The folding of dimeric cytoplasmic malate dehydrogenase Equilibrium and kinetic studies
IR@IICB: CSIR-Indian Institute of Chemical Biology, Kolkata
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Title |
The folding of dimeric cytoplasmic malate dehydrogenase
Equilibrium and kinetic studies
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Creator |
Sanyal, Suparna C
Bhattacharyya, Debasish Das Gupta, Chanchal |
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Subject |
Structural Biology & Bioinformatics
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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.
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Publisher |
Blackwell Publishing
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Date |
2002
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Type |
Article
PeerReviewed |
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Format |
application/pdf
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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. |
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Relation |
http://dx.doi.org/10.1046/j.1432-1033.2002.03085.x
http://www.eprints.iicb.res.in/755/ |
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