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 i s 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 i s to obtain protein crystals of diffraction quality and to obtain a structure of a dimeric Class II RNR.
The third aim i s 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. ? ?
| Hamill, Michael J; Chobot, Sarah E; Hernandez, Hector H et al. (2008) Direct electrochemical analyses of a thermophilic thioredoxin reductase: interplay between conformational change and redox chemistry. Biochemistry 47:9738-46 |
| Hernandez, Hector H; Jaquez, Orlando A; Hamill, Michael J et al. (2008) Thioredoxin reductase from Thermoplasma acidophilum: a new twist on redox regulation. Biochemistry 47:9728-37 |
| Chobot, Sarah E; Hernandez, Hector H; Drennan, Catherine L et al. (2007) Direct electrochemical characterization of archaeal thioredoxins. Angew Chem Int Ed Engl 46:4145-7 |
