Multi-Wavelength Analytical Ultracentrifugation of Biopolymer Mixtures and Interactions

Abstract

Multi-wavelength analytical ultracentrifugation (MW-AUC) is a recent development made possible by new analytical ultracentrifuge optical systems. MW-AUC is suitable for a wide range of applications and biopolymer systems and is poised to become an essential tool to characterize macromolecular interactions. It adds an orthogonal spectral dimension to the traditional hydrodynamic characterization by exploiting unique chromophores in analyte mixtures that may or may not interact. Here we illustrate the utility of MW-AUC for representative classes of challenging biopolymer systems, including interactions between mixtures of different sized proteins with small molecules, mixtures of loaded and empty viral AAV capsids contaminated with free DNA, and mixtures of different proteins, where some have identical hydrodynamic properties, all of which are difficult to resolve with traditional AUC methods. We explain the improvement in resolution and information content obtained by this technique compared to traditional single- or dual-wavelength approaches. We discuss experimental design considerations and limitations of the method, and address the advantages and disadvantages of the two MW optical systems available today, and the differences in data analysis strategies between the two systems.