1. Justin Biel Dr. Elisar Barbar HHMI Summer Research 2011 September 20, 2011

2. 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.  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. 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.  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. RunLC8 wtIC3Temp °CKdKd ΔHΔH-TΔSΔGΔGN 10.672 mM0.062 mM3014.64-13.626.91-6.711.02 20.672 mM0.062 mM104.39-9.7012.75-6.951.04

7. RunLC8 Y65EICTemp °CKdKd ΔHΔH-TΔSΔGΔGN 1 0.690 mM0.0680 mM3019.69-16.7210.18-6.540.82 20.690 mM0.0680 mM103.34-13.296.20-7.090.79

8. RunLC8 wtIC3Temp °CKdKd ΔHΔH-TΔSΔGΔGN 10.672 mM0.062 mM3014.97-13.086.39-6.690.85 20.672 mM0.062 mM3014.64-13.626.91-6.711.02 30.672 mM0.062 mM124.85-10.173.25-6.921.07 40.672 mM0.062 mM104.39-9.7012.75-6.951.04 RunLC8 Y65EIC3Temp °CKdKd ΔHΔH-TΔSΔGΔGN 50.690 mM0.0680 mM3020.58-16.6710.15-6.520.78 60.690 mM0.0680 mM3019.69-16.7210.18-6.540.82 70.690 mM0.0680 mM103.34-13.296.20-7.090.79 80.690 mM0.0680 mM103.16-13.196.06-7.130.72

9.  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.  Dr. Elisar Barbar  HHMI program  Dr. Kevin Ahern  URISC