Structural Studies of Human Mitochondrial Aconitase
Transcript of Structural Studies of Human Mitochondrial Aconitase
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Structural Studies of Human
Mitochondrial Aconitase
Maggie Adams, Harsimran Singh
Dr. Laura Busenlehner
Department of Chemistry
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Friedreich’s Ataxia (FRDA)
Inherited neurodegenerative disease
Symptoms:
◦ Muscle weakness
◦ Loss of coordination, vision, and hearing
◦ Diabetes
◦ Heart disorders
Caused by mutation in gene for frataxin
◦ Creates a deficiency
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Frataxin
Protein localized to mitochondria
Primarily in cardiac and neuronal tissue
Function not entirely clear
Deficiency causes iron build up in mitochondria
N
C
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The Problem
How does frataxin deficiency lead to
FRDA?
◦ What are the direct effects on the
mitochondria?
◦ Examine frataxin pathways
◦ Study protein interactions
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Our Research
Many proteins
contain iron-sulfur
clusters
◦ Clusters can lose iron
◦ Iron loss inactivates
cluster
Frataxin as an “iron
chaperone” or shield
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Mitochondrial Aconitase
Enzyme in
Tricarboxylic Acid
(TCA) cycle
Citrate to Isocitrate
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Aconitase
Iron-sulfur protein
[Fe4S4]2+
Is an oxidative stress
“sensor”
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Research Goals
Examine structure of aconitase
Study interactions with frataxin
◦ Hydrogen/Deuterium Exchange Mass
Spectrometry
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Process
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1. Cloning
◦ polymerase chain reaction (PCR)
◦ digestion
◦ ligation
2. Propagation
◦ insert into E. coli strain (HB101)
◦ purify plasmid from E. coli
3. Protein Expression
◦ insert into different E. coli strain (Rosetta)
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Process
4. Peptide Identification
◦ Digest with pepsin
◦ Mass Spectrometry
5. Structure and Interaction Studies
◦ Hydrogen/Deuterium Amide Exchange Mass Spectrometry
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Summary of Results
Cloned and expressed human
mitochondrial aconitase in E. coli
Purified protein with Nickel-Affinity
Chromatography
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Cloning
Had trouble with PCR
◦ Redesigned primers
◦ Experimented with
thermocycler settings
◦ New stocks to avoid
endonuclease
contamination
◦ Finally successful when
added adjuvant
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PCR Product
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PCR Results
PCR with bad primers PCR without adjuvant
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PCR with Adjuvants
PCR with adjuvant SUCCESS!!
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Protein Expression
Used Carbenicillin and Chloramphenicol
(antibiotics)
Induced protein expression with IPTG
(sugar)
Tried several different media
◦ Seemed to resist growth
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Culture Media
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LB LB +
glucose
2xYT Circle
Grow
Circle
Grow +
glucose
Over
Night
Express
Super
Rich
Media
Auto-
Induce
Saw
Growth
Expressed
Protein
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Glycolysis and the TCA Cycle
Appears that extra fuel
(i.e. sugars) are
required for E. coli
growth during
expression of aconitase
May be related to
glycolytic pathway
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Nickel column purification
Aconitase engineered with six additional Histidine
residues
Histidines specifically bind protein to Nickel resin
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Nickel column purification
Use low concentration imidazole to wash
Use high concentration imidazole to elute aconitase
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Purification SDS-PAGE Gel
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Future Steps
Measure protein activity with coupled enzymatic assay
Digest protein with pepsin
Use Mass Spectrometry to identify peptides
Study structure and interactions
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Acknowledgments
Dr. Laura Busenlehner
Harsimran Singh
Busenlehner Laboratory
University of Alabama
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Sources
Nelson, David L. and Michael M. Cox,
Lehninger: Principles of Biochemistry. Fifth
Edition, W.H. Freeman & Co.: New York
http://bama.ua.edu/~lsbusenlehner/
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