Mesodynamics in the SARS nucleocapsid measured by NMR field cycling

Clarkson, Michael W.; Lei, Ming; Eisenmesser, Elan Z.; Labeikovsky, Wladimir; Redfield, Alfred; Kern, Dorothee

The SARSN construct was a gift from Dr. Hualiang Jiang (Luo et al. 2004 ). E. Coli BL21 (DE3) cells were grown in 15 N M9 medium to an OD of 0.6 and induced with 1 mM IPTG. Cells were harvested by centrifugation and resuspended in lysis buffer (0.1 M Tris, 0.5 M NaCl, pH 7.0) with a protease inhibitor cocktail (Calbiochem). Following lysis by sonication, cellular debris was removed by centrifugation. The lysate was then dialyzed against 50 mM Na 4 H 2 PO 4 , 50 mM NaCl pH 7.0 and purified over SP-sepharose equilibrated in the same buffer, eluting with 2 M NaCl. Following a repeat dialysis step, the protein was purified over Q-sepharose using the same buffer system. Following dialysis into the NMR buffer (50 mM HEPES, 150 mM NaCl, 0.02% NaN 3 pH 7.0), the protein was purified over S-100 sepharose and concentrated to 1 mM. NMR samples contained 10% D 2 O. All separation media were purchased from GE Healthcare.