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

Bhatt, Anant Narayan ; Shukla, Nidhi ; Aliverti, Alessandro ; Zanetti, Giuliana ; Bhakuni, Vinod (2005) Modulation of cooperativity in Mycobacterium tuberculosis NADPH-ferredoxin reductase: cation- and pH-induced alterations in native conformation and destabilization of the NADP+-binding domain Protein Science, 14 (4). pp. 980-992. ISSN 0961-8368

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Official URL: http://www3.interscience.wiley.com/journal/1216027...

Related URL: http://dx.doi.org/10.1110/ps.041162705

Abstract

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 μ M 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 apoenzyme. 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.

Item Type:Article
Source:Copyright of this article belongs to Cold Spring Harbor Laboratory Press.
Keywords:Flavoprotein; Mycobacterium tuberculosis; Cooperativity; Thermal Denaturation; Cation
ID Code:20969
Deposited On:20 Nov 2010 09:27
Last Modified:17 May 2016 05:13

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