title: N-terminal domain of Pyrococcus furiosus l-asparaginase functions as a non-specific, stable, molecular chaperone.
abstract: The enzyme l-asparaginase of Pyrococcus furiosus (PfA) functions as a dimer with each monomer consisting of distinct N- and C-terminal domains (NPfA and CPfA, respectively), connected by a linker. Here we present data to show that NPfA functions as a non-specific molecular chaperone. Independently expressed NPfA refolded spontaneously whereas CPfA formed insoluble aggregates. However, when mixed and refolded together, NPfA augmented CPfA to fold with ~90% recovery. NPfA also protected a variety of substrate proteins from thermal and refolding-mediated aggregation as monitored by a reduction in light scattering. The co-appearance of substrate protein with NPfA in antibody pull-down assays as well as in eluted gel filtration peaks indicated direct protein-protein interaction. These interactions were hydrophobic in nature as determined by 8-anilino-1-naphthalene sulfonic acid fluorescence. NPfA inhibited polyglutamine-mediated amyloid formation and also facilitated disintegration of preformed amyloid fibrils of amyloid-β (1-42) as determined by reverse-phase HPLC-based sedimentation assay and thioflavin T binding assays, respectively. Dynamic light scattering experiments suggested that NPfA readily assembled into polydispersed oligomeric species. With no sequence similarity to α-crystallin or any known molecular chaperone, we present here NPfA as a novel molecular chaperone.
authors.affiliation: Kusuma School of Biological Sciences, Indian Institute of Technology Delhi, New Delhi 110016, India.
authors.forename: Rachana
authors.initials: R
authors.lastname: Tomar
authors.forename: Dushyant K
authors.initials: DK
authors.lastname: Garg
authors.forename: Rahul
authors.initials: R
authors.lastname: Mishra
authors.forename: Ashwani K
authors.initials: AK
authors.lastname: Thakur
authors.forename: Bishwajit
authors.initials: B
authors.lastname: Kundu
mesh_terms: D001215:Asparaginase / Q000737:chemistry* / Q000378:metabolism*; D004795:Enzyme Stability; D018832:Molecular Chaperones / Q000737:chemistry* / Q000378:metabolism*; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D020027:Pyrococcus furiosus / Q000201:enzymology*
chemical_list: D018832:Molecular Chaperones; D001215:Asparaginase