Abstract
It has been known for decades that proteins undergo conformational changes in response to binding ligands. Such changes are usually accompanied by a loss of entropy by the protein, and thus conformational changes are integral to the thermodynamics of ligand association. Methods to detect these alterations are numerous; here, we focus on the sedimentation velocity (SV) mode of AUC, which has several advantages, including ease of use and rigorous data-selection criteria. In SV, it is assumed that conformational changes manifest primarily as differences in the sedimentation coefficient (the s-value). Two methods of determining s-value differences were assessed. The first method used the widely adopted c(s) distribution to gather statistics on the s-value differences to determine whether the observed changes were reliable. In the second method, a decades-old technique called “difference SV” was revived and updated to address its viability in this era of modern instrumentation. Both methods worked well to determine the extent of conformational changes to three model systems. Both simulations and experiments were used to explore the strengths and limitations of the methods. Finally, software incorporating these methodologies was produced.
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Acknowledgements
The authors wish to thank Dr. Peter Schuck for helpful discussions and the use of one of the analytical ultracentrifuges in his lab to collect some of the data presented herein. Parts of this research were supported by a Grant (AI056305) to M.V.N. from the National Institutes of Health.
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NIH Grant No. AI056305 to M.V.N.
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Brautigam, C.A., Tso, SC., Deka, R.K. et al. Using modern approaches to sedimentation velocity to detect conformational changes in proteins. Eur Biophys J 49, 729–743 (2020). https://doi.org/10.1007/s00249-020-01453-w
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DOI: https://doi.org/10.1007/s00249-020-01453-w