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. Author manuscript; available in PMC: 2010 May 15.
Published in final edited form as: J Mol Biol. 2009 Mar 28;388(4):851–864. doi: 10.1016/j.jmb.2009.03.059

Figure 6.

Figure 6

Ability of various metal ions to support NS3h_1b(con1) catalyzed ATP hydrolysis and NTP analog binding. Inorganic phosphate was measured after 15 min at 37 °C in reactions containing 2 mM ATP and either 100 nM (a) or 15 nM (b) NS3h in 25 mM MOPS pH 6.5 with the indicated amounts of metal ions. (a) ATP hydrolysis in the absence of RNA. (b) ATP hydrolysis in the presence of 0.1 mg/ml poly(U) RNA (Sigma, average length 2500 nt). (c) FRET analysis of mantADP(BeF3) binding to HCV helicase. A solution containing 0.5 μM NS3h 1b (con1), 5 mM NaF, 0.5 mM BeF2, 25 mM MOPS pH 6.25, 2 μM mantADP was titrated with various metal ions. Data are fit to a one site binding equation to determine Kd: (FRET=Bmax*[M]/(Kd+[M]) with shared a Bmax of 88 a.u. The following Kd’s were determined: MgCl2 = 153 μM, MnCl2 = 40 μM, and ZnCl2 = 90 μM.