Unveiling the structure of the primary caseinate particle using small-angle X-ray scattering and simulation methodologies.

The low-resolution structure of casein (CN) clusters in sodium caseinate (NaCas) solution and its conformational dynamics were obtained by size-exclusion chromatography (SEC), analytical ultracentrifugation (AUC), small-angle X-ray scattering (SAXS), and molecular dynamics (MD) simulations. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and native PAGE revealed that the casein clusters consisted predominantly of α- and β-CN complexes, and a trace amount of κ-CN. The AUC analysis indicated that the casein clusters were composed of 34.6% of casein monomers, 19.2%, 20.4%, and 25.8% of complexes with molar weight (M) of ~50.3, ~70.6, and ~133 kDa, respectively. The volume fractions of components in casein clusters were quantified as 64.3% of α-β-α-CN, 22.3% of α-CN, 8.5% of α-CN, and 4.4% of α-α-CN, respectively. The ensemble optimization method (EOM) gave a fitting result where α-β-α-CN species coexisted in ~35.3% under compact conformation and ~64.7% in elongated conformation in solution. The three-dimensional structures of α-β-α-CN from EOM showed a good overlay on the casein clusters ab initio model obtained from DAMMIN and DAMMIX program. MD simulations revealed that α-β-α-CN underwent a conformational change from the elongated state into the compact state within the initial 200 ns of simulations. The addition of nonionic surfactants affected little the backbone-to-backbone interactions in the formation of the casein clusters. We propose that α-CN, β-CN, α-CN, and κ-CN associated in consecutive steps into casein clusters, and a trace of κ-CN may be located at the surface of the assemblies limiting the growth of casein aggregates.