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

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Presentation transcript:

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

Drugs

AhpC Ox AhpF Ox NADH NAD + AhpC Red AhpF Red ROOH ROH + H 2 O AhpR = Alkyl hydroperoxide Reductase What is AhpC? A 2-cys peroxiredoxin.A 2-cys peroxiredoxin.

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

NADH NAD + AhpF ROH AhpC SRSRSRSR SPSPSPSP AhpC SRHSRHSRHSRH SPHSPH H2OH2OH2OH2O ROOH SRHSRHSRHSRH S P OH AhpC ROOHROH SPO2HSPO2HSPO2HSPO2H SRHSRHSRHSRH AhpC (Inactivated) High levels of hydroperoxides Catalytic cycle of AhpC

Of Decamers and Dimers

NADH NAD + AhpF SRHSRHSRHSRH S P OH AhpC AhpC SRSRSRSR SPSPSPSP AhpC SRHSRHSRHSRH SPHSPHSPHSPH H2OH2OH2OH2O ROH ROOH Decamer to Dimer The decamer to dimer conformation is directly related to the state of the redox system.

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

Do you really need that doughnut? NADH NAD + AhpF SRHSRHSRHSRH S P OH AhpC AhpC SRSRSRSR SPSPSPSP AhpC SRHSRHSRHSRH SPHSPHSPHSPH H2OH2OH2OH2O ROH ROOH ? ?

Threonine 77 Dude, where’s the mutation?

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

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

Wavelength: Å Red Violet RadiowavesInfrared Ultra Violet X-rayGamma Rays

Model Fitting CH 3 C CH CH CH 2 NH NH O O C CH

Monitoring Progress

AhpC T77V

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

What’s next? Structure? T77D Structure? T77I

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