Multi-Method Approaches for the Study of Complex Protein Interactions

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We have continued to expand the global multi-method analysis (GMMA) software SEDPHAT, which offers a variety of models and specific statistical functions for the analysis of multi-component interactions in a multi-method approach. This includes analytical ultracentrifugation (sedimentation velocity and sedimentation equilibrium), isothermal titration microcalorimetry, spectroscopic methods, and optical biosensing. Specifically, we have implemented visualization tools for all data types to show experimental data in a space of binding isotherms, or experimental observables, as a function of concentration. This can highlight the information content of data and help examining experimental design. Further, we have implemented extensive simulation tools that allow the investigator to explore whether the acquisition of data from a certain biophysical technique would be promising to aid in the characterization of the molecular parameter of interest. In order to disseminate this tool and facilitate its application by colleagues, we have held workshops at NIH in Bethesda, at the Dallas UT Southwestern Medical Center, and at the Medizinische Hochschule Hannover. This has generated positive feedback and software improvements. Experimentally, we have significantly extended the potential for GMMA with the development of fluorescence-detected sedimentation velocity (FDS-SV) method for the study of of high-affinity interactions. FDS-SV offers now a possibility to study of interactions with affinities in the low nM range, complementary to isothermal titration calorimetry and surface plasmon resonance biosensing.