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Thermodynamic Determinants and Binding Diversity of a Hub Protein

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

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Thermodynamic Determinants and Binding Diversity of a Hub Protein

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  1. Thermodynamic Determinants and Binding Diversity of a Hub Protein Justin Biel Dr. Elisar Barbar HHMI Summer Research 2011 September 20, 2011

  2. 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.

  3. Lc8 as a Hub Protein • 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

  4. 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.

  5. ITC: Isothermal calorimetry • 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.

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

  7. LC8 Y65E (syringe) + IC3(cell) at 30°C & 10°C

  8. Thermodynamics LC8 wt vs. Y65EIC ligand

  9. Other Work • 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

  10. Acknowledgements • Dr. Elisar Barbar • HHMI program • Dr. Kevin Ahern • URISC

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