Justin Biel
Dr. Elisar Barbar
HHMI Summer Research 2011
September 20, 2011
•Lc8 is involved in the assembly of Dynein.
•Intercellular motor protein
•Carries packages around the cell
•Critical in spindle formation for cellular division.
•The binding of Lc8, to its binding partner IC, allows for a 50 fold
enhancement of binding of neighboring proteins during assembly.
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IC – Dynein Intermediate Chain
Swa – Swallow
◦ Involved in maintaining polarity during oogenesis
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nNOS – Neuronal Nitric Oxide Synthase
Bim – Apoptosis protein factor
Kibra 1 – Kidney and brain protein
•Single amino acid
substitutions are usually either
loss of function or neutral
mutations.
•It is our hypothesis that the
position 65 Tyrosine plays a
role in determining binding
ability to LC8’s binding
partners.
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Technique used to measure the small
changes in heat given or taken by the system
when proteins create bonding interactions.
Method:
LC8’s binding partners will be placed in the
cell in a solution of ITC phosphate buffer
LC8 will be titrated in through a series of
injections.
Run
LC8 wt
IC3
Temp °C
Kd
ΔH
-TΔS
ΔG
N
1
0.672 mM
0.062 mM
30
14.64
-13.62
6.91
-6.71
1.02
2
0.672 mM
0.062 mM
10
4.39
-9.701
2.75
-6.95
1.04
Run
LC8 Y65E
IC
Temp °C
Kd
ΔH
-TΔS
ΔG
N
1
0.690 mM
0.0680 mM
30
19.69
-16.72
10.18
-6.54
0.82
2
0.690 mM
0.0680 mM
10
3.34
-13.29
6.20
-7.09
0.79
Run
LC8 wt
IC3
Temp °C
Kd
ΔH
-TΔS
ΔG
N
1
0.672 mM
0.062 mM
30
14.97
-13.08
6.39
-6.69
0.85
2
0.672 mM
0.062 mM
30
14.64
-13.62
6.91
-6.71
1.02
3
0.672 mM
0.062 mM
12
4.85
-10.17
3.25
-6.92
1.07
4
0.672 mM
0.062 mM
10
4.39
-9.701
2.75
-6.95
1.04
Run
LC8 Y65E
IC3
Temp °C
Kd
ΔH
-TΔS
ΔG
N
5
0.690 mM
0.0680 mM
30
20.58
-16.67
10.15
-6.52
0.78
6
0.690 mM
0.0680 mM
30
19.69
-16.72
10.18
-6.54
0.82
7
0.690 mM
0.0680 mM
10
3.34
-13.29
6.20
-7.09
0.79
8
0.690 mM
0.0680 mM
10
3.16
-13.19
6.06
-7.13
0.72
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ITC with other LC8 binding partners:
◦ Swa
◦ Bim
◦ nNos
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Crystallography
◦ Setting up crystal screens for LC8 + ligands that
bind
◦ Investigating any possible structural differences in
binding between mutant and wild type LC8
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Dr. Elisar Barbar
HHMI program
Dr. Kevin Ahern
URISC