Insight into the Regulation of Dynein by Dynactin

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