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Journal of Biochemistry Advance Access originally published online on February 23, 2009
Journal of Biochemistry 2009 145(6):721-731; doi:10.1093/jb/mvp029
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© The Authors 2009. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved

Guanidine Hydrochloride- and Urea-Induced Unfolding of Toxoplasma gondii Ferredoxin-NADP+ Reductase: Stabilization of a Functionally Inactive Holo-Intermediate

Kulwant Singh* and Vinod Bhakuni

Division of Molecular and Structural Biology, Central Drug Research Institute, Lucknow 226 001, India

*To whom correspondence should be addressed: Tel: +91-522-2612411–18; Fax: +91-522-223405; E-mail: ksinghcdri{at}gmail.com

Received January 3, 2009; Accepted February 16, 2009


  Abstract

Usually during the folding/unfolding of flavoproteins, an apo-intermediate is stabilized before global unfolding of the enzymes occurs. However, stabilization of a holo-intermediate has also been reported for a few flavoproteins. We have studied the unfolding of Toxoplasma gondii ferredoxin-NADP+ reductase (TgFNR) using GdnHCl and urea. A functionally inactive holo-intermediate of the enzyme was found to be stabilized during this unfolding process. The intermediate species had cofactor FAD bound to it, but it showed free movement due to which the stabilized intermediates were functionally inactive. The native TgFNR behaves cooperatively with the two structural domains interacting strongly with each other. The denaturants GdnHCl and urea, at low concentrations, were found to interact selectively with the NADP+-binding domain of TgFNR and to induce structural modifications in it. These selective modifications in the protein molecule lead to loss of interactions between two domains and the enzyme behaved non-cooperatively resulting in stabilization of an intermediate species. Significant differences in the structural properties of the GdnHCl- and urea-stabilized holo-intermediates of TgFNR were observed. Comparison of the unfolding pathway of TgFNR (a plant-type FNR) with that of FprA (a GR-type FNR) demonstrates that they follow very different pathways of unfolding.

Key Words: flavin adenine dinucleotide, flavoproteins, folding/unfolding, holo-intermediate, protein stabilization/destabilization

Abbreviations: AdR, adrenodoxin reductase; ANS, 1-anilino-8-naphthalene sulfonate; CD, circular dichroism; Cm, midpoint of chemical denaturation; FAD, flavin adenine dinucleotide; FNR, ferredoxin-NADP+ reductase; FprA, Mycobacterium tuberculosis NADPH-ferredoxin reductase; GdnHCl, guanidine hydrochloride; GR, glutathione reductase; ONFR, oxygenase-coupled NADH-ferredoxin reductase; SEC, size exclusion chromatography; TgFNR, Toxoplasma gondii ferredoxin-NADP+ reductase; Tm, midpoint of thermal denaturation


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