title: Integration and global analysis of isothermal titration calorimetry data for studying macromolecular interactions.
abstract: Isothermal titration calorimetry (ITC) is a powerful and widely used method to measure the energetics of macromolecular interactions by recording a thermogram of differential heating power during a titration. However, traditional ITC analysis is limited by stochastic thermogram noise and by the limited information content of a single titration experiment. Here we present a protocol for bias-free thermogram integration based on automated shape analysis of the injection peaks, followed by combination of isotherms from different calorimetric titration experiments into a global analysis, statistical analysis of binding parameters and graphical presentation of the results. This is performed using the integrated public-domain software packages NITPIC, SEDPHAT and GUSSI. The recently developed low-noise thermogram integration approach and global analysis allow for more precise parameter estimates and more reliable quantification of multisite and multicomponent cooperative and competitive interactions. Titration experiments typically take 1-2.5 h each, and global analysis usually takes 10-20 min.
authors.affiliation: Department of Biophysics, The University of Texas Southwestern Medical Center, Dallas, Texas, USA.
authors.forename: Chad A
authors.initials: CA
authors.lastname: Brautigam
authors.affiliation: Dynamics of Macromolecular Assembly Section, Laboratory of Cellular Imaging and Macromolecular Biophysics, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Huaying
authors.initials: H
authors.lastname: Zhao
authors.affiliation: Molecular Biophysics, University of Kaiserslautern, Kaiserslautern, Germany.
authors.forename: Carolyn
authors.initials: C
authors.lastname: Vargas
authors.affiliation: Molecular Biophysics, University of Kaiserslautern, Kaiserslautern, Germany.
authors.forename: Sandro
authors.initials: S
authors.lastname: Keller
authors.affiliation: Dynamics of Macromolecular Assembly Section, Laboratory of Cellular Imaging and Macromolecular Biophysics, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Peter
authors.identifier: http://orcid.org/0000-0002-8859-6966
authors.initials: P
authors.lastname: Schuck
mesh_terms: D002151:Calorimetry / Q000379:methods* / Q000706:statistics & numerical data; D003627:Data Interpretation, Statistical; D046911:Macromolecular Substances / Q000737:chemistry; D012815:Signal Processing, Computer-Assisted; D012984:Software; D013269:Stochastic Processes
chemical_list: D046911:Macromolecular Substances