Structural Analysis of Cytoplasmic Dynein

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Structural Analysis of Cytoplasmic Dynein
Marcus A. Chiodo Dr. Elisar Barbar Biochemistry
and Biophysics Department
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Importance

• Dynein transports cargo throughout the cell
• Assists organization of cellular components
• Plays a major function in cellular division
• Segregation of chromosomes

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Motor Proteins

• Motor proteins are the cells transportation
  system
• Dynein and Kinesin are the two primary classes of
  motor proteins
• They are powered by ATP and walk along
  microtubules transporting their cargo
• Dynein and Kinesin transport cargo in opposing
  directions and are different structures

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Dynein

• Two classes of dynein
• Axonemal dynein
• propels beating of cilia and flagella
• Cytoplasmic dynein
• transports membrane bound vesicles, protein
  complexes, chromosomes
• Cellular organization
• Cell Division

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Cytoplasmic Dynein

• A large multi-subunit molecular motor protein
• Heavy chains contain the ATP and microtubule
  binding sites
• Cytoplasmic dynein is responsible for
  transporting cellular cargo to the minus end of
  microtubules (i.e. toward the centrosome)

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Light Chain 8 (LC8)

• 10.3 kDa subunit of Dynein found in all
  eukaryotes
• Connects the cargo binding proteins to the mobile
  proteins (heavy chains)
• Assists in assemblage of Dynein complex
• Free form structure is known

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Drosophila Swallow (Swa)

• Swa is an example of a natural occurring cargo
  protein in Drosophila cells
• Role in localizing bicoid mRNA of the oocyte
  during oogenesis
• Focused on the 206 to 297 amino acid domain of Swa

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Project Objective

• Grow, purify and collect 30mg of LC8/Swa protein
• Screen LC8/Swa protein for promising
  crystallization conditions
• Optimize conditions of promising leads from
  screens
• X-Ray diffraction on LC8/Swa crystal to determine
  protein structure

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Recombinant Protein Growth
Insertion of LC8 Swa DNA into vector
(coexpression)
Vector inserted into E. coli bacterium
Cell Replication
1. Induction with IPTG
Centrifuge
2. Lyse Cells
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Purification Affinity Column

• The protein has a His-tag that has an affinity
  towards the divalent Ni ions in the columns
  resin
• Untagged proteins either have a weaker or no
  affinity for the Ni compared to the His-tagged
  protein
• Imidazole also has an affinity for Ni and can
  compete with the protein with the His-tag

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Affinity Column Process
www.bio.davidson.edu/Courses/genomics/method/
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Affinity Column Data
FLOW
FLOW
350 mM
350 mM
WASH
50 mM
100 mM
WASH
50 mM
100 mM
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Size Exclusion Column (SEC)

• SECs take advantage of porous particles to
  separate molecules by different sizes and shapes
• Smaller molecules can enter the porous particles
  and therefore have a longer travel path and
  elution time

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LC8/Swa SEC Data
3600 uV
63 minutes
Intensity (uV)
71 minutes
2000 uV
60 minutes
Time (minutes)
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Crystal Screens

• Used Hampton Crystal Screens I and II
• Allows 96 different combinations of various salt,
  precipitant and buffer types, concentrations and
  pH
• Assists in determines starting point for
  crystallization

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Crystal Screen Optimization

• Determine best lead from the screen
• Optimize the selected condition
• Let crystallize
• Locate best crystal
• X-Ray Diffraction

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What Remains?

• Check for crystal on optimized crystal condition
• Perform X-Ray Diffraction study
• Determine 3-D structure of the LC8/Swa complex

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Thank You!

• Howard Hughes Medical Institute
• Dr. Kevin Ahern
• Dr. Elisar Barbar
• Dr. Gregory Benson
• Gretchen Clark-Scannel
• Yujuan Song
• Dr. Karplus Lab
• Grant Farr