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Modulation of cooperativity in Mycobacterium tuberculosis NADPH-ferredoxin reductase: Cation-and pH-induced alterations in native conformation and destabilization of the NADP⁺-binding domain

¹ Division of Molecular and Structural Biology, Central Drug Research Institute, Lucknow 226 001, India
² Dipartimento di Scienze Biomolecolarie e Biotecnologie, Università degli Studi di Milano, Milano, Italy

(RECEIVED October 6, 2004; FINAL REVISION November 25, 2004; ACCEPTED December 23, 2004)

FprA, a Mycobacterium tuberculosis NADPH-ferredoxin reductase, consists of two structural domains, a FAD-binding and a NADP-binding domain, respectively. For the first time, we demonstrated that native FprA, on thermal treatment underwent partial denaturation with unfolding of only the FAD-binding domain and release of the protein-bound flavin. The NADP-binding domain of this protein is highly resistant to denaturation under these conditions. However, the presence of either 150 mM NaCl or KCl or 10 µM MgCl₂ or CaCl₂ or slightly acidic pH of 6.0 resulted in a highly cooperative and complete thermal unfolding of the protein. Physicochemical investigations showed that the monovalent cations or low concentrations of divalent cations induced compaction of the protein conformation. However, divalent cations at higher concentrations resulted in FAD release leading to stabilization of an enzymatically inactive apo-enzyme. Detailed thermal denaturation studies on the native protein and the isolated NADP-binding domain showed that cations and pH 6.0 destabilized only the heat-stable NADP-binding domain. The experimental studies demonstrate that modulation of intramolecular ionic interactions induce significant conformational changes in the NADP-binding domain of FprA, resulting in a substantial increase in the structural cooperativity of the whole molecule. The results presented in this paper are of importance as they demonstrate alterations in the native three-dimensional structure of FprA and cooperativity in protein molecule on slight alteration of pH or modification of ionic interactions in protein.

Keywords: flavoprotein; Mycobacterium tuberculosis; cooperativity; thermal denaturation; cation

Abbreviations: FprA, NADPH-ferredoxin reductase • T_m, midpoint of thermal denaturation

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.041162705.

Reprint requests to: Vinod Bhakuni, Division of Molecular and Structural Biology, Central Drug Research Institute, Lucknow 226 001, India; e-mail: bhakuniv{at}rediffmail.com; fax: +91-522-223405.

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