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Insight into the Regulation of Dynein by Dynactin Drew Calhoun Dr. Elisar Barbar Biochemistry & Biophysics Dept.
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Motor Proteins
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LC8: (LC), 10 kDa, dimer Tctex-1: (LC), 12.5 kDa, dimer Heavy chain: 530 kDa, motor domain Light intermediate chain: 50-60 kDa Microtubule binding domain Intermediate chain (IC 74): 74 kDa THE CYTOPLASMIC DYNEIN COMPLEX Roadblock: (LC), 10.9 kDa, dimer
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Intermediate Chain, 74 kDa: IC74 UL34, PLAC24… Cargo: UL34, PLAC24… Important interaction between IC (1-143) and p150 glued. WD p150 glued binding region WD Roadblock binding region Tctex-1 LC8 N-terminal domainC-terminal domain IC74
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p150 glued – A Subunit of Dynactin Dynactin
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What is the Point? Maybe… Controlling dynein = controlling cancer & viral diseases Cells cannot divide without dynein Dynein transports viruses
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Objective Characterize individual subunits Characterize individual subunits Structure and interactions of the p150 Glued subunit of dynactin, residues 250-538 Structure and interactions of IC(1-143) of IC74, a subunit of dynein Characterize the association between subunits Characterize the association between subunits Investigate interaction between IC(1-143) of IC74 and Glued 250-538 of p150 Glued Understand mechanism of regulation of dynein by dynactin
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Recombinant Protein Preparation 1. INDUCE 2. CENTRIFUGE CENTRIFUGE Protein of Interest Lyse Cells
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Protein Purification Affinity Column Chromatography Protein Poly-histidine tag
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Circular Dichroism Unfolded p150 Folded: -Helical CD: p150 Wavelength
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Circular Dichroism (cont.) CD: p150 Temperature-Dependence Midpoint ~ 31˚C Unfolded p150 Folded p150
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Differential Scanning Calorimetry (DSC) – Glued (250-538) CpCp Unfolded Folded
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Size Exclusion Chromatography (SEC)
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Size Exclusion Chromatography p150 & IC1; 40:80 p150 & IC1, 40:40 p150, 40 uM IC1, 90 uM IC1, 45 uM
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Light Scattering
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SEC & LS - p150 (alone) Red = UV absorbance Black = Light Scattering Aggregates Average molecular weight = 97.8 kDa Trimer Dimer
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SEC & LS – p150 & IC1 Aggregates Red = UV absorbance Black = Light Scattering Molecular weight = 86.2 kDa Glued dimer + IC1 monomer
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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) Glued dimer + IC1 monomer
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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
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Thank you! Dr. Elisar Barbar HHMI NSF Special thanks to… Dr. Afua Nyarko Protein preparation Gretchen Clark-Scannel SEC & LS
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