides to deoxynucleotides required for DNA replication and repair in all organisms. Class I RNRs are composed of two subunits: ? and ?. ? binds the NDP substrates and dNTP (ATP) effectors that govern substrate specificity and turnover rates. ? contains the essential diferric-tyrosyl radical cofactor. The active form of RNR is a complex of the two subunits and the quaternary structure differs between organisms (?n?n, where n = 2, 4 or 6). The interaction between the subunits is dramatically effected by substrate/effector binding to ? and plays a key role in maintaining the balanced dNTP pools essential for fidelity of DNA replication and repair. A better understanding of subunit interactions in RNR could be exploited to develop novel therapeutic agents. We have initiated a systematic study of the subunit interactions using E. coli RNR, a model for mammalian RNRs. Previous studies have shown that the C-terminus (350-375) of ? is largely responsible for subunit interactions. We have site specifically incorporated cysteines at position 341-375 of ? to attach an environmentally sensitive fluorophore (6-bromoacetyl-2-dimethyl aminonaphthalene -BADAN) or a cross-linker (N-(4-Benzoyl-phenyl)-2-iodo-acetamide-BPI). The scanning of the interface with BADAN-? in titrations with ? in the presence and absence of NDP/dNTP pairs have provided insight about the environment at the subunit interface and allowed determination of the Kds for subunit interactions. To trap the transient complex between ? and ? and to identify the interacting residues at the interface, we have used ?s with site-specifically incorporated BPIs. The cross-linking efficiency matches the regions of hydrophobicity observed with the BADANs titrations. Methods to identify the sites of cross-linking using a mixture of protonated and deuteriated-BPIs will be presented using HPLC separation of tyrpsinized protein, followed by MALDI-TOF. |
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Total Synthesis, Materials, Devices and Switches, Molecular Recognition and Self-Assembly, Biologically-Related Molecules and Processes
8:00 PM-10:00 PM, Sunday, August 19, 2007 BCEC -- Exhibit Hall - B2, Poster
Division of Organic Chemistry |