Transcript Dr. Elisar Barbar`s Lab - Oregon State University

Structural Analysis of
Cytoplasmic Dynein
Marcus A. Chiodo
Dr. Elisar Barbar
Biochemistry and Biophysics Department
Importance
• Dynein transports cargo
throughout the cell
• Assists organization of
cellular components
• Plays a major function in
cellular division
– Segregation of
chromosomes
Motor Proteins
• Motor proteins are the
cell’s 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
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
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)
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
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
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
Recombinant Protein Growth
Insertion of LC8 &
Swa DNA into vector
(coexpression)
Vector inserted into
E. coli bacterium
Cell
Replication
Centrifuge
1. Induction with IPTG
2. Lyse Cells
Purification: Affinity Column
• The protein has a His-tag that
has an affinity towards the
divalent Ni ions in the
column’s 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
Affinity Column Process
www.bio.davidson.edu/Courses/genomics/method/
Affinity Column Data
Size Exclusion Column (SEC)
•SEC’s 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
LC8/Swa SEC Data
Intensity (uV)
3600 uV
63 minutes
71 minutes
2000 uV
60 minutes
Time (minutes)
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
Crystal Screen Optimization
•Determine best lead from the screen
•Optimize the selected condition
•Let crystallize
•Locate best crystal
•X-Ray Diffraction
What Remains?
• Check for crystal on optimized
crystal condition
• Perform X-Ray Diffraction
study
• Determine 3-D structure of the
LC8/Swa complex
Thank You!
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Howard Hughes Medical Institute
Dr. Kevin Ahern
Dr. Elisar Barbar
Dr. Gregory Benson
Gretchen Clark-Scannel
Yujuan Song
Dr. Karplus’ Lab
Grant Farr