Transcript Derek Youngblood - Oregon State University

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
NADH
AhpFOx
AhpCRed
ROOH
NAD+
AhpFRed
AhpCOx
ROH +
H2O
• A 2-cys peroxiredoxin.
Why is AhpC important?
•
Peroxiredoxins protect cells of all
organisms from toxic oxygen species.
•
Peroxiredoxins regulate hydroperoxide
signaling in eukaryotes.
Catalytic cycle of AhpC
High levels of
hydroperoxides
H2O
NADH
AhpC
SR
ROOH
SP
AhpC
AhpF
SRH
NAD+
SRH
SPOH
ROOH
AhpC
SP O 2 H
SRH
(Inactivated)
AhpC
SP H
ROH
ROH
Of Decamers and Dimers
Decamer to Dimer
H2O
NADH
AhpC
SR
SP
AhpC
SRH
ROH
SPOH
AhpF
NAD+
SRH
AhpC
ROOH
SP H
The decamer to dimer conformation is directly related to the
state of the redox system.
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
Do you really need that doughnut?
H2O
NADH
AhpC
SR
SP
AhpF
AhpC
?
SRH
SPOH
ROH
SRH
NAD+
AhpC
?
ROOH
SP H
Dude, where’s the mutation?
Threonine 77
Threonine (T) 77
Breaking up is hard to do
Valine (V)
Isoleucine (I)
Aspartic Acid (D)
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.
Radiowaves
1012
109
Infrared
Ultra
Violet
106
103
X-ray
1
Gamma Rays
10-3
10-6
Wavelength: Å
Red
Violet
Model Fitting
CH3
CH3
CHCH2
NH
C OCH
O
NH
C
CH
Monitoring Progress
0.4000
0.3500
0.3000
0.2500
0.2000
0.1500
0.1000
0.0500
0.0000
1
2
3
4
5
6
7
8
Rounds of Refinement
R
R-free
9
R
10
11
R-factor
AhpC T77V
The Preliminary Results Are In!
Mutant
Ultracentrifugation Activity
T77V
Oligomerization redoxsensitivity is lost
T77D
Mostly dimeric in the
reduced form
T77D
What’s next?
Structure?
T77I
Structure?
Thank you
Howard Hughes Medical Institute
Dr. P. Andrew Karplus
Ganapathy Sarma
Rick Faber
Zac Wood
Dr. Leslie Poole
Derek Parsonage
Kevin Ahern
YOU!