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Multi-Wavelength Analytical Ultracentrifugation of Biopolymer Mixtures and Interactions

Henrickson, Amy and Gorbet, Gary E. and Savelyev, Alexey and Kim, Minji and Schultz, Sarah K. and Ding, Xiaozhe and Hargreaves, Jason and Gradinaru, Viviana and Kothe, Ute and Demeler, Borries (2021) Multi-Wavelength Analytical Ultracentrifugation of Biopolymer Mixtures and Interactions. . (Unpublished)

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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.

Item Type:Report or Paper (Discussion Paper)
Related URLs:
URLURL TypeDescription Paper
Ding, Xiaozhe0000-0002-0267-0791
Gradinaru, Viviana0000-0001-5868-348X
Kothe, Ute0000-0002-2744-7334
Demeler, Borries0000-0002-2414-9518
Additional Information:The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. This version posted December 30, 2021. This work was supported by the Canada 150 Research Chairs program (C150-2017-00015), the Canada Foundation for Innovation (CFI-37589), the National Institutes of Health (1R01GM120600) and the Canadian Natural Science and Engineering Research Council (DG-RGPIN-2019-05637). UltraScan supercomputer calculations were supported through NSF/XSEDE grant TG-MCB070039N, and University of Texas grant TG457201. Computational resources and support from the University of Montana’s Griz Shared Computing Cluster (GSCC) contributed to this research (all grants awareded to BD). The Canadian Natural Science and Engineering Research Council supports AH through a scholarship grant, and UK through RGPIN-2020-04965. The AAV work was supported by National Institutes of Health grant UF1MH128336 (to VG). Plasmids encoding fluorescent proteins were kindly provided by Dr. Robert Cambpell, University of Alberta. Author contributions: AH performed and analyzed all AUC experiments, and edited the manuscript. GEG, AS, and MK contributed to the multi-wavelength analysis modules software development in UltraScan. JH prepared and contributed the oil seed protein samples, SKS and UK prepared and contributed the fluorescent proteins, and VG and XD prepared and contributed the AAV samples. The authors have declared no competing interest.
Funding AgencyGrant Number
Canada Research Chairs ProgramC150-2017-00015
Canada Foundation for InnovationCFI-37589
Natural Sciences and Engineering Research Council of Canada (NSERC)DG-RGPIN-2019-05637
University of TexasTG457201
Natural Sciences and Engineering Research Council of Canada (NSERC)RGPIN-2020-04965
Record Number:CaltechAUTHORS:20220104-249193900
Persistent URL:
Official Citation:Multi-Wavelength Analytical Ultracentrifugation of Biopolymer Mixtures and Interactions. Amy Henrickson, Gary E. Gorbet, Alexey Savelyev, Minji Kim, Sarah K. Schultz, Xiaozhe Ding, Jason Hargreaves, Viviana Gradinaru, Ute Kothe, Borries Demeler. bioRxiv 2021.12.29.474408; doi:
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:112660
Deposited By: Tony Diaz
Deposited On:04 Jan 2022 19:52
Last Modified:05 Jan 2022 00:01

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