insight into the regulation of dynein by dynactin
DESCRIPTION
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: - PowerPoint PPT PresentationTRANSCRIPT
Insight into the Regulation of Dynein by Dynactin
Drew CalhounDr. Elisar Barbar
Biochemistry & Biophysics Dept.
Motor Proteins
LC8: (LC), 10 kDa, dimerLC8: (LC), 10 kDa, dimer
Tctex-1: (LC), 12.5 kDa, dimerTctex-1: (LC), 12.5 kDa, dimer
Heavy chain: 530 Heavy chain: 530 kDa, kDa, motor domainmotor domain
Light intermediate chain:Light intermediate chain:50-60 kDa50-60 kDa
Microtubule Microtubule binding domainbinding domain
Intermediate chainIntermediate chain (IC 74):(IC 74): 74 kDa74 kDa
THE CYTOPLASMIC DYNEIN COMPLEXTHE CYTOPLASMIC DYNEIN COMPLEX
Roadblock: (LC),Roadblock: (LC),10.9 kDa, dimer10.9 kDa, dimer
Intermediate Chain, 74 kDa: IC74
Cargo: UL34, PLAC24… UL34, PLAC24… Important interaction between IC (1-143) and p150glued.
WD
p150glued binding region
WD WDWD WD WDWD
Roadblock binding region
Tctex-1
LC8
N-terminal domain C-terminal domain
IC74
p150glued – 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 subunitsCharacterize 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 subunitsCharacterize 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
Recombinant Protein Preparation
1. INDUCE
2. CENTRIFUGE
CENTRIFUGE
Protein of Interest
Lyse Cells
Protein PurificationAffinity 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 (250-538)
ΔH= 235.8 kJ/molΔS= 0.783 kJ/(K mol)Tt= 28.2 C
OSU Sample (Baseline Subtracted)
kJ/Mole K
Temperature (°C)0 5 10 15 20 25 30 35 40 45 50
303540455055606570758085
ΔCp
Unfolded
Folded
Size Exclusion Chromatography (SEC)
Size Exclusion Chromatography
p150 & IC1; 40:80p150 & IC1, 40:40
p150, 40 uM
IC1, 90 uM
IC1, 45 uM
Light Scattering
SEC & LS - p150 (alone)
Red = UV absorbanceBlack = Light Scattering
Aggregates
Average molecular weight = 97.8 kDa
Trimer Dimer
SEC & LS – p150 & IC1
Aggregates
Red = UV absorbanceBlack = Light Scattering
Molecular weight = 86.2 kDa
Glued dimer +
IC1 monomer
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
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