LAUSR

Nuclear-export-protein (NEP) plays multiple-functions during influenza virus replication-cycle and shows unique pattern of conserved residues, which altogether make NEP a potential target for developing novel anti-influenza drugs. However, the mechanistic structural biology of NEP has not been fully characterized so far owing to its tendency to aggregate in solution. As structural information is important to guide rational drug-discovery process; therefore, procedural optimization efforts are going on to achieve properly folded NEP in sub-millimolar concentrations for solution-NMR investigations. As a first step in this direction, the refolding-cum-aggregation behavior of recombinant-NEP with N-terminal purification-tag (referred here as NEPN) at different urea-concentrations has been investigated here by NMR-based methods. Several attempts were made to refold denatured NEP-N through step-dialysis. However, owing to its strong tendency to aggregate, excessive precipitation was observed at sub-higher levels of urea concentration (5.0 ± 1.0 M). Finally, we used drip-dilution method with 10.5 M urea-denatured NEP-N and were able to refold NEP-N instantly. The amide 1H dispersion of 3.6 ppm (6.6-10.2 ppm) in the 15N-HSQC-spectra of instantly refolded NEP-N confirmed the folded state. This successful instant-refolding of NEP-N has been reported for the first-time and the underlying mechanism has been rationalized through establishing the complete backbone-resonance-assignments of NEP-N at 9.7 M urea-denatured state.