We have further developed our global multi-method analysis approach, which combines independent observation of different observables from different techniques. We have further improved our data analysis program SEDPHAT for this purpose. It currently allows the seamless combination of sedimentation velocity, sedimentation equilibrium, dynamic light scattering, isothermal titration calorimetry, surface binding, and different spectroscopies. Key new developments are the statistical tools of global analysis. We have developed consistency matrices, which report to what degree experimental data sets are consistent and/or contribute new orthogonal information, and information content matrices, which report the improvement in binding parameters arising from the inclusion of each data set. Furthermore, we improved our strategies for weighting different data sets. We have applied these tools to a comprehensive model system with two-site binding, studied by surface plasmon resonance, isothermal titration calorimetry, sedimentation velocity, and fluorescence polarization. Since isothermal titration calorimetry is a uniquely useful tool for the study of macromolecular binding energetics, we have embarked on the improvement of data quality in this technique. In collaboration with Dr. Chad Brautigam, we have developed a noval approach for extracting heats of reaction at each titration step from the time course of the power signal that constitutes the raw recorded data, based on peak-shape analysis and singular value decomposition. This resulted in a new public domain software NITPIC, which was developed as a standalone tool for the integration of thermograms.

Project Start
Project End
Budget Start
Budget End
Support Year
6
Fiscal Year
2012
Total Cost
$303,195
Indirect Cost
Name
National Institute of Biomedical Imaging and Bioengineering
Department
Type
DUNS #
City
State
Country
Zip Code
Zhao, Huaying; Boyd, Lisa F; Schuck, Peter (2017) Measuring Protein Interactions by Optical Biosensors. Curr Protoc Protein Sci 88:20.2.1-20.2.25
Brautigam, Chad A; Zhao, Huaying; Vargas, Carolyn et al. (2016) Integration and global analysis of isothermal titration calorimetry data for studying macromolecular interactions. Nat Protoc 11:882-94
Chaires, Jonathan B; Hansen, Lee D; Keller, Sandro et al. (2015) Biocalorimetry. Methods 76:1-2
Zhao, Huaying; Piszczek, Grzegorz; Schuck, Peter (2015) SEDPHAT--a platform for global ITC analysis and global multi-method analysis of molecular interactions. Methods 76:137-148
Zhao, Huaying; Schuck, Peter (2015) Combining biophysical methods for the analysis of protein complex stoichiometry and affinity in SEDPHAT. Acta Crystallogr D Biol Crystallogr 71:3-14
Gustchina, Elena; Li, Mi; Ghirlando, Rodolfo et al. (2013) Complexes of neutralizing and non-neutralizing affinity matured Fabs with a mimetic of the internal trimeric coiled-coil of HIV-1 gp41. PLoS One 8:e78187
Keller, Sandro; Vargas, Carolyn; Zhao, Huaying et al. (2012) High-precision isothermal titration calorimetry with automated peak-shape analysis. Anal Chem 84:5066-73
Coussens, Nathan P; Schuck, Peter; Zhao, Huaying (2012) Strategies for assessing proton linkage to bimolecular interactions by global analysis of isothermal titration calorimetry data. J Chem Thermodyn 52:95-107
Zhao, Huaying; Schuck, Peter (2012) Global multi-method analysis of affinities and cooperativity in complex systems of macromolecular interactions. Anal Chem 84:9513-9
Gondeau, Claire; Corradin, Giampietro; Heitz, Frédéric et al. (2009) The C-terminal domain of Plasmodium falciparum merozoite surface protein 3 self-assembles into alpha-helical coiled coil tetramer. Mol Biochem Parasitol 165:153-61