TAILIEUCHUNG - Báo cáo Y học: The folding of dimeric cytoplasmic malate dehydrogenase Equilibrium and kinetic studies

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-anilino8-napthalene sulfonic acid binding, dynamic light scattering, and chromatographic (HPLC) techniques. The unfolding and refolding transitions are reversible and show the presence of two equilibrium intermediate states. The first one is a compact monomer (MC) formed immediately after subunit dissociation and the second one is an expanded monomer (ME), which is little less compact than the native monomer and has most of the characteristic features. | Eur. J. Biochem. 269 3856-3866 2002 FEBS 2002 doi The folding of dimeric cytoplasmic malate dehydrogenase Equilibrium and kinetic studies Suparna C. Sanyal1 Debasish Bhattacharyya2 and Chanchal Das Gupta1 1 Department of Biophysics Molecular Biology and Genetics University of Calcutta Kolkata India 2Indian Institute of Chemical Biology Kolkata India Porcine heart cytoplasmic malate dehydrogenase s-MDH is a dimeric protein 2 X 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 light scattering and chromatographic HPLC techniques. The unfolding and refolding transitions are reversible and show the presence of two equilibrium intermediate states. The first one is a compact monomer MC formed immediately after subunit dissociation and the second one is an expanded monomer ME which is little less compact than the native monomer and has most of the characteristic features of a molten globule state. The equilibrium transition is fitted in the model 2U 2Me 2McoD. The time course of kinetics of self- refolding of s-MDH revealed two parallel folding pathways Rudolph R. Fuchs I. Jaenicke R. 1986 Biochemistry 25 16621669 . The major pathway 70 is 2Ufi2M fi2MfiD the rate limiting step being the isomerization of the monomers K1 X 10-3 s-1 . The minor pathway 30 involves an association step leading to the incorrectly folding dimers prior to the very slow D fiD 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. Keywords equilibrium denaturation folding unfolding molten globule malate .

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