title: Purification and structural characterization of Mce4A from Mycobacterium tuberculosis.
abstract: The mce4A gene of Mycobacterium tuberculosis encodes a 400 amino acid residues protein of 43kDa, 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 150mM, 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.
authors.affiliation: Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi 110025, India.
authors.forename: Shagufta
authors.initials: S
authors.lastname: Khan
authors.affiliation: Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi 110025, India. Electronic address: aislam@jmi.ac.in.
authors.forename: Asimul
authors.initials: A
authors.lastname: Islam
authors.affiliation: Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi 110025, India.
authors.forename: Md Imtaiyaz
authors.initials: MI
authors.lastname: Hassan
authors.affiliation: Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi 110025, India.
authors.forename: Faizan
authors.initials: F
authors.lastname: Ahmad
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics; D009169:Mycobacterium tuberculosis / Q000235:genetics; D017433:Protein Structure, Secondary; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; C510664:Mce4A protein, Mycobacterium tuberculosis
keywords: Cell invasion; Cholesterol utilization; MCE-family protein; Mycobacterium tuberculosis