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)

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

PCR Results

PCR with bad primers PCR without adjuvant

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5000

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5000

4000

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2000

1500

PCR with Adjuvants

PCR with adjuvant SUCCESS!!

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5000

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3000

2500

2000

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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

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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