Low stability of nucleocapsid protein in SARS virus

The nucleocapsid protein (N protein) is one of the major virion structural proteins of a newly identified coronavirus, which has been confirmed as the causative agent of severe acute respiratory syndrome (SARS). The major function of N protein is to assemble the RNA of coronavirus. In the present study, the gene encoding the N protein was cloned and the protein was expressed, purified, and refolded as shown by H-1 NMR measurement. The maximal Trp emission wavelength occurs near 331 nm, suggesting substantial burial of Trp residues. Circular dichroism measurements indicate that N protein contains little alpha-helical structure. Acid titration shows that N protein begins to unfold near pH 5 and is fully denatured near pH 2.7, and the acid unfolding process is reversible. The physical and chemical properties of N protein indicate that its stability is low. N protein is denatured reversibly at pH 7.4 either by urea (with C-m of 2.77 M and m value of 2.74 kcal mol(-1) M-1) or GdmCl (with C-m of 1.46 M and m value of 4.50 kcal mol(-1) M-1). In the heat-induced denaturation in phosphate-buffered saline buffer, N-protein starts to unfold at 35 degreesC and is completely denatured at 55 degreesC, where SARS virus was also reported to be inactivated. We propose that the low stability of N protein may be critical for the stability and function of SARS virus.