High-Precision Isothermal Titration Calorimetry with Automated Peak-Shape Analysis

Abstract

Isothermal titration calorimetry (ITC) is a powerful classical method that enables researchers in many fields to study the thermodynamics of molecular interactions. Primary ITC data comprise the temporal evolution of differential power reporting the heat of reaction during a series of injections of aliquots of a reactant into a sample cell. By integration of each injection peak, an isotherm can be constructed of total changes in enthalpy as a function of changes in solution composition, which is rich in thermodynamic information on the reaction. However, the signals from the injection peaks are superimposed by the stochastically varying time-course of the instrumental baseline power, limiting the precision of ITC isotherms. Here, we describe a method for automated peak assignment based on peak-shape analysis via singular value decomposition in combination with detailed least-squares modeling of local pre- and post-injection baselines. This approach can effectively filter out contributions of short-term noise and adventitious events in the power trace. This method also provides, for the first time, statistical error estimates for the individual isotherm data points. In turn, this results in improved detection limits for high-affinity or low-enthalpy binding reactions and significantly higher precision of the derived thermodynamic parameters.

Biophysical and Structural Characterization of the Centriolar Protein Cep104 Interaction Network

“…Titrations were performed at 0.2-min intervals at a stirring speed of 1000 s Ϫ1 . The resulting heats were integrated using NITPIC software (32) and isotherms were fitted using a 1:1 bimolecular interaction model in program SEDPHAT (31). All final figures were prepared using the GUSSI software package.…”

Cardiolipin Prevents Membrane Translocation and Permeabilization by Daptomycin

“…Data were collected by the MicroCal customized Origin software. Baselines were normalized using Nitpic (14), and the results were analyzed with the Origin "one binding site" model.…”

Multipoint Binding of the SLP-76 SH2 Domain to ADAP Is Critical for Oligomerization of SLP-76 Signaling Complexes in Stimulated T Cells

“…Titrations were carried out using a MicroCal VP-ITC or ITC 200 titration microcalorimeter (Northampton, MA). Raw thermograms were integrated with automated shape analysis using NITPIC (31) and then imported into the software SEDPHAT (32) for individual analysis or global analysis of multiple titrations, using models for 1:1 and 2:1 association schemes and nonlinear least-squares fitting. In addition to parameters for binding constants, change in enthalpy (⌬H), and baseline offsets, where necessary, a parameter to account for local incompetent fractions was included and refined.…”