The Dynein Motor Protein Complex: Structure and Dynamics HHMI Summer Research 2009 with Dr. Elisar Barbar By: Jonathan Yih .

The Dynein Motor Protein Complex: Structure and Dynamics

HHMI Summer Research 2009

with Dr. Elisar Barbar

By: Jonathan Yih

http://genome.gsc.riken.go.jp/hgmis/graphics/slides/images/Dynein_web.jpg

GoalImprove understanding of dynein’s structure

http://www.physics.berkeley.edu/research/yildiz/DyneinNew.jpg

Can lead to greater understanding of basic biological processes

http://dms.dartmouth.edu/compton/photos/photos/Mitotic_spindle_sm.jpg

Mitosis Motor Neuron Disease

http://www.physorg.com/newman/gfx/news/28-researcherss.jpg

Relevance

Dynein

Motor protein– Chemical energy (ATP) to

mechanical work

Functions– Transport– Organelle positioning– Cell division

http://cjournal.concordia.ca/journalarchives/2006-07/feb_8/008627.shtml

Made with 3DS Max 9

Background

A short domain of intermediate chain, IC3, is shown to be disordered but still able to bind LC8 and Tctex-1.

IC3 = IC84-143 Heavy Heads

Heavy Chains

LC8 Tctex-1

IC3

Hypothesis

The interaction of light chains with IC3 causes small changes in ordered structure that are not limited to the binding site of IC3.

Barbar 08 Biochemistry

Hypothesis

Learning the basics

Protein steps:

– Expression/Growth

– Purification

– Cleaning

http://www.biochem.arizona.edu/classes/bioc462/462a/NOTES/Protein_Properties/Fig5_18aCationExchColumn.GIF

Preparing LC8

LC 8 Purification

1 2 3 4 5 6Lane Description1 FT2 Buffer3 50 mM

Imidazole4 100 mM

Imidazole5 350 mM

Imidazole6 MW markers

Learning the basics

Make Tctex-1 unlabeled

Tctex-1 Purification

1 2 3 4 5 6

Lane Description1 FT2 Buffer3 50 mM

Imidazole4 100 mM

Imidazole5 350 mM

Imidazole6 MW markers

Make IC3 labeled

1 2 3 4 5 6

15N IC3 Purification

Lane Description1 MW markers2 FT3 Buffer4 50 mM

Imidazole5 100 mM

Imidazole6 350 mM

Imidazole

IC3/Tctex-1 complex

Titrate IC3 with unlabeled Tctex-1 and collect Nuclear Magnetic Resonance (NMR) dynamics experiments

+ 2

IC3 Tctex-1

?

Analyzing dynamics data

http://www.chem.unsw.edu.au/research/groups/field/images/two-D-NMR.gif

1H and 15N

Peak 1

Peak 2

HSQC - correlation of 1H and 15N

Unphased Phased

1H 1H

15N 15N

Free IC3 IC3 bound to LC8

HSQC interactions

Nuclear Overhauser Effect data

-6.009-9.380

N HHow “flexible” the N/H bond is

Acknowledgements

Special Thanks to:– Dr. Elisar Barbar– The Barbar lab group

– Dr. Kevin Ahern– Howard Hughes Medical Institute

Yujuan Song Afua NyarkoGreg Benison Justin HallJessica Morgan