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

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 A short domain of intermediate chain, IC3, is shown to be disordered but still able to bind LC8 and Tctex-1. IC3 = IC 84-143 Heavy Heads Heavy Chains LC8Tctex-1 IC3

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. Barbar 08 Biochemistry Hypothesis

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 LC 8 Purification 1 2 3 4 5 6 LaneDescription 1FT 2Buffer 350 mM Imidazole 4100 mM Imidazole 5350 mM Imidazole 6MW markers Learning the basics

11. Make Tctex-1 unlabeled Tctex-1 Purification 1 2 3 4 5 6 LaneDescription 1FT 2Buffer 350 mM Imidazole 4100 mM Imidazole 5350 mM Imidazole 6MW markers

12. Make IC3 labeled 1 2 3 4 5 6 15 N IC3 Purification LaneDescription 1MW markers 2FT 3Buffer 450 mM Imidazole 5100 mM Imidazole 6350 mM Imidazole

13. IC3/Tctex-1 complex Titrate IC3 with unlabeled Tctex-1 and collect Nuclear Magnetic Resonance (NMR) dynamics experiments + 2 IC3Tctex-1 ?

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

15. HSQC - correlation of 1 H and 15 N Unphased Phased 1H1H 1H1H 15 N

16. Free IC3IC3 bound to LC8 HSQC interactions

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

18. Acknowledgements Special Thanks to: – Dr. Elisar Barbar – The Barbar lab group – Dr. Kevin Ahern – Howard Hughes Medical Institute Yujuan SongAfua Nyarko Greg BenisonJustin Hall Jessica Morgan