Fig. 2: Interaction of Spy and native apoflavodoxin.
From: Mechanism of the small ATP-independent chaperone Spy is substrate specific
a Change in intrinsic tryptophan fluorescence of 0.09 µM AnFld WT with time in the absence and presence of various concentrations of Spy (0–54.8 μM after mixing). Time axis is shown in logarithmic scale. AU arbitrary units. The black lines shown for each Spy concentration are best fits of each dataset to a single exponential equation. b AnFld WT-Spy binding as monitored by intrinsic tryptophan fluorescence of AnFld. Spy dimer was titrated into 17.2 µM native AnFld WT at 25 °C. The binding isotherm is plotted with change in fluorescence (F) as normalized by fluorescence intensity relative to native AnFld fluorescence (F0) in the y-axis. Black line shows the fit to a one-site binding model. c, d SV-AUC experiments showing frictional ratios (f/f0) and sedimentation coefficients for c AnFld WT alone and d AnFld WT in the presence of 2.5-fold excess of Spy dimer in 40 mM HEPES-KOH (pH 7.5), 25 mM NaCl. Data were analyzed by two-dimensional sedimentation analysis (2DSA) followed by analysis with a genetic algorithm, which was further validated by a Monte Carlo analysis. e, f ITC analysis for the interaction of Spy and AnFld F98N at 10 °C. The low temperature minimizes enthalpy changes due to the binding-induced unfolding of AnFld. Five hundred and fifty micromolar Spy dimer in the syringe was titrated into 50 μM AnFld F98N in the cell. The thermogram in e was integrated and fit to a one-site binding model, as shown in f to obtain thermodynamic binding parameters. N stoichiometry of binding, K association constant, ΔH enthalpy change, ΔS entropy change. Values reported are the mean ± s.e.m. of the fit. Source data are provided as a Source Data file.
