Saponin Chemistry Controls Anionic Lipid Tolerance and Divalent Metal Ion Responses in Magnetically Alignable Bicelles

Saponin-phospholipid bicelles have emerged as promising membrane-mimetic systems for anisotropy-based NMR studies, but their utility depends on their ability to accommodate physiologically relevant lipid compositions and ionic environments. Here, we systematically investigated how saponin chemistry governs three key properties of magnetically alignable bicelles: tolerance to anionic lipid incorporation, responsiveness to lanthanide-induced alignment reorientation, and stability in the presence of divalent metal ions. Using 31P NMR spectroscopy as a sensitive probe of phase behavior and alignment, we compared bicelles formed with glycyrrhizic acid (GA), hederacoside C (HC), and crude Quillaja saponins (CQS). While all saponins effectively solubilized the anionic lipid DMPG, only HC supported magnetically aligned bicelles with high anionic lipid fractions (up to 70%), whereas GA was limited to low incorporation (10%). Both GA- and HC-based bicelles underwent lanthanide-induced alignment flipping, though with significant spectral broadening and intermediate alignment states indicative of increased heterogeneity. Divalent cation effects were strongly ion- and saponin-dependent; HC bicelles were robust to the presence of Ca2+ but were destabilized by Mg2+, while GA bicelles were disrupted by both ions at low concentrations. Together, these results demonstrate that saponin identity critically determines bicelle compatibility with charged lipids and ionic conditions, establishing design principles for tailoring saponin-based bicelles as versatile, biomimetic alignment media for membrane-protein structural studies.