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

Introduction pH levels Solvent Temperature Concentration Time X

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

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.

Background (cont.) Small ubiquitin-like modifier (SUMO). Protein modifier which attaches itself to other protein substrates. Discovered in Helps protein regulate cellular processes.

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

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

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

SEC Chromatogram for SUMO protease where Abs280 nm is plotted against elution time on 45 mL SEC Column. Buffer used was common affinity buffer. 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 90 mL SEC Column.

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. 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 90mL SEC Column. Buffer used was prescribed by Structural Biology Program at Cornell University. 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 90 mL SEC Column. Buffer used was original buffer. Affinity Buffer Content: 20mM Na-Phosphate 500mM NaCl 5% Glycerol 10mM Imidazole 1mM Na-azide pH=9.0

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

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

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

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.

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

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.