The Dynein Motor Protein Complex:
Structure and Dynamics
http://genome.gsc.riken.go.jp/hgmis/graphics/slides/images/Dynein_web.jpg
HHMI Summer Research 2009
with Dr. Elisar Barbar
By: Jonathan Yih
Goal
Improve understanding of dynein’s structure
http://www.physics.berkeley.edu/research/yildiz/DyneinNew.jpg
Relevance

Can lead to greater understanding of basic
biological processes
Mitosis
http://dms.dartmouth.edu/compton/photos/photos/Mitotic_spindle_sm.jpg
Motor Neuron Disease
http://www.physorg.com/newman/gfx/news/28-researcherss.jpg
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
Heavy
Chains

IC3 = IC84-143
Heavy
Heads
LC8 Tctex-1
Hypothesis

The interaction of light chains with IC3 causes
small changes in ordered structure that are not
limited to the binding site of IC3.
Hypothesis
Barbar 08 Biochemistry
Learning the basics

Protein steps:
–
Expression/Growth
–
Purification
–
Cleaning
http://www.biochem.arizona.edu/classes/bioc462/462a/NOTES/Protein_Properties/Fig5_18aCationExchColumn.GIF
Learning the basics

Preparing LC8
1
2
3
4
LC 8 Purification
5
6
Lane
1
2
3
4
5
6
Description
FT
Buffer
50 mM Imidazole
100 mM Imidazole
350 mM Imidazole
MW markers
Make Tctex-1 unlabeled
1
2
3
4
5
6
Lane
1
2
3
4
5
6
Tctex-1 Purification
Description
FT
Buffer
50 mM Imidazole
100 mM Imidazole
350 mM Imidazole
MW markers
Make IC3 labeled
1
2
3
4
5
6
Lane
1
2
3
4
5
6
15N
IC3 Purification
Description
MW markers
FT
Buffer
50 mM Imidazole
100 mM Imidazole
350 mM Imidazole
IC3/Tctex-1 complex

Titrate IC3 with unlabeled Tctex-1 and collect
Nuclear Magnetic Resonance (NMR) dynamics
experiments
+
IC3
?
2
Tctex-1
Analyzing dynamics data
1H
and 15N
Peak 1
Peak 2
http://www.chem.unsw.edu.au/research/groups/field/images/two-D-NMR.gif
HSQC - correlation of 1H and 15N
Unphased
Phased
15N
1H
15N
1H
HSQC interactions
Free IC3
IC3 bound to LC8
Nuclear Overhauser Effect data
How “flexible” the N/H bond is
N
NOE – IC3 Bound to LC8
0.0
-0.5
-1.0
-1.5
-2.0
-2.5
-3.0
-3.5
-4.0
0.2
0.0
-0.2
NOE
NOE
84
87
91
94
97
100
103
106
110
113
116
119
124
127
130
133
136
139
142
84
87
91
94
97
100
103
106
110
113
116
119
124
127
130
133
136
139
142
NOE – IC3 Free
H
-0.4
-0.6
-0.8
Residue
-6.009
-1.0
-9.380
Residue
Acknowledgements

Special Thanks to:
–
–
Dr. Elisar Barbar
The Barbar lab group
Yujuan Song
Greg Benison
Jessica Morgan
–
–
Afua Nyarko
Justin Hall
Dr. Kevin Ahern
Howard Hughes Medical Institute