Origin 7

Data are expressed as means ± SEM. The differences among treatment groups were analyzed by One-Way ANOVA, followed by Duncan test, using Origin 7 Software (MicroCal Software, Northampton, MA, USA). Values of P < 0.05 were considered to indicate statistical significance.

Protein samples in HEPES buffer (50 mM, pH 7.5), 200 mM NaCl and 0.5 mM TCEP were adjusted to 100 μM, and NADP+ added to 1 mM. ITC measurements were carried out at 25°C using a MicroCal Auto-iTC200 instrument (GE-Healthcare). Aliquots (2 μL) of potential ligands (1 mM) were injected into protein samples until binding appeared saturated (18 cycles, at 150 s intervals). Titrations of ligand buffer-only samples were performed to provide baseline readings. The corrected heat change was fitted in Origin 7 software (Microcal Inc., U.S.A) to obtain binding affinity constants (K_a) and enthalpy of binding (ΔH).

ITC was used to measure the GDP-binding affinities of wild-type EcNFeoB and mutant proteins. Protein (approximately 0.1 mM) in buffer (20 mM Tris pH 8.0 and 100 mM NaCl) was equilibrated for 1 min at 25 °C with stirring (1000 rpm) in the sample cell of a MicroCal iTC₂₀₀ Isothermal Titration Calorimeter. GDP (2.5–5 mM) was titrated into the sample cell in 0.5–2 μl injections over 0.8 s with 150 s spacing between injections. Power input (μcal s⁻¹) required to maintain equal temperatures between the sample and reference cells in response to each addition of ligand was plotted versus time (min). The data were integrated and plotted versus the molar ratio of ligand to protein. Non-linear regression was used to obtain the thermodynamic parameters (including GDP-binding affinity, K_a). Data were fitted to a one-site binding model using the Origin 7 Software (MicroCal) to obtain stoichiometry (N), enthalpy (ΔH), entropy (ΔS) and association rate constant (K_a). The dissociation constant (K_d) was calculated from Equation 1 (K_d=1/K_a). All reported values are the average of three or more independent titrations. Due to interdiffusion of the solutions during the insertion of the syringe into the sample chamber, the first injection is not useful for analysis and was omitted from all calculations.

Binding of the 8-mer RNA containing the 2′-O-methyl group at its 3′ end (ACCGACUU_m) to the SUMO-tagged Piwi PAZ domain and the SUMO protein (as a control) was measured at 20 °C, using a MicroCal iTC200 (GE Healthcare). The 8-mer RNA was purchased from GeneDesign, and dissolved in the gel filtration buffer (10 mm Tris-HCl (pH 8.0) and 500 mm NaCl). The 8-mer RNA (0.50 mm) was injected 18 times (0.4 µl for injection 1 and 2 µl for injections 2–18) into the protein solution (20 µm SUMO-PAZ or 20 µm SUMO in the gel filtration buffer), with 150 s intervals between injections. The concentrations of the protein and RNA samples were determined using the BCA protein assay kit (TaKaRa) and the absorbance at 260 nm, respectively. Data were analyzed using the Origin7 software (MicroCal). Data obtained from injections into the buffer were subtracted from the sample data before data analysis. Measurements were repeated at least twice, and similar results were obtained.