Abstract
A recent investigation was aimed at obtaining structural information on a highly extended protein via SEC-MALS-SAXS. Significantly broadened elution peaks were observed, reminiscent of a phenomenon known as viscous fingering. This phenomenon is usually observed above 50 mg/mL for proteins like bovine serum albumin (BSA). Interestingly, the highly extended protein (Brpt5.5) showed viscous fingering at concentrations lower than 5 mg/mL. The current study explores this and other non-ideal behavior, emphasizing the presence of these effects at relatively low concentrations for extended proteins. BSA, Brpt5.5, and a truncated form of Brpt5.5 referred to as Brpt1.5 are studied systematically using size-exclusion chromatography (SEC), sedimentation velocity analytical ultracentrifugation (AUC), and viscosity. The viscous fingering effect is quantified using two approaches and is found to correlate well with the intrinsic viscosity of the proteins—Brpt5.5 exhibits the most severe effect and is the most extended protein tested in the study. By AUC, the hydrodynamic non-ideality was measured for each protein via global analysis of a concentration series. Compared to BSA, both Brpt1.5 and Brpt5.5 showed significant non-ideality that could be easily visualized at concentrations at or below 5 mg/mL and 1 mg/mL, respectively. A variety of relationships were examined for their ability to differentiate the proteins by shape using information from AUC and/or viscosity. Furthermore, these relationships were also tested in the context of hydrodynamic modeling. The importance of considering non-ideality when investigating the structure of extended macromolecules is discussed.
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Work was performed using National Institutes of Health funding from the National Institute of General Medical Sciences (R01-GM094363) awarded to A.B.H.
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AEY conceived the project, designed experiments, performed experiments, analyzed data, and wrote the manuscript. VD designed experiments, performed experiments, and analyzed data. SEH guided analysis and reviewed the manuscript. ABH designed experiments and provided funding. All authors reviewed the manuscript and contributed significantly to the final product.
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A.B.H. serves as a Scientific Advisory Board member for Hoth Therapeutics, Inc., holds equity in Hoth Therapeutics and Chelexa BioSciences, LLC, and was a co-inventor on seven patents broadly related to proteins described in this study.
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Yarawsky, A.E., Dinu, V., Harding, S.E. et al. Strong non-ideality effects at low protein concentrations: considerations for elongated proteins. Eur Biophys J 52, 427–438 (2023). https://doi.org/10.1007/s00249-023-01648-x
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DOI: https://doi.org/10.1007/s00249-023-01648-x