Unfolding and refolding of glucose/xylose isomerase from Streptomyces sp. NCIM 2730

Ghatge, Mohini S.; Phadatare, Sangita U.; Bodhe, A.M.; Deshpande, Vasanti

doi:10.1016/0141-0229(94)90174-0

Cited by 11 publications

(8 citation statements)

References 21 publications

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“…Light scattering studies indicated an absence of aggregates in the molten globule state. Partially folded dimeric intermediates were also detected in the case of oligomeric proteins such as phosphoglucose isomerase [38], alkaline phosphatase [39], creatinase [40] and during the GdnHCl‐induced denaturation of glucose/xylose isomerase [41].…”

Section: Discussionmentioning

confidence: 99%

Characterization of acid‐induced unfolding intermediates of glucose/xylose isomerase

Pawar

Deshpande

2000

European Journal of Biochemistry

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Acid-induced unfolding of the tetrameric glucose/xylose isomerase (GXI) from Streptomyces sp. NCIM 2730 has been investigated using intrinsic fluorescence, fluorescence quenching, second derivative spectroscopy, hydrophobic dye (1-anilino-8-naphthalene-sulfonate) binding and CD techniques. The pH dependence of tryptophanyl fluorescence of GXI at different temperatures indicated the presence of two stable intermediates at pH 5.0 and pH 3.0. The pH 3.2 intermediate was a dimer and exhibited molten globule-like characteristics, such as the presence of native-like secondary structure, loss of tertiary structure, increased exposure of hydrophobic pockets, altered microenvironment of tyrosine residues and increased accessibility to quenching by acrylamide. Fluorescence and CD studies on GXI at pH 5.0 suggested the involvement of a partially folded intermediate state in the native to molten globule state transition. The partially folded intermediate state retained considerable secondary and tertiary structure compared to the molten globule state. This state was characterized by its hydrophobic dye binding capacity, which is smaller than the molten globule state, but was greater than that of the native state. This state shared the dimeric status of the molten globule state but was prone to aggregate formation as evident by the Rayleigh light scattering studies. Based on these results, the unfolding pathway of GXI can be illustrated as: N3PFI3MG3U; where N is the native state at pH 7.5; PFI is the partially folded intermediate state at pH 5.0; MG is the molten globule state at pH 3.2 and U is the monomeric unfolded state of GXI obtained in the presence of 6 m GdnHCl. Our results demonstrate the existence of a partially folded state and molten globule state on the unfolding pathway of a multimeric a/b barrel protein. A variety of proteins has been observed that exist in stable conformations that are neither fully folded nor unfolded, and are said to be in the`molten globule' state [5]. These partly folded intermediates can be made to accumulate in equilibrium by mild concentrations of chemical denaturants, low pH, covalent trapping or by protein engineering [6]. The molten globule state represents a third conformational state and possesses several characteristic features such as: (a) high content of native-like secondary structure; (b) overall compactness with buried but highly mobile aromatic side-chains; (c) exposure of hydrophobic surface, as indicated by dye binding ability and susceptibility to aggregation; (d) lack of cooperative interactions; (e) rapid equilibration with the fully unfolded states; and (f ) an increase in the hydrodynamic volume compared to the folded state. Interest in the molten globule state has also been due to its physiological role as it is recognized by the chaperone GroEL during insertion of the protein into the cell membrane [7,8]. Previous studies with monomeric proteins have contributed significantly towards our understanding of the protein folding problem [9±14]. However, similar studies...

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Section: Discussionmentioning

confidence: 99%

Characterization of acid‐induced unfolding intermediates of glucose/xylose isomerase

Pawar

Deshpande

2000

European Journal of Biochemistry

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“…strain NCIM 2730 revealed that the tetramer and the dimer are the active species whereas the monomer is inactive. The occurrence of a molten-globule-like intermediate in the folding pathway of GI was demonstrated for the first time (76). Intact tertiary rather than secondary structure was shown to be responsible for the biological activity of GI.…”

Section: Subunit Structurementioning

confidence: 94%

Molecular and industrial aspects of glucose isomerase.

Bhosale

Rao

Deshpande

1996

Microbiological Reviews

333

205

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“…A substrate stabilization effect has also been reported for immobilized Streptomyces murinus enzyme (Converti and Del Borghi, 1997), although it was not as dramatic as in the study of Volkin and Klibanov (1989). Other studies carried out in various inactivating conditions suggest that Streptomyces glucose isomerases are irreversibly inactivated when the tetrameric enzyme is converted to monomeric form (Callens et al, 1988;Ghatge et al, 1994). For glucose isomerase from Thermoanaerobacterium thermosulfurigens, the controlling factor of inactivation in high temperature was incorrect protein folding (Meng et al, 1993).…”

Section: Introductionmentioning

confidence: 97%