Multi-Signal Sedimentation Velocity Analysis with Mass Conservation for Determining the Stoichiometry of Protein Complexes

摘要: Multi-signal sedimentation velocity analytical ultracentrifugation (MSSV) is a powerful tool for the determination of number, stoichiometry, and hydrodynamic shape reversible protein complexes in two- three-component systems. In this method, evolution profiles macromolecular mixtures recorded simultaneously using multiple absorbance refractive index signals globally transformed into both spectrally diffusion-deconvoluted component coefficient distributions. For reactions with complex lifetimes comparable to time-scale sedimentation, MSSV reveals number stoichiometry co-existing complexes. systems short lifetimes, composition reaction boundary coupled reaction/migration process, which we show here may be used directly determine an association constant. A prerequisite that interacting components are distinguishable, result, example, extrinsic chromophores or different abundances aromatic amino acids contributing UV absorbance. poorly resolved, introduce method additional regularization spectral deconvolution by exploiting approximate knowledge total loading concentrations. While novel mass conservation principle does not discriminate contributions species, it can effectively combined constraints range uncomplexed species. We theory, computer simulations, experiment, how as implemented SEDPHAT enhance even substitute discrimination components. This should broaden applicability analysis