Insight into the Regulation of Dynein by Dynactin

Drew Calhoun

Dr. 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

CEN

TRIFU

GE

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 5030

35

40

45

50

55

60

65

70

75

80

85

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 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

Thank you!

Dr. Elisar Barbar HHMI NSF

Special thanks to… Dr. Afua Nyarko

Protein preparation Gretchen Clark-Scannel

SEC & LS