Sumit K. Chaturvedi | Searcharxiv

arXiv2017

Sedimentation of rapidly interacting multicomponent systems

The biophysical analysis of dynamically formed multi-protein complexes in solution presents a formidable technical challenge. Sedimentation velocity (SV) analytical ultracentrifugation achieves strongly size-dependent hydrodynamic resolution of different size species, and can be combined with multi-component detection by exploiting different spectral properties or temporally modulated signals from photoswitchable proteins. Coexisting complexes arising from self- or hetero-associations that can be distinguished in SV allow measurement of their stoichiometry, affinity, and cooperativity. However, assemblies that are short-lived on the time-scale of sedimentation (t1/2 < 100 sec) will exhibit an as of yet unexplored pattern of sedimentation boundaries governed by coupled co-migration of the entire system. Here, we present a theory for multi-component sedimentation of rapidly interacting systems, which reveals simple underlying physical principles, offers a quantitative framework for analysis, thereby extending the dynamic range of SV for studying multi-component interactions

q-bio.QM↗

arXiv2017

Sedimentation of Reversibly Interacting Macromolecules with Changes in Fluorescence Quantum Yield

Sedimentation velocity analytical ultracentrifugation with fluorescence detection has emerged as a powerful method for the study of interacting systems of macromolecules. It combines picomolar sensitivity with high hydrodynamic resolution, and can be carried out with photoswitchable fluorophores for multi-component discrimination, to determine the stoichiometry, affinity, and shape of macromolecular complexes with dissociation equilibrium constants from picomolar to micromolar. A popular approach for data interpretation is the determination of the binding affinity by isotherms of weight-average sedimentation coefficients, sw. A prevailing dogma in sedimentation analysis is that the weight-average sedimentation coefficient from the transport method corresponds to the signal- and population-weighted average of all species. We show that this does not always hold true for systems that exhibit significant signal changes with complex formation - properties that may be readily encountered in practice, e.g., from a change in fluorescence quantum yield. Coupled transport in the reaction boundary of rapidly reversible systems can make significant contributions to the observed migration in a way that cannot be accounted for in the standard population-based average. Effective particle theory provides a simple physical picture for the reaction-coupled migration process. On this basis we develop a more general binding model that converges to the well-known form of sw with constant signals, but can account simultaneously for hydrodynamic co-transport in the presence of changes in fluorescence quantum yield. We believe this will be useful when studying interacting systems exhibiting fluorescence quenching, enhancement or Forster resonance energy transfer with transport methods.

q-bio.BM↗