Multi-wavelength analytical ultracentrifugation of biopolymer mixtures and interactions

• Published in: Analytical biochemistry 2022-09, Vol.652, p.114728-114728, Article 114728
• Main Authors: Henrickson, Amy, Gorbet, Gary E., Savelyev, Alexey, Kim, Minji, Hargreaves, Jason, Schultz, Sarah K., Kothe, Ute, Demeler, Borries
• Format: Article
• Language: English
• Subjects: Biopolymers; Composition analysis; Hydrodynamics; Macromolecular hetero-interactions; Multi-wavelength analytical ultracentrifugation; Spectral decomposition; Ultracentrifugation - methods; UltraScan AUC software
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2022.114728

Multi-wavelength analytical ultracentrifugation (MW-AUC) is a recent development made possible by new analytical ultracentrifuge optical systems. MW-AUC extends the basic hydrodynamic information content of AUC and provides access to a wide range of new applications for biopolymer characterization, and is poised to become an essential analytical tool to study 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 experimental investigations where the benefit of the added spectral dimension provides critical information that is not accessible, and impossible to resolve with traditional AUC methods. We demonstrate the improvements in resolution and information content obtained by this technique compared to traditional single- or dual-wavelength approaches, and discuss experimental design considerations and limitations of the method. We further address the advantages and disadvantages of the two MW optical systems available today, and the differences in data analysis strategies between the two systems. [Display omitted] •MW-AUC adds a second, spectral characterization dimension to the traditional hydrodynamic dimension.•MW-AUC significantly improves resolution of samples with similar hydrodynamic properties.•MW-AUC provides new tools for the study of hetero-mixtures of molecules with unique chromophores.•MW-AUC provides orthogonal information to hydrodynamic analysis by elucidating stoichiometry and composition of complex mixtures.•MW-AUC characterizes of unknown mixtures by providing spectral signatures for individual components.