Poster abstracts

Poster number 56 submitted by Wen-I Luo

Kinetic and Structural Characterization of Human Mortalin

Wen-I Luo (Ohio State Biochemistry Program), Eric Dizin (Department of Chemistry), Taejin Yoon (Department of Chemistry), James A. Cowan (Ohio State Biochemistry Program and Department of Chemistry)

Abstract:
Human mortalin, an Hsp70 chaperone that has been implicated in cancer and Parkinson’s disease, is engaged in iron-sulfur cluster biogenesis; however, the lack of isolable samples has prohibited its studies. Herein, we describe the cloning, expression, purification and biochemical characterization of mortalin. The purified recombinant mortalin possesses ATPase activity and a characteristic blue-shift in the fluorescence emission maximum following ATP addition. This protein was characterized through spectroscopic methods: UV-visible absorbance, fluorescence, and circular dichroism spectroscopies. Steady-state and single turnover kinetic experiments were performed and compared with another Hsp70 protein, T. maritima DnaK. Both enzymes show slow ATP turnover (mortalin: k_hyd=0.00060 s^-1; Tm DnaK: k_hyd=0.00036 s^-1) and similar secondary structures (~30% alpha-helix and 15% beta-strand). While Tm DnaK displays Michaelis-Menten behavior, mortalin shows a non-Michaelian profile. This work demonstrates key similarities and differences with other chaperones that are of functional significance.

References:
1. Mansy, S. S., and Cowan, J. A., Acc Chem Res 37, 719 (2004).
2. Kaul, S. C., Deocaris, C. C., and Wadhwa, R., Exp Gerontol 42, 263 (2007).
3. Schilke, B., Williams, B., Knieszner, H., Pukszta, S., D^,Silva, P., Craig, E. A., and Marszalek, J., Curr Biol 16, 1660 (2006).
4. Hoff, K. G., Silberg, J. J., and Vickery, L. E., Proc Natl Acad Sci U S A 97, 7790 (2000).
5. Silberg, J. J., Hoff, K. G., Tapley, T. L., and Vickery, L. E., J Biol Chem 276, 1696 (2001).

Keywords: Hsp70, mortalin, iron-sulfur cluster