Characterization of Dynein Solubility using Small Ubiquitin-like Modifiers.

1. Characterization of Dynein Solubility using Small Ubiquitin-like Modifiers. Brian Phan Dr. Elisar Barbar HHMI Summer 2009

2. Introduction X • pH levels • Solvent • Temperature • Concentration • Time

4. Microtubule ATP Converts chemical energy from ATP into mechanical energy. Transports cellular cargo along microtubules. Cellular Cargo

6. Relevance Structural and mechanistic dysfunction in dynein can provide understanding to diseases and disorders. Provide insight into producing pharmaceutical agents that prevent aggregation. Leads into further studies for protein chemistry.

7. Background (cont.) Small ubiquitin-like modifier (SUMO). Protein modifier which attaches itself to other protein substrates. Discovered in 1996. Helps protein regulate cellular processes. http://www.mskcc.org/mskcc/_assets/content-image/215901.jpg

8. Hypothesis: SUMO helps the dynein intermediate chain become more soluble in solution.

9. Methods • Grow and purify SUMO-protease. • Use SUMO protease to cleave off SUMO from ICdel • Use gel to identify if enzyme is active in cleaving off SUMO

10. Results Affinity Buffer Content: 20mM Na-Phosphate 500mM NaCl 5mM B-mercaptoethanol 5% Glycerol 10mM Imidazole 1mM Na-azide SDS Gel of SUMO-protease after Affinity purification. • Molecular Marker • Flowthrough • Wash • 50mM Imidazole • 350mM Imidazole MW 55kDa 43kDa 34kDa 26kDa 17kDa 1 2 3 4 5

12. Affinity Buffer Content: 20mM Na-Phosphate 500mM NaCl 5mM B-mercaptoethanol 5% Glycerol 10mM Imidazole 1mM Na-azide pH=9.0 • SEC Chromatogram for SUMO protease where Abs280 nm is plotted against elution time on 45 mL SEC Column. Buffer used was common affinity buffer.

13. SEC Chromatogram for SUMO protease where Abs280 nm is plotted against elution time on 90 mL SEC Column.

14. Aff. Buffer Content: 50mM Tris-Base 350mM NaCl 10mM Imidazole 1mM B-mercaptoethanol 20% sucrose pH=8.0 • SEC Chromatogram for SUMO protease where Abs280 nm is plotted against elution time on 90 mL SEC Column. Buffer used was prescribed by Structural Biology Program at Cornell University.

15. Aff. Buffer Content: 50mM Tris-Base 350mM NaCl 10mM Imidazole 1mM B-mercaptoethanol No sucrose pH=9.0 • SEC Chromatogram for SUMO protease where Abs280 nm is plotted against elution time on 90mL SEC Column. Buffer used was prescribed by Structural Biology Program at Cornell University.

16. Affinity Buffer Content: 20mM Na-Phosphate 500mM NaCl 5% Glycerol 10mM Imidazole 1mM Na-azide pH=9.0 • SEC Chromatogram for SUMO protease where Abs280 nm is plotted against elution time on 90 mL SEC Column. Buffer used was original buffer.

17. Results MW 43kDa 34kDa 26kDa 17kDa 1 hr at 30oC SUMO-CAT: ~39kDa CAT: ~25kDa SUMO: ~15kDa Lane 1: Molecular Marker Lane 2: 1:500 Lane 3: 1:1000 Lane 4: 1:3000 Lane 5: 1:5000

18. Results MW 55kDa 43kDa 34kDa 26kDa 17kDa 24 hours at 4oC SUMO-CAT: ~39kDa CAT: ~25kDa SUMO: ~15kDa Lane 1: Molecular Marker Lane 2: 1:500 Lane 3: 1:1000 Lane 4: 1:3000 Lane 5: 1:5000

19. Results MW 43kDa 34kDa 26kDa 17kDa 48 hours at 4oC SUMO-CAT: ~39kDa CAT: ~25kDa SUMO: ~15kDa Lane 1: Molecular Marker Lane 2: 1:500 Lane 3: 1:1000 Lane 4: 1:3000 Lane 5: 1:5000

20. Conclusions • Use of sucrose leads to possible protein aggregation during purification. • Higher pH levels change protein solubility. • SUMO-protease is active in cleaving off SUMO from substrate.

21. Future Research Grow and purify an ICdel (92-261) construct. Grow and purify a SUMO ICdel( 92-261) construct. Analyze solubility levels of ICdel modified by SUMO and solubility levels of ICdel not modified by SUMO.

22. Acknowledgements • Dr. Elisar Barbar • Dr. Kevin Ahern • Barbar Lab Staff • Yujuan Song • Afua Nyarko • Justin Hall • Greg Benison • Jessica Morgan • Invitrogen • HHMI • URISC • OSU Biochemistry and Biophysics Dept.