N-terminal domain of Pyrococcus furiosus l-asparaginase functions as a non-specific, stable, molecular chaperone.

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.