A New Temporal Dimension for Multisignal Sedimentation Velocity as a Tool to Analyze Multicomponent Interactions

Abstract

Multisignal sedimentation velocity (MSSV) analysis is a powerful method for determining the stoichiometry of reversibly formed multi-protein complexes. For example, it has been used to unravel the number and stoichiometry of co-existing complexes, for example, in receptor-ligand interactions and in systems of adaptor proteins. By globally analyzing absorbance data acquired simultaneously at different wavelengths, differences in the component spectra can be exploited to obtain information on the composition of all hydrodynamically resolved species. For fluorescence data that are acquired only at a single excitation wavelength, it was recently shown that the characteristic quantum yield of photoswitching of reversibly photoswitchable fluorescent proteins leads to a temporal fluorescence signal change on the time-scale of sedimentation, which can substitute for spectral discrimination of components in an analysis analogous to MSSV. In the present work, we have embarked on combining the two concepts by considering the temporal extinction coefficient changes associated with reversible photoswitching in the context of spectral decomposition of components. To this end, we report pilot experiments, for the first time acquiring and analyzing multi-wavelength absorbance data with temporal modulation of extinction amplitudes. Theoretically, the combination of spectral and temporal signal domain is expected to greatly enhance the discrimination of sedimenting components, thereby facilitating the study of multi-protein complexes with a higher number of components in solution.