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Mobility measurements by EPR of the anthracis repressor protein by Katie Schwarz

Mobility measurements by EPR of the anthracis repressor protein by Katie Schwarz. Background of AntR Purpose Methodology Preliminary Results. MntR, a homologue of AntR. Background of AntR. Actual structure is not known Binds to metal and target DNA

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Mobility measurements by EPR of the anthracis repressor protein by Katie Schwarz

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  1. Mobility measurements by EPR of the anthracis repressor proteinby Katie Schwarz

  2. Background of AntR • Purpose • Methodology • Preliminary Results MntR, a homologue of AntR

  3. Background of AntR • Actual structure is not known • Binds to metal and target DNA • The DNA and metal binding may be linked to conformational changes. • Folded form is a dimer in solution

  4. Native protein Mutate AntR to include Cysteine Labeled protein

  5. Purpose • To measure mobility changes between sites on the protein as a function of metal binding and DNA binding. • The mobility changes will help us understand the protein’s conformational changes.

  6. Expression of the mutant protein

  7. Preparation of the protein for EPR(Electron Paramagnetic Resonance) • Purification of the protein • Addition of spin labels • Dialysis to remove excess spin labels

  8. EPR • Requires unpaired electron • Two main types: 1. Continuous Wave Electron paramagnetic resonance (CW EPR) 2.Pulsed EPR

  9. Double labeled Dipolar EPR Non-interacting spins

  10. Continuous wave EPR: mobility Correlation time tr = 100 nsec tr = 20 nsec tr = 10 nsec tr = 1 nsec tr = 0.001 nsec

  11. CW for AntR 37C, MSL Black: apo Red: holo Blue: holo + DNA

  12. CW for AntR 37C, MTSSL Black: holo +DNA Red: holo Blue:apo

  13. CW for AntR 37C, IASL Black: holo+ DNA Red: holo Blue: apo

  14. CW for AntR 104C, MSL Black: holo+ DNA Red: holo Blue: apo

  15. Preliminary Results • Results of the CW experiments: 124C

  16. Conclusions • Mobility at sites 37 and 104 is decreased by addition of Zn+2 but appears unaffected by addition of DNA • Mobility at site 124 decreases with the addition of Zn+2 • Further experiments are needed to determine whether or not DNA causes conformational changes in AntR

  17. Acknowledgements Special thanks to: Fajer Lab Logan Lab Ilker Sen The REU program

  18. Distance measurements from DEER: Apo 124 Mtssl

  19. Distance measurements from DEER: Zn 124 and Mtssl

  20. Pulsed EPR: the DEER experiment • Double electron-electron resonance (DEER) • Pulsed EPR- the bell analogy • Pulse sequence

  21. Ca-Ca (124) = 18.3A

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