1. Determination of Dynein Light Chain LC7 Stability and Folding using Circular Dichroism Rachel Rasberry HHMI Summer 2010 Working with Dr. Elisar Barbar

2. Background Cytoplasmic dynein  a large protein complex LC7  a ubiquitous component of cytoplasmic dynein  binds to Intermediate Chain (IC) http://www.nature.com/nrm/journal/v10/n12/box/nrm2804_BX1.html

3. Background Cytoplasmic dynein  is a motor protein that converts chemical energy (ATP) into mechanical energy  transports cellular cargo by “walking” along microtubules

4. Relevance Dynein plays a role in:  Mitosis  Vesicular transport  Development and maintenance of neurons  Diseases resulting from dynein dysfunction Lissencephaly Neural degeneration Male infertility

5. LC7 a. Two structural views of LC7 b. IC-LC7 complex Barbar ’10 JOURNAL OF BIOLOGICAL CHEMISTRY

6. Bound IC Completes the Fold LC7 belongs to an ancient protein superfamily. The position of bound IC in the IC-LC7 complex mimics a helix that is integrated into the primary structure in distantly related LC7 homologs. a. IC-LC7 complex b. MgI complex c. MP1_p14 complex Barbar ’10 JOURNAL OF BIOLOGICAL CHEMISTRY

7. Hypothesis IC binding increases the stability of LC7.

8. Circular Dichroism (CD) Spectroscopy CD  measures differences in the absorption of left-handed polarized light versus right-handed polarized light which arise due to structural asymmetry  Can be used to: determine whether a protein is folded, and if so characterize its secondary structure study the conformational stability of a protein under stress -- thermal stability, pH stability, and stability to denaturants (i.e. urea)

9. Determination of Protein Secondary Structure by Circular Dichroism Need to collect data in the "far-UV" spectral region (wavelengths of 190-250 nm) Alpha-helix, beta-sheet, and random coil structures each give rise to a characteristic spectral profile of the CD spectrum

10. Determination of Protein Secondary Structure by Circular Dichroism An approximate fraction of each secondary structure type that is present in any protein can be determined  For example, CD can determine that a protein contains about 50% alpha-helix; however, it cannot determine where the alpha-helical portions are located in the molecule.

11. Experiment LC7 concentrations tested  30.0 μM  16.7 μM  9.0 μM  6.0 μM  3.3 μM  1.4 μM

15. Far UV-CD spectra of LC7 wt (-)His6X Urea denaturation Protein concentration: 3.3 μM 0.5 cm cell path length 30°C

16. Far UV-CD spectra of LC7 wt (-)His6X Urea denaturation Protein concentration: 16.7 μM 0.1 cm cell path length 30°C

17. Urea unfolding profiles monitored by far-UV CD for LC7 wt (-) His6X protein 30°C protein concentrations: 16.7 μM (monitored at 220 nm, cell path length = 0.1 cm) 3.3 μM (monitored at 222 nm, cell path length = 0.5 cm)

18. Purifying the IC-LC7 complex Residues 212-260 of IC (IC 212-260 ) contain the region of IC that binds to LC7 Add SUMO protein and His-tag to IC 212-260 Run through nickel column and Size Exclusion Column to purify 260212 SUMOHis tagIC

19. Purifying the IC-LC7 complex Run a SDS-PAGE gel to check purity Mixed with LC7 Run through a nickel column  His-tag allows binding to nickel  Cut with SUMO protease  Elute IC-LC7 complex His-tag_SUMO_IC 212-260 _LC7 Add SUMO protease Elute IC_LC7 Add 350μM Imidazole His-tag_SUMO

20. Continuation of project… Perform CD experiments on the IC-LC7 complex Analyze data  Compare CD plots for free and bound LC7

21. Acknowledgements Special thanks to: Dr. Elisar Barbar The Barbar Lab Group  Jessica Morgan  Yujuan Song  Afua Nyarko Dr. Kevin Ahern Howard Hughes Medical Institute