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
Dec 22, 2015
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