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The Dynein Motor Protein Complex: Structure and Dynamics

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. Goal. Improve understanding of dynein’s structure.

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The Dynein Motor Protein Complex: Structure and Dynamics

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  1. 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

  2. Goal Improve understanding of dynein’s structure http://www.physics.berkeley.edu/research/yildiz/DyneinNew.jpg

  3. Relevance • Can lead to greater understanding of basic biological processes Mitosis Motor Neuron Disease http://dms.dartmouth.edu/compton/photos/photos/Mitotic_spindle_sm.jpg http://www.physorg.com/newman/gfx/news/28-researcherss.jpg

  4. 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

  5. Made with 3DS Max 9

  6. Background IC3 LC8 Tctex-1 Heavy Chains Heavy Heads A short domain of intermediate chain, IC3, is shown to be disordered but still able to bind LC8 and Tctex-1. IC3 = IC84-143

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

  8. Hypothesis Barbar 08 Biochemistry

  9. Learning the basics • Protein steps: • Expression/Growth • Purification • Cleaning http://www.biochem.arizona.edu/classes/bioc462/462a/NOTES/Protein_Properties/Fig5_18aCationExchColumn.GIF

  10. Preparing LC8 Learning the basics 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

  11. 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

  12. Make IC3 labeled 1 2 3 4 5 6 Lane Description 1 MW markers 2 FT 3 Buffer 4 50 mMImidazole 5 100 mMImidazole 6 350 mMImidazole 15N IC3 Purification

  13. IC3/Tctex-1 complex • Titrate IC3 with unlabeled Tctex-1 and collect Nuclear Magnetic Resonance (NMR) dynamics experiments ? + 2 IC3 Tctex-1

  14. Analyzing dynamics data 1H and 15N Peak 1 Peak 2 http://www.chem.unsw.edu.au/research/groups/field/images/two-D-NMR.gif

  15. HSQC - correlation of 1H and 15N Unphased Phased 15N 15N 1H 1H

  16. HSQC interactions Free IC3 IC3 bound to LC8

  17. Nuclear Overhauser Effect data How “flexible” the N/H bond is N H -6.009 -9.380

  18. 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

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