Structural Studies of Archaeal Ribonucleotide Reductases

Massachusetts Institute Of Technology, Cambridge MA

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Abstract

[unreadable] DESCRIPTION (provided by applicant): The primary objective of this research proposal is to obtain structural insight into the molecular details of the allosteric regulation of ribonucleotide reductases (RNRs). Dimeric Class II RNRs from several archaeal organisms will be used as our model system for the RNR enzyme family. The first aim is to clone, express, and characterize several Class II RNRs. The activity and allosteric regulation of the RNRs will be determined and compared to literature values for other RNR enzymes. The second aim is to obtain protein crystals of diffraction quality and to obtain a structure of a dimeric Class II RNR. The third aim is to obtain a co-crystal structure of a Class II RNR with substrate and effector bound. Radioactive assays, circular dichroism, analytical ultracentifugation, differential scanning calorimetry, and dynamic light scattering will be used as the primary methods to characterize the expressed RNRs. High through-put format crystallization screens will be used to determine initial crystallization conditions followed by optimization of the conditions. X-ray structures will be solved by molecular replacement or Seleomethionine-MAD phasing techniques. [unreadable] [unreadable]