Analytical Ultracentrifugation as a Tool for Exploring COSAN Assemblies.

Abstract

The self-assembly of the cobalta bis(dicarbollide) (COSAN) anionic boron clusters into micelles above a critical micelle concentration (cmc) of 10 - 20 mM and its behavior as "sticky nano-ions" facilitating controlled protein aggregation have been previously investigated using scattering techniques, particularly small-angle neutron and X-ray scattering. These techniques effectively provide average structural parameters but, when applied to colloidal systems, often rely on models assuming polydispersity or anisotropic shapes. Thus, complementary techniques are required to confirm or infirm the proposed analyses. Here, we employed sedimentation velocity analytical ultracentrifugation (SV-AUC), which offers the ability to resolve discrete species. We revisited two key questions: (1) the aggregation behavior of COSAN into micelles, a topic still under debate and due to its unconventional amphiphilic nature, and (2) the nature of the protein assemblies induced by COSAN, specifically their size/shape distribution and aggregation number. Our findings confirm the cmc of COSAN of 16 mM and reveal that COSAN micelles exhibit low aggregation numbers (8 in water and 14 in dilute salt), consistent with recent hypotheses. Furthermore, SV-AUC showed that COSAN promotes myoglobin aggregation into discrete oligomeric species with well-defined aggregation numbers, such as dimers, tetramers, and higher-order assemblies, depending on the COSAN-to-protein ratio. These results provide clarity on the discrete nature of COSAN micelle aggregation and protein assembly. This study highlights the complementary role of SV-AUC in understanding supramolecular assemblies, offering useful insights into the behavior of COSAN nano-ions and their interactions with biomacromolecules.