Computational protocol: Stabilization by Fusion to the C-terminus of Hyperthermophile Sulfolobus tokodaii RNase HI: A Possibility of Protein Stabilization Tag

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[…] Circular dichroism (CD) spectra measurements, equilibrium and kinetic guanidine hydrochloride (GdnHCl)-induced, and heat-induced unfolding were as previously described . Buffers were 20 mM glycine-HCl pH 3.0 for Sto-RNase HI, 10 mM acetate pH 5.5 with 1 M GdnHCl for So-RNase HI, 10 mM glycine-HCl pH 3.0 for Ec-RNase HI, and 25 mM CHES-NaOH pH 9.0 with 1 M GdnHCl for Sto-esterase.Equilibrium experiments on GdnHCl-induced unfolding were examined by monitoring the CD at 220 nm. Protein solutions were incubated in GdnHCl at different concentrations and at different temperatures for unfolding. The GdnHCl-induced unfolding curves were determined, and a nonlinear least-squares analysis was used to fit the data to(1)(2)where y is the observed CD signal at a given concentration of GdnHCl, [D] is the concentration of GdnHCl, b0n is the CD signal for the native state, b0u is the CD signal for the unfolded states, an is the slope of the pre-transition of the baseline, and au is the slope of the posttransition of the baseline. ΔG(H2O) is the Gibbs energy change (ΔG) of the unfolding in the absence of GdnHCl, m is the slope of the linear correlation between ΔG and the GdnHCl concentration [D], and Cm is the GdnHCl concentration at the midpoint of the curve. Two or three replicates were measured for each condition. The raw experiment data were directly fitted to Eq. (1) using SigmaPlot (Jandel Scientific).Stability profiles (temperature dependence of ΔG(H2O)) were fitted to the Gibbs-Helmholtz equation, Eq. (3).(3)where ΔH(To) and ΔS(To) are the enthalpy and entropy of unfolding at the reference temperature To, and ΔCp is the difference in heat capacity between the native and unfolded states.Kinetic experiments on GdnHCl-induced unfolding were followed by CD spectra measurement at 220 nm. The unfolding reactions of proteins were induced by a concentration jump in GdnHCl, with various differing concentrations. The kinetic data were analyzed using Eq. (4).(4)Here, A(t) is the value of the CD signal at a given time t, A(∞) is the value when no further change is observed, k is the apparent rate constant, and A is the amplitude. Two or three replicates were measured for each condition. The GdnHCl concentration dependence of the logarithms of the apparent rate constant (kapp) for unfolding was also examined. The rate constants for unfolding in the absence of GdnHCl (ku(H2O)) were calculated by fitting to Eq. (5):(5)where [D] is the concentration of GdnHCl and mu represents the slopes of the linear correlations of ln ku with the GdnHCl concentration.Heat-induced unfolding was examined by monitoring the CD at 220 nm. All experiments were carried out at a scan rate of 1°C min−1. A nonlinear least-squares analysis was used to fit the data to(6)where y is the observed CD signal at a given temperature [T], bn is the CD signal for the native state, bu is the CD signal for the unfolded states, an is the slope of the pretransition of the baseline, au is the slope of the posttransition of the baseline, ΔHm is the enthalpy of unfolding at the transition midpoint temperature (Tm), T is the temperature, and R is the gas constant. Curve fitting was performed using SigmaPlot. Two or three replicates were measured for each condition. [...] Crystals of ΔC6 Sto-RNase HI were grown in 20% PEG 3000, 0.1 M citrate pH 5.5, including 6–7 mg mL−1 protein at 4°C. All full diffraction sets were collected at 100 K without cryoprotectants on a SPring-8 BL38B1. Diffraction data were indexed, integrated, and scaled using the HKL2000 program suite . The crystal structure was solved by the molecular replacement method using MOLREP in the CCP4 program suite , with the wild-type structure (2EHG) as a starting model. Structure refinement was with the programs Coot and REFMAC in the CCP4 program suite , . Progress in structure refinement was evaluated at each stage by the free R-factor and by inspecting stereochemical parameters calculated by the program PROCHECK . Collected and refined data are in . Figures were prepared using PyMol (http://www.pymol.org). […]

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