Multi-Method Approaches for the Study of Complex Protein Interactions

National Institute Of Biomedical Imaging And Bioengineering, Bethesda

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Abstract

The study of reversible multi-protein complexes is significantly enhanced by global multi-method analysis (GMMA), which combines independent data sets from biophysical methods with different observables into one hybrid analysis. To facilitate the practical application of this approach, we have previously developed the software SEDPHAT, which is now widely used for studying protein interactions by a variety of methods, including sedimentation velocity and sedimentation equilibrium analytical ultracentrifugation, isothermal titration microcalorimetry, circular dichroism and fluorescence spectroscopy, dynamic light scattering, and optical biosensing. While the software originally featured several pre-defined interaction models, including ternary and quaternary multi-site models, more recently we have also implemented a general model editor that permits inclusion of multi-site models with an arbitrary number of sites, indefinite self-association, and nonideality. In the review period we have further tested the application of this model editor with modules for different biophysical techniques, prior to the planned release of this software extension. We have also gained significant experience with the new biophysical technique of mass photometry (MP), which is based on interferometric scattering mass spectrometry. In this technique, individual adsorption events of macromolecules to a glass substrate are observed, recording the accompanying change in local reflectivity to determine the macromolecular mass. Accumulation of many adsorption events provides a size-distribution of particles in the sample, to the extent that surface adsorption is an unbiased representation of the species in solution. Using protein-nucleic acid complexes as model systems, we have compared the size-distribution measured by MP with that in sedimentation velocity analytical ultracentrifugation (SV) and dynamic light scattering (DLS), and found them to be highly complementary. While MP measures a high-resolution mass distribution in units of relative particle numbers, SV provides a signal-weighted hydrodynamic sedimentation coefficient distribution that is typically of lower resolution, but can reveal interactions and composition of particles. DLS offers only a low-resolution z-average Stokes radius distribution, but the data can be combined with sedimentation coefficients from SV to determine molar masses, as measured by MP. We believe there is great potential for quantitative combination of data from MP, SV, and DLS to obtain high-resolution particle size-and-shape distributions. In the review period we have addressed some existing limitations of MP. Using ribonucleoprotein complexes we were able to develop a protocol for using MP, for the first time, for the measurement of dissociation kinetics of multi-molecular complexes. This work is currently under review for publication. Furthermore, we have embarked on the application of surface coatings to reduce irreversible surface binding that may lead to time-dependent adsorption frequencies and selective bias. Following published methods, we have explored different protocols for vapor deposition of perfluoroalkane brushes onto MP substrates. Results so far have been promising but also indicative of uncontrolled variables. We envision this effort will ultimately allow us to minimize surface bias in MP for the study protein assemblies, and potentially pave the way for a more quantitative global analysis of MP with SV and DLS data. Finally, in the reporting period we have further pursued the combination of SV with NMR, in collaboration with the laboratory of Lewis Kay (University of Toronto). In different applications to bacterial virulence factors, we have previously shown the great potential of this hybrid approach to unravel large dynamic multi-molecular complexes. In the reporting period we have used this approach to study Caspase-11 interactions with gasdermin D, an inflammasome complex implicated in autoimmune diseases. Publications: The joint analysis of MP, SV, and DLS is described in a manuscript currently under review in eLife and published as a preprint in bioRxiv (10.1101/2025.04.26.650775).