thermodynamic determinants and binding diversity of a hub protein
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Thermodynamic Determinants and Binding Diversity of a Hub Protein. Justin Biel Dr. Elisar Barbar HHMI Summer Research 2011 September 20, 2011. LC8: Involvement in Dynein Assembly. Lc8 is involved in the assembly of Dynein. Intercellular motor protein Carries packages around the cell - PowerPoint PPT PresentationTRANSCRIPT
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 N1 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