Abstract
Human mortalin, an Hsp70 chaperone that has been implicated in cancer and Parkinson's disease, is engaged in Fe‐S 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: khyd=0.00060 s−1; Tm DnaK: khyd=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 importance.
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