Title
Purification and Structural Characterization of Mce4A fromMycobacterium tuberculosis
Author
Shagufta Khan, Asimul Islam, Md. Imtaiyaz Hassan, Faizan Ahmad
Year
2016
Journal
International Journal of Biological Macromolecules
Abstract
The mce4A gene of Mycobacterium tuberculosis encodes a 400 amino acid residues protein of 43 kDa, which is a mammalian cell entry protein (Mce4A) and plays important role in host cell invasion. Mce4A helps in long-term survival of M. tuberculosis by cholesterol utilization. Host cholesterol utilization mechanism by Mce4A is not clearly understood. In order to investigate the role of Mce4A in M. tuberculosis pathogenesis, we purified the recombinant protein by affinity chromatography, analyzed by SDS-PAGE and confirmed by western blot. We performed structural studies of Mce4A as function of pH and salt concentration by using different spectroscopic techniques. This protein was found to be stable over the wide range of pH 5.5 ≤ pH ≤ 11.5. An addition of sodium chloride up to the concentration of 150 mM, shows no significant change in the secondary structure content of the protein. To confirm its activity, we performed isothermal titration calorimetry measurements of Mce4A in the presence of cholesterol. This is the first report of binding of cholesterol to Mce4A in vitro. Binding of cholesterol to Mce4A is sequential four-step and entropy driven process. The structural studies of this protein will help to understand the mechanism of pathogenesis of M. tuberculosis.
Instrument
J-1500
Keywords
Circular dichroism, Secondary structure, Biochemistry