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Structural Analysis of Cytoplasmic Dynein

Structural Analysis of Cytoplasmic Dynein

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Structural Analysis of Cytoplasmic Dynein

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

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

  3. 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

  4. 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

  5. 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)

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. Affinity Column Process www.bio.davidson.edu/Courses/genomics/method/

  12. Affinity Column Data FLOW FLOW 350 mM 350 mM 100 mM 50 mM 100 mM WASH 50 mM WASH

  13. 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

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

  15. 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

  16. Crystal Screen Optimization • Determine best lead from the screen • Optimize the selected condition • Let crystallize • Locate best crystal • X-Ray Diffraction

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

  18. 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