The Dynein Motor Protein Complex: Structure and Dynamics

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The Dynein Motor Protein Complex Structure and
Dynamics
http//genome.gsc.riken.go.jp/hgmis/graphics/slide
s/images/Dynein_web.jpg

• HHMI Summer Research 2009
• with Dr. Elisar Barbar
• By Jonathan Yih

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Goal

• Improve understanding of dyneins structure

http//www.physics.berkeley.edu/research/yildiz/Dy
neinNew.jpg
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Relevance

• Can lead to greater understanding of basic
  biological processes

Mitosis
Motor Neuron Disease
http//dms.dartmouth.edu/compton/photos/photos/Mit
otic_spindle_sm.jpg
http//www.physorg.com/newman/gfx/news/28-research
erss.jpg
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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
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Made with 3DS Max 9
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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

IC3
LC8
Tctex-1
Heavy Chains
Heavy Heads
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Hypothesis

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

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Hypothesis

Barbar 08 Biochemistry
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Learning the basics

• Protein steps
• Expression/Growth
• Purification
• Cleaning

http//www.biochem.arizona.edu/classes/bioc462/462
a/NOTES/Protein_Properties/Fig5_18aCationExchColum
n.GIF
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Learning the basics

• Preparing LC8

1 2 3 4 5 6
Lane Description 1 FT 2 Buffer 3 50 mM
Imidazole 4 100 mM Imidazole 5 350 mM
Imidazole 6 MW markers
LC 8 Purification
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Make Tctex-1 unlabeled
1 2 3 4 5 6
Lane Description 1 FT 2 Buffer 3 50 mM
Imidazole 4 100 mM Imidazole 5 350 mM
Imidazole 6 MW markers
Tctex-1 Purification
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Make IC3 labeled
1 2 3 4 5 6
Lane Description 1 MW markers 2 FT 3 Buffer 4
50 mM Imidazole 5 100 mM Imidazole 6 350 mM
Imidazole
15N IC3 Purification
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IC3/Tctex-1 complex

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

?

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IC3
Tctex-1
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Analyzing dynamics data
1H and 15N
Peak 1
Peak 2
http//www.chem.unsw.edu.au/research/groups/field/
images/two-D-NMR.gif
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HSQC - correlation of 1H and 15N
Unphased Phased
15N
15N
1H
1H
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HSQC interactions

• Free IC3 IC3 bound to LC8

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Nuclear Overhauser Effect data
N
H
How flexible the N/H bond is
-6.009
-9.380
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Acknowledgements

• Special Thanks to
• Dr. Elisar Barbar
• The Barbar lab group
• Dr. Kevin Ahern
• Howard Hughes Medical Institute

Yujuan Song Afua Nyarko Greg Benison Justin
Hall Jessica Morgan