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BIAcore control software 3.2 was used to analyze the data, and the best 1 1 Langmuir binding fit was used to derive kinetic constants.
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The titration data of JAZ ZF2 were initially fitted to a one-site model, and the Kd value obtained from this initial fit was used to approximate the dissociation constant of the primary binding site (Kd1); the program was then used to fit the data to a two-site binding model to calculate the dissociation constant of the secondary binding site (Kd2).
A ligand-depleted binding equation was used to fit the fluorescent quenching data (Equation 1).
A one-site binding model was used to fit the data and infer binding affinity in Graphpad Prism.
Best-fit analysis was used to calculate the Vd and with the latter model also the binding potential (BP).
A one site total binding fit using Prism 5.04 for Windows was used to fit the curves and determine the dissociation constants.
For the competitive binding assay, a one site-fit logIC50 curve was used.
DynaFit was used to fit some of the kinetic and binding data, e.g., substrate and product binding (Supporting Information).
Origin 5.0 (Microcal Software, Inc., Northampton, MA) was used to fit thermodynamic parameters (single binding site class) to the heat profiles.
Specifically, hydrophobic surface area and curvature of the binding pocket were used to fit the binding affinities of a training set of protein-ligand binding complexes.
Origin 7.0 was used for data fitting with a single-binding site model.
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