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. ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Predoctoral Individual National Research Service Award (F31)
Project #
5F31GM073569-02
Application #
6959261
Study Section
Special Emphasis Panel (ZRG1-F05 (29))
Program Officer
Gaillard, Shawn R
Project Start
2004-09-10
Project End
2006-09-09
Budget Start
2005-09-10
Budget End
2006-09-09
Support Year
2
Fiscal Year
2005
Total Cost
$43,346
Indirect Cost
Name
Massachusetts Institute of Technology
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
001425594
City
Cambridge
State
MA
Country
United States
Zip Code
02139
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