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Derek Youngblood Sophomore - Oregon State University Biochemistry and Biophysics

Do you really need that doughnut? Structural studies of peroxiredoxin mutants. Derek Youngblood Sophomore - Oregon State University Biochemistry and Biophysics Dr. P. Andrew Karplus’ Lab. Drugs. What is AhpC?. AhpR = Alkyl hydroperoxide Reductase. A 2-cys peroxiredoxin. NADH. AhpF Ox.

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Derek Youngblood Sophomore - Oregon State University Biochemistry and Biophysics

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  1. Do you really need that doughnut? Structural studies of peroxiredoxin mutants Derek Youngblood Sophomore - Oregon State University Biochemistry and Biophysics Dr. P. Andrew Karplus’ Lab

  2. Drugs

  3. What is AhpC? AhpR = Alkyl hydroperoxide Reductase • A 2-cys peroxiredoxin. NADH AhpFOx AhpCRed ROOH ROH + H2O NAD+ AhpFRed AhpCOx

  4. Why is AhpC important? • Peroxiredoxins protect cells of all organisms from toxic oxygen species. • Peroxiredoxins regulate hydroperoxide signaling in eukaryotes.

  5. High levels of hydroperoxides ROOH ROH SRH SPO2H AhpC AhpC SPOH SRH (Inactivated) Catalytic cycle of AhpC H2O SR NADH AhpC SP AhpF SRH AhpC NAD+ SPH ROH ROOH

  6. Of Decamers and Dimers

  7. SR AhpC SP Decamer to Dimer H2O SRH AhpC NADH ROH SPOH AhpF SRH NAD+ AhpC ROOH SPH The decamer to dimer conformation is directly related to the state of the redox system.

  8. Ultracentrifugation results Oxidized AhpC [2.5 M] (Dimer) Reduced AhpC [2.5 M] (Strong Decamer) Wood, et al. (2002) Biochemistry, Vol 41, No. 17, pp 5493-5504

  9. Do you really need that doughnut? H2O SRH SR AhpC NADH ROH AhpC SPOH SP ? AhpF SRH NAD+ AhpC ROOH SPH ?

  10. Dude, where’s the mutation? Threonine 77

  11. Threonine (T) 77 Breaking up is hard to do Isoleucine (I) Valine (V) Aspartic Acid (D)

  12. My job • Find conditions that grow crystals for all three mutants. • Collect x-ray diffraction data from those crystals. • Build and refine a model for each mutant to solve their structures.

  13. 1012 109 106 103 1 10-3 10-6 Wavelength: Å Ultra Violet Radiowaves Infrared X-ray Gamma Rays Red Violet

  14. CH3 CH3 CH2 CH NH C CH O NH C O CH Model Fitting

  15. Monitoring Progress

  16. AhpC T77V

  17. Oligomerization redox- sensitivity is lost T77V Mostly dimeric in the reduced form T77D The Preliminary Results Are In! Activity Mutant Ultracentrifugation

  18. What’s next? T77D Structure? T77I Structure?

  19. Thank you Howard Hughes Medical Institute Kevin Ahern Dr. P. Andrew Karplus Ganapathy Sarma Rick Faber Zac Wood Dr. Leslie Poole Derek Parsonage YOU!

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