The folding of dimeric cytoplasmic malate dehydrogenase

Abstract

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 (M_C) formed immediately after subunit dissociation and the second oneis an expanded monomer (M_E), 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⇆2M_E⇆2M_C⇆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.