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Insight into the Regulation of Dynein by Dynactin

Insight into the Regulation of Dynein by Dynactin. Drew Calhoun Dr. Elisar Barbar Biochemistry & Biophysics Dept. Motor Proteins. THE CYTOPLASMIC DYNEIN COMPLEX. Microtubule binding domain. Heavy chain: 530 kDa, motor domain. Roadblock: (LC), 10.9 kDa, dimer. Light intermediate chain:

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Insight into the Regulation of Dynein by Dynactin

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  1. Insight into the Regulation of Dynein by Dynactin Drew Calhoun Dr. Elisar Barbar Biochemistry & Biophysics Dept.

  2. Motor Proteins

  3. THE CYTOPLASMIC DYNEIN COMPLEX Microtubule binding domain Heavy chain: 530 kDa, motor domain Roadblock: (LC), 10.9 kDa, dimer Light intermediate chain: 50-60 kDa Intermediate chain(IC 74):74 kDa LC8: (LC), 10 kDa, dimer Tctex-1: (LC), 12.5 kDa, dimer

  4. Tctex-1 Roadblock binding region p150glued binding region LC8 WD WD WD WD WD WD WD N-terminal domain C-terminal domain Intermediate Chain, 74 kDa: IC74 • Cargo: UL34, PLAC24… • Important interaction between IC (1-143) and p150glued. IC74

  5. p150glued – A Subunit of Dynactin Dynactin

  6. What is the Point? • Cells cannot divide without dynein • Dynein transports viruses • Maybe… • Controlling dynein = controlling cancer & viral diseases

  7. Objective • Characterize individual subunits • Structure and interactions of the p150Glued subunit of dynactin, residues 250-538 • Structure and interactions of IC(1-143) of IC74, a subunit of dynein • Characterize the association between subunits • Investigate interaction between IC(1-143) of IC74 and Glued 250-538 of p150Glued • Understand mechanism of regulation of dynein by dynactin

  8. Recombinant Protein Preparation Protein of Interest 1. INDUCE CENTRIFUGE 2. CENTRIFUGE Lyse Cells

  9. Protein Purification Affinity Column Chromatography Protein Poly-histidine tag

  10. Circular Dichroism CD: p150 Unfolded p150 Folded: -Helical Wavelength

  11. Circular Dichroism (cont.) CD: p150 Temperature-Dependence Unfolded p150 Midpoint ~ 31˚C Folded p150

  12. Differential Scanning Calorimetry (DSC) – Glued (250-538) Unfolded Folded Cp

  13. Size Exclusion Chromatography (SEC)

  14. Size Exclusion Chromatography p150 & IC1; 40:80 p150 & IC1, 40:40 p150, 40 uM IC1, 45 uM IC1, 90 uM

  15. Light Scattering

  16. SEC & LS - p150 (alone) Red = UV absorbance Aggregates Black = Light Scattering Average molecular weight = 97.8 kDa Trimer Dimer

  17. SEC & LS – p150 & IC1 Aggregates Red = UV absorbance Black = Light Scattering Molecular weight = 86.2 kDa Glued dimer + IC1 monomer

  18. Conclusions • CD & DSC results • Glued (250-538) • -helical secondary structure when folded • Thermodynamically, relatively unstable • SEC & LS • Glued (250-538) • Trimer Dimer • IC (1-143) • Monomer Dimer Trimer • Glued (250-538) and IC (1-143) • Glueddimer + IC1 monomer

  19. Future… • Verification of results • Other methods to help verify accuracy of results • Chemical cross-linking • Mass spectrometry • Repeat experiments to ensure accuracy • More Circular Dichroism • Temperature-dependence with varying concentrations • More Size Exclusion Chromatography • Concentration dependence studies • Varying ratios of IC1:p150

  20. Thank you! • Dr. Elisar Barbar • HHMI • NSF Special thanks to… • Dr. Afua Nyarko • Protein preparation • Gretchen Clark-Scannel • SEC & LS

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