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Integration and global analysis of isothermal titration calorimetry data for studying macromolecular interactions

Nature Protocols volume 11, pages 882–894 (2016)Cite this article

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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.

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Figure 1: Workflow of global ITC analysis using NITPIC, SEDPHAT/ITCsy and GUSSI.
Figure 2: The Experimental Parameter window in SEDPHAT.
Figure 3: Removing an outlying data point in SEDPHAT.
Figure 4: An experiment before and after fitting.
Figure 5: A GUSSI plot of a ten-titration global analysis of randomly methylated β-cyclodextrin (mβCD) binding to the nonionic detergent n-octyl-β-D-maltopyranoside (OM), acquired at different temperatures to determine ΔCp°.
Figure 6: Example of global ITC analysis of a three-protein interaction analyzed in SEDPHAT and plotted in GUSSI.
Figure 7: Global multimethod analysis (GMMA) in SEDPHAT of the two-site interaction of α-chymotrypsin (CT) binding to soybean trypsin inhibitor (SBTI), complementing ITC data with surface plasmon resonance (SPR) surface competition isotherm data, sedimentation velocity isotherms and fluorescence polarization data (not shown).

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Acknowledgements

This work was supported by the Deutsche Forschungsgemeinschaft (DFG) through International Research Training Group 1830, the Stiftung Rheinland–Pfalz für Innovation and the Intramural Research Program of the National Institute of Biomedical Imaging and Bioengineering, US National Institutes of Health.

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  1. Peter.Schuck@nih.gov

Authors and Affiliations

  1. Department of Biophysics, The University of Texas Southwestern Medical Center, Dallas, Texas, USA

    Chad A Brautigam

  2. 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

    Huaying Zhao & Peter Schuck

  3. Molecular Biophysics, University of Kaiserslautern, Kaiserslautern, Germany

    Carolyn Vargas & Sandro Keller

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Contributions

C.V. and S.K. collected data. C.A.B., H.Z., C.V., S.K. and P.S. performed data analysis. C.A.B., H.Z., C.V., S.K. and P.S. prepared the manuscript.

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Correspondence to Sandro Keller or Peter Schuck.

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Integrated supplementary information

Supplementary Figure 1 Screenshot of the SEDPHAT window after importing the data from NITPIC.

Shown are the isotherms from the different binding experiments, each in a separate graphics with subpanels for the raw data (top, circles and error bars) and the residuals (bottom). Initially, the residuals are meaningless due to the lack of a fitting model. In each experiment graph, a set of pushbuttons in the upper right corner provides access to individual experimental parameters (blue numbered button) and functions for data management (small buttons labeled i and x to inactivate or delete the titration). To get a quick overview, the identity of each titration along with local experimental parameters can be printed as text over the respective panels by pressing control-O.

Supplementary Figure 2 Screenshot of the SEDPHAT window after the fit has converged.

In each experiment panel, the best-fit titration model is now drawn as a solid line. It should be noted that the residuals are now on a reasonable scale, and for most experiments lack significant systematicity. The overlay of textual output reporting global and local fit parameters can be toggled on and off with the keys ESC and control-O. The best-fit parameters can be obtained also in the model parameter box (control-P), or be summarized in a displayed ASCII text-file (control-T).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1 and 2 (PDF 460 kb)

Supplementary Data

Example Data Sets from the duplicate titration experiments of CAII with TFMSA in five different buffers. (ZIP 168 kb)

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Brautigam, C., Zhao, H., Vargas, C. et al. Integration and global analysis of isothermal titration calorimetry data for studying macromolecular interactions. Nat Protoc 11, 882–894 (2016). https://doi.org/10.1038/nprot.2016.044

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