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!