title: Functional intermediate in the refolding pathway of a large and multidomain protein malate synthase G.
abstract: Despite their prevalence in biological systems, information about the folding pathways of large and multidomain proteins is meager, as they often unfold irreversibly under in vitro conditions which make their folding studies difficult or even impossible. The folding mechanism of a large (82 kDa) and multidomain protein Malate synthase G (MSG) has been demonstrated in the present study using intrinsic tryptophan fluorescence, enzymatic activity, and extrinsic fluorophore ANS as probes for monitoring the refolding process. Refolding of MSG is found to occur in three kinetic phases. Denatured MSG forms a collapsed state in the burst phase of refolding, which then gives rise to an active intermediate having the same tryptophan fluorescence and enzymatic activity as native MSG in the slow phase. Native topology of MSG is formed from the active intermediate in the very slow phase of refolding which is silent to tryptophan fluorescence change and is susceptible to aggregation at higher protein concentrations. Dependence of rates of very slow phase on GdnHCl concentration suggests that it is not solely a cis/trans proline isomerization limited process but might involve an additional folding event of the domains, not forming the active site of the protein. In light of the above findings, the appearance of a functional intermediate during refolding of MSG was predicted to be an instance of weak interdomain cooperativity. This work has significant implications in the characterization of the refolding intermediates of multidomain proteins in general and MSG in particular, where weak interdomain cooperativity might contribute toward generation of a functional intermediate during its refolding.
authors.affiliation: Kusuma School of Biological Sciences, Indian Institute of Technology Delhi, Hauz Khas, New Delhi-110016, India.
authors.forename: Vinay
authors.initials: V
authors.lastname: Dahiya
authors.forename: Tapan K
authors.initials: TK
authors.lastname: Chaudhuri
mesh_terms: D002942:Circular Dichroism; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology*; D005453:Fluorescence; D007700:Kinetics; D008292:Malate Synthase / Q000737:chemistry*; D011392:Proline / Q000737:chemistry; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013816:Thermodynamics
chemical_list: D011392:Proline; D008292:Malate Synthase