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

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Presentation transcript:

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

Motor Proteins

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

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

p150 glued – A Subunit of Dynactin Dynactin

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

Objective Characterize individual subunits Characterize individual subunits Structure and interactions of the p150 Glued subunit of dynactin, residues 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 of p150 Glued Understand mechanism of regulation of dynein by dynactin

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

Protein Purification Affinity Column Chromatography Protein Poly-histidine tag

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

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

Differential Scanning Calorimetry (DSC) – Glued ( ) CpCp Unfolded Folded

Size Exclusion Chromatography (SEC)

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

Light Scattering

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

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

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

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

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