Thermodynamic Determinants and Binding Diversity of a Hub Protein

Justin BielDr. Elisar Barbar

HHMI Summer Research 2011September 20, 2011

LC8: Involvement in Dynein Assembly

•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.

IC – Dynein Intermediate Chain Swa – Swallow

◦ Involved in maintaining polarity during oogenesis nNOS – Neuronal Nitric Oxide Synthase Bim – Apoptosis protein factor Kibra 1 – Kidney and brain protein

Lc8 as a Hub Protein

LC8 Y65E•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.

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.

ITC: Isothermal calorimetry

LC8 wt (syringe) + IC (cell) at 30°C & 10°C

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

LC8 Y65E (syringe) + IC3 (cell)

at 30°C & 10°C

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

Thermodynamics LC8 wt vs. Y65EIC ligand

ITC with other LC8 binding partners:◦ Swa◦ Bim◦ nNos

Crystallography◦ Setting up crystal screens for LC8 + ligands that

bind◦ Investigating any possible structural differences in

binding between mutant and wild type LC8

Other Work

Dr. Elisar Barbar HHMI program Dr. Kevin Ahern URISC

Acknowledgements