Transcript Structural Determination of StAhpC2 and MpGlpO

Structural Studies of StAhpC2
and MpGlpO
By: Callia K. Palioca
Mentor: Dr. P. Andrew Karplus
Department of Biochemistry & Biophysics, OSU
HHMI Summer Program 2010
Proteins are IMPORTANT
 Enzymes are vital in all of life’s functions
 Most drugs interact with proteins
Myosin
Actin
Protein Structure Matters
 Function flows from structure
 Structure-based drug design
HIV protease drug
complex
X-Ray Crystallography Methods
 Goal: Obtain single, high-quality diffracting crystals and
use the data to solve the structure
Grow
Grow
Crystal
Crystal
Diffraction&
Diffraction
&Data
Data
Processing
Processing
Solve
the
Solve the
Phase
Phase
Problem
Problem
Electron
Electron
Density
Density Map
Map
Refinement
Refinement
of
of Structure
Structure
Food Poisoning
Macrophage attacking bacteria
StAhpC2 and the Peroxiredoxins
 Catalytic cycle of this family of proteins
 Function: uses cysteine residues to breakdown peroxides
How Motifs Relate to Sensitivity
 Correlation
Sensitive
between YF and
a helical
structure
 Leads to
sensitive
enzymes
Robust
AhpC2 C50S
 YL vs. YF
 Seen in significant
human pathogens that
cause African sleeping
sickness, ulcers, malaria,
and Beaver Fever
Hypothesis
 StAhpC2 is a representative of the proteins in
deadly human pathogens
 Structure gives insights into pathogen survival in
humans and forms basis for drug design
 YF motif significance can also be studied
Crystals!
 Pure protein from Dr. Leslie Poole’s
lab, WFU
 Optimizing conditions
 (NH4)2HPO4
 pH
Grow Crystal
Diffraction &
Data Processing
Solve the Phase
Problem
Electron
Density Map
Refinement of
Structure
Crystals!
Grow Crystal
Diffraction &
Data Processing
Solve the Phase
Problem
Electron
Density Map
Refinement of
Structure
Crystals!
 Predictions do
not line up
 Stalled progress
Grow Crystal
Diffraction &
Data Processing
Solve the Phase
Problem
Electron
Density Map
Refinement of
Structure
The Mycoplasma Diseases
 Mycoplasma pneumoniae
 Walking pneumonia
 Mycoplasma mycoides
 Contagious bovine pleuropnemonia
GlpO
(Glycerolphosphate oxidase)
What is known
 Structure of GlpO
 No substrates able to be
bound
 DHAP,
tartaric acid,
2-phosphoglycerate,
menadione,
G3P,
phosphoenolpyruvate
Hypothesis
 Drug target
 Substrates, products and inhibitors in GlpO
 Insight into Mycoplasma and other organisms cause
disease
 Basis for structure-based drug design
Crystallization
 Pure protein from Dr. Al Claiborne’s
lab, WFU
 NaCl and neutral-basic pH buffer
Grow Crystal
Diffraction &
Data Processing
Solve the Phase
Problem
Electron
Density Map
Refinement of
Structure
Diffraction and Data Processing
 Resolution:
enough to define
atomic structure
(3.0 Å)
Grow Crystal
Diffraction &
Data Processing
Solve the Phase
Problem
Electron
Density Map
Refinement of
Structure
Molecular Replacement
 The Phase Problem
 Structure of related protein (3DME)
Height
Grow Crystal
Diffraction &
Data Processing
Solving the
Phase Problem
Electron
Density Map
Refinement of
Structure
A Good Solution
 Differences between model and map
Grow Crystal
Diffraction &
Data Processing
Solving the
Phase Problem
Electron
Density Map
Refinement of
Structure
Structure Refinement
 Process of manually changing sequence
 Avoiding model bias
R-free vs. Round of
Refinement
R-free
0.6
0.4
0.2
0
1
Grow Crystal
Diffraction &
Data Processing
Solve the Phase
Problem
2
3
4
5
6
Round of Refinement
Electron
Density Map
7
Refinement of
Structure
Future Work
 Continue refinement of
structure
 Soaking of substrates
 Optimization of
cryo-protectant
R-factor vs. Round of
Refinement
0.6
0.4

0.2
0
1 2 3 4 5 6 7 8 9 10 11 12
 15% glycerol
0% glycerol
10% glycerol
15% glycerol
Acknowledgements
 Dr. P. Andrew Karplus
 The rest of the Karplus lab
 Dr. Andrea Hall, Camden Driggers, Ian Winter
 HHMI, Cripps
 URISC
 Our collaborators
at Wake Forest
University
 Dr. Leslie Poole
 Dr. Al Claiborne
 Dr. Kevin Ahern