CSIR Central

The folding of dimeric cytoplasmic malate dehydrogenase Equilibrium and kinetic studies

IR@IICB: CSIR-Indian Institute of Chemical Biology, Kolkata

View Archive Info
 
 
Field Value
 
Title The folding of dimeric cytoplasmic malate dehydrogenase Equilibrium and kinetic studies
 
Creator Sanyal, Suparna C
Bhattacharyya, Debasish
Das Gupta, Chanchal
 
Subject Structural Biology & Bioinformatics
 
Description 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 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«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) Biochemistry 25, 1662– 1669]. The major pathway (70%) is 2Ufi2M*fi2MfiD, the rate limiting step being the isomerization of the monomers (K1 ¼ 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*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.
 
Publisher Blackwell Publishing
 
Date 2002
 
Type Article
PeerReviewed
 
Format application/pdf
 
Identifier http://www.eprints.iicb.res.in/755/1/EUROPEAN_JOURNAL_OF_BIOCHEMISTRY__269_(15)_3856%2D3866;2002[35].pdf
Sanyal, Suparna C and Bhattacharyya, Debasish and Das Gupta, Chanchal (2002) The folding of dimeric cytoplasmic malate dehydrogenase Equilibrium and kinetic studies. European Journal Of Biochemistry, 269 (15). pp. 3856-3866.
 
Relation http://dx.doi.org/10.1046/j.1432-1033.2002.03085.x
http://www.eprints.iicb.res.in/755/