Insight into the Regulation of Dynein by Dynactin Drew Calhoun Dr. Elisar Barbar Biochemistry &...
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Transcript of Insight into the Regulation of Dynein by Dynactin Drew Calhoun Dr. Elisar Barbar Biochemistry &...
Insight into the Regulation of Dynein by Dynactin
Drew Calhoun
Dr. Elisar Barbar
Biochemistry & Biophysics Dept.
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
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
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 5030
35
40
45
50
55
60
65
70
75
80
85
ΔCp
Unfolded
Folded
SEC & LS - p150 (alone)
Red = UV absorbance
Black = Light ScatteringAggregates
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 (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