derek youngblood sophomore - oregon state university biochemistry and biophysics dr. p. andrew...
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Derek YoungbloodDerek YoungbloodSophomore - Oregon State UniversitySophomore - Oregon State University
Biochemistry and Biophysics Biochemistry and Biophysics
Dr. P. Andrew Karplus’ LabDr. P. Andrew Karplus’ Lab
Do you really need that doughnut?
Structural studies of peroxiredoxin mutants
DrugsDrugs
AhpCAhpCOxOx
AhpFAhpFOxOxNADHNADH
NADNAD++
AhpCAhpCRedRed
AhpFAhpFRedRed
ROOHROOH
ROH + ROH + HH22OO
AhpR = Alkyl hydroperoxide ReductaseAhpR = Alkyl hydroperoxide Reductase
What is AhpC?What is AhpC?
• A 2-cys peroxiredoxin.A 2-cys peroxiredoxin.
Why is AhpC important?Why is AhpC important?
• Peroxiredoxins regulate hydroperoxide Peroxiredoxins regulate hydroperoxide signaling in eukaryotes.signaling in eukaryotes.
• Peroxiredoxins protect cells of all Peroxiredoxins protect cells of all organisms from toxic oxygen species.organisms from toxic oxygen species.
NADHNADH
NADNAD++
AhpFAhpF
ROHROH
AhpCAhpCSSRR
SSPP
AhpCAhpCSSRRHH
SPH
HH22OO
ROOHROOH
SSRRHH
SSPPOHOHAhpCAhpC
ROOHROOH ROHROH
SSPPOO22HH
SSRRHHAhpCAhpC
(Inactivated)(Inactivated)
High levels of High levels of hydroperoxideshydroperoxides
Catalytic cycle of AhpCCatalytic cycle of AhpC
Of Decamers and DimersOf Decamers and Dimers
NADHNADH
NADNAD++
AhpFAhpF
SSRRHH
SSPPOHOHAhpCAhpCAhpCAhpC
SSRR
SSPP
AhpCAhpCSSRRHH
SSPPHH
HH22OO
ROHROH
ROOHROOH
Decamer to DimerDecamer to Dimer
The decamer to dimer conformation is directly related to the The decamer to dimer conformation is directly related to the state of the redox system.state of the redox system.
Oxidized AhpC [2.5 Oxidized AhpC [2.5 M] M] (Dimer)(Dimer)
Reduced AhpC [2.5 Reduced AhpC [2.5 M] M] (Strong Decamer)(Strong Decamer)
Ultracentrifugation resultsUltracentrifugation results
Wood, et al. (2002) Wood, et al. (2002) BiochemistryBiochemistry, Vol 41, No. 17, pp 5493-5504, Vol 41, No. 17, pp 5493-5504
Do you really need that doughnut?Do you really need that doughnut?
NADHNADH
NADNAD++
AhpFAhpF
SSRRHH
SSPPOHOHAhpCAhpCAhpCAhpC
SSRR
SSPP
AhpCAhpCSSRRHH
SSPPHH
HH22OO
ROHROH
ROOHROOH
?
?
Threonine 77Threonine 77
Dude, where’s the mutation?Dude, where’s the mutation?
Valine (V)Valine (V) Isoleucine (I)Isoleucine (I) Aspartic Acid (D)Aspartic Acid (D)
Breaking up is hard to doBreaking up is hard to do
Threonine (T) 77Threonine (T) 77
My jobMy job
• Collect x-ray diffraction data from Collect x-ray diffraction data from those crystals.those crystals.
• Build and refine a model for each Build and refine a model for each mutant to solve their structures.mutant to solve their structures.
• Find conditions that grow crystals Find conditions that grow crystals for all three mutants.for all three mutants.
Wavelength: Å
103 1 10-3 10-6 1012 109 106
Red Violet
Radiowaves InfraredUltra Violet X-ray Gamma Rays
Model FittingModel Fitting
CHCH33
CHCH33
CC
CHCH
CHCHCHCH22
NHNH
NHNHOO
OO
CC
CHCH
1 2 3 4 5 6 7 8 9 10 11
R
0.0000
0.0500
0.1000
0.1500
0.2000
0.2500
0.3000
0.3500
0.4000
Rounds of Refinement
R-factor
R R-free
Monitoring ProgressMonitoring Progress
AhpC T77VAhpC T77V
The Preliminary Results Are In!The Preliminary Results Are In!
UltracentrifugationUltracentrifugation ActivityActivity
T77VT77V Oligomerization redox- Oligomerization redox- sensitivity is lostsensitivity is lost
T77DT77D Mostly dimeric in the Mostly dimeric in the reduced formreduced form
MutantMutant
What’s next?What’s next?
Structure?Structure?
T77DT77D
Structure?Structure?
T77IT77I
Thank youThank youHoward Hughes Medical InstituteHoward Hughes Medical Institute
Dr. P. Andrew KarplusDr. P. Andrew Karplus
Ganapathy SarmaGanapathy Sarma
Rick FaberRick Faber
Zac WoodZac Wood
Dr. Leslie PooleDr. Leslie Poole
Derek ParsonageDerek Parsonage
Kevin AhernKevin Ahern
YOU!YOU!