Multi-Method Approaches for the Study of Complex Protein Interactions

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Since a single biophysical technique is limited in the number of observable properties, one promising approach is the simultaneous consideration of data from multiple biophysical techniques. Therefore, one important goal is develop a robust and general computational framework for the global analysis of binding studies of triple (or higher) protein mixtures conducted with different techniques. In the past, we have developed for this purpose a data analysis program SEDPHAT for the global analysis of data from sedimentation velocity, sedimentation equilibrium, dynamic light scattering, isothermal titration calorimetry, and surface binding. It is now widely used in the biophysical community for data analysis of protein interactions. However, the combined analysis of data from different techniques is still very rare, and the full potential has not yet been explored. One of the difficulties is that a detailed understanding of the limitations and systematic errors for each of the applied techniques is essential. To this end, we have conducted experiments with interacting protein systems to serve as model for the study of the detailed compatibility of data from surface binding, calorimetry, circular dichroism spectroscopy, and sedimentation. We have continued our efforts to determine detailed size-and-shape distributions of macromolecular complexes by combined sedimentation and light scattering approaches.