structural analysis of cytoplasmic dynein marcus a. chiodo dr. elisar barbar biochemistry and...
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Structural Analysis of Cytoplasmic Dynein
Marcus A. ChiodoDr. Elisar BarbarBiochemistry 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)
Cell Replication
Vector inserted into E. coli bacterium
1. Induction with IPTG
2. Lyse Cells
Centrifuge
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
FLOWW
ASH
50 mM
100 mM
350 mM
FLOWW
ASH
50 mM
100 mM
350 mM
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
1009080706050403020100
3,600
3,400
3,200
3,000
2,800
2,600
2,400
2,200
2,000
1,800
1,600
1,400
1,200
1,000
800
600
400
200
SPW 0.20
STH 10.00
RT [min]
LC8swa_081806_mac2.DATAµV
60 minutes
Time (minutes)
Inte
nsity
(uV
)
63 minutes
71 minutes
3600 uV
2000 uV
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!• Howard Hughes Medical Institute• Dr. Kevin Ahern• Dr. Elisar Barbar• Dr. Gregory Benson• Gretchen Clark-Scannel• Yujuan Song• Dr. Karplus’ Lab• Grant Farr