structural analysis of cytoplasmic dynein marcus a. chiodo dr. elisar barbar biochemistry and...

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

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Page 1: Structural Analysis of Cytoplasmic Dynein Marcus A. Chiodo Dr. Elisar Barbar Biochemistry and Biophysics Department

Structural Analysis of Cytoplasmic Dynein

Marcus A. ChiodoDr. Elisar BarbarBiochemistry and Biophysics Department

Page 2: 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

Page 3: Structural Analysis of Cytoplasmic Dynein Marcus A. Chiodo Dr. Elisar Barbar Biochemistry and Biophysics Department

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

Page 4: Structural Analysis of Cytoplasmic Dynein Marcus A. Chiodo Dr. Elisar Barbar Biochemistry and Biophysics Department

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

Page 5: Structural Analysis of Cytoplasmic Dynein Marcus A. Chiodo Dr. Elisar Barbar Biochemistry and Biophysics Department

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)

Page 6: Structural Analysis of Cytoplasmic Dynein Marcus A. Chiodo Dr. Elisar Barbar Biochemistry and Biophysics Department

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

Page 7: Structural Analysis of Cytoplasmic Dynein Marcus A. Chiodo Dr. Elisar Barbar Biochemistry and Biophysics Department

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

Page 8: Structural Analysis of Cytoplasmic Dynein Marcus A. Chiodo Dr. Elisar Barbar Biochemistry and Biophysics Department

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

Page 9: Structural Analysis of Cytoplasmic Dynein Marcus A. Chiodo Dr. Elisar Barbar Biochemistry and Biophysics Department

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

Page 10: Structural Analysis of Cytoplasmic Dynein Marcus A. Chiodo Dr. Elisar Barbar Biochemistry and Biophysics Department

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

Page 11: Structural Analysis of Cytoplasmic Dynein Marcus A. Chiodo Dr. Elisar Barbar Biochemistry and Biophysics Department

Affinity Column Process

www.bio.davidson.edu/Courses/genomics/method/

Page 12: Structural Analysis of Cytoplasmic Dynein Marcus A. Chiodo Dr. Elisar Barbar Biochemistry and Biophysics Department

Affinity Column Data

FLOWW

ASH

50 mM

100 mM

350 mM

FLOWW

ASH

50 mM

100 mM

350 mM

Page 13: Structural Analysis of Cytoplasmic Dynein Marcus A. Chiodo Dr. Elisar Barbar Biochemistry and Biophysics Department

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

Page 14: Structural Analysis of Cytoplasmic Dynein Marcus A. Chiodo Dr. Elisar Barbar Biochemistry and Biophysics Department

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

Page 15: Structural Analysis of Cytoplasmic Dynein Marcus A. Chiodo Dr. Elisar Barbar Biochemistry and Biophysics Department

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

Page 16: Structural Analysis of Cytoplasmic Dynein Marcus A. Chiodo Dr. Elisar Barbar Biochemistry and Biophysics Department

Crystal Screen Optimization•Determine best lead from the screen

•Optimize the selected condition

•Let crystallize

•Locate best crystal

•X-Ray Diffraction

Page 17: Structural Analysis of Cytoplasmic Dynein Marcus A. Chiodo Dr. Elisar Barbar Biochemistry and Biophysics Department

What Remains?• Check for crystal on optimized

crystal condition

• Perform X-Ray Diffraction study

• Determine 3-D structure of the LC8/Swa complex

Page 18: Structural Analysis of Cytoplasmic Dynein Marcus A. Chiodo Dr. Elisar Barbar Biochemistry and Biophysics Department

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