The folding of dimeric cytoplasmic malate dehydrogenase. Equilibrium and kinetic studies.
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 <--> 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) Biochemistry 25, 1662-1669]. The major pathway (70%) is 2U-->2M*-->2M-->D, the rate limiting step being the isomerization of the monomers (K1 = 1.7 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*-->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.
Sanyal
Suparna C
SC
Department of Biophysics, Molecular Biology and Genetics, University of Calcutta, Kolkata, India. suparna.sanyal@icm.uu.se
Bhattacharyya
Debasish
D
Das Gupta
Chanchal
C
D000817:Anilino Naphthalenesulfonates / Q000737:chemistry / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003593:Cytoplasm / Q000201:enzymology*; D005453:Fluorescence; D019791:Guanidine; D007700:Kinetics; D008027:Light; D008291:Malate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism*; D011489:Protein Denaturation; D017510:Protein Folding; D012542:Scattering, Radiation; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D013997:Time Factors
D000817:Anilino Naphthalenesulfonates; C027132:1-anilino-8-naphthalenesulfonate; D008291:Malate Dehydrogenase; D019791:Guanidine