No 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-b (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 a-crystallin or any known molecular chaperone, we present here NPfA as a novel molecular chaperone. Structured digital abstract• Ab amyloid 1-42 and Ab amyloid 1-42 bind by fluorescence technology (View interaction)• alpha-amylase and alpha-amylase bind by light scattering (View interaction)• Ab amyloid 1-42 and Ab amyloid 1-42 bind by transmission electron microscopy (View interaction)• NPfa binds to BCA II by anti tag coimmunoprecipitation (View interaction)• MSG and MSG bind by light scattering (View interaction)

We have shown the conversion of an amyloid fiber forming nucleation pathway of polyglutamine (polyGln) to a non-nucleated pathway, generating nanospherical amyloid particles. This is achieved by engineering an intermolecular salt bridge interaction between the positively charged lysine and the negatively charged glutamate residues, in two polyGln rich peptides. The mechanism of their formation is characterized by chromatography, infrared, fluorescence and imaging methods.