The folding of dimeric cytoplasmic malate dehydrogenase

Sanyal, Suparna C. ; Bhattacharyya, Debasish ; Das Gupta, Chanchal (2002) The folding of dimeric cytoplasmic malate dehydrogenase European Journal of Biochemistry, 269 (15). pp. 3856-3866. ISSN 0014-2956

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Porcine heart cytoplasmic malate dehydrogenase (s-MDH) is a dimeric protein (2 × 35 kDa). We have studied equilibrium unfolding and refolding of s-MDH using activity assay, fluorescence, far-UV and near-UV circular dichroism (CD) spectroscopy, hydrophobic probe-1-anilino-8-napthalene sulfonic acid binding, dynamic lightscattering, and chromatographic (HPLC) techniques. The unfolding and refolding transitions are reversible andshow the presence of two equilibrium intermediate states. The first one is a compact monomer (MC) formed immediately after subunit dissociation and the second oneis an expanded monomer (ME), which is little less compact than the native monomer and has most of thecharacteristic features of a 'molten globule' state. Theequilibrium transition is fitted in the model: 2U⇆2ME⇆2MC⇆D. The time course of kinetics of self- refolding of s-MDH revealed two parallel folding pathways [Rudolph, R., Fuchs, I. & Jaenicke, R. (1986) Biochemistry25, 1662-1669]. The major pathway (70%) is 2U→2M*→ 2M→D, the rate limiting step being the isomerization of the monomers (K1 = 1.7 × 10-3 s-1). The minor pathway (30%) involves an association step leading to the incorrectly folding dimers, prior to the very slow D*→D folding step. In this study, we have characterized the folding-assembly pathway of dimeric s-MDH. Our kinetic and equilibrium experiments indicate that the folding of s-MDH involves the formation of two folding intermediates. However, whether the equilibrium intermediates are equivalent to the kinetic ones is beyond the scope of this study.

Item Type:Article
Source:Copyright of this article belongs to John Wiley and Sons.
Keywords:Equilibrium Denaturation; Folding; Unfolding; Molten Globule; Malate Dehydrogenase
ID Code:65848
Deposited On:19 Oct 2011 09:10
Last Modified:19 Oct 2011 09:10

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