Du, Zhongyao; Xu, Ning; Yang, Ying; Li, Guimei; Tai, Zhonghong; Li, Na; Sun, Yang

INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES

2022, 224, 0141-8130

Du, Zhongyao; Xu, Ning; Yang, Ying; Li, Guimei; Tai, Zhonghong; Li, Na; Sun, Yang

In the current study, the internal structure of casein micelles (CMs), primary casein cluster, has been studied by size-exclusion chromatography (SEC) coupled with small-angle X-ray scattering (SAXS), isothermal titration calorimetry (ITC), transmission electron microscopy (TEM) and molecular dynamics (MD) simulations. The casein cluster featured a hierarchical structure predominately consisting of alpha(s)-CN and beta-CN molecules and a trace of x-CN. ITC profile showed a typical enthalpogram of CMs with a critical micelle concentration (CMC) of similar to 0.85 mu g/mL. The casein cluster exhibited apparent characteristics of intrinsically disordered proteins (IDPs) with a secondary structure content of 24 % in alpha-helix, 35.4 % in antiparallel-parallel, 20.2 % in 8 -turn and 20.4 % in random coil. SAXS results revealed a slightly elongated and tortuous worm-like conformation for the casein cluster in solution with an aspect ratio of 1.36 and an estimated molecular weight of 162.7 kDa. Further scat-tering data analysis proposed three coexisted species (alpha(s1)-beta-alpha(s2)-CN, alpha s1-CN and alpha(s1)-beta-alpha(s2)-CN dimer) with a volume fraction of 57.4 %, 30.1 %, and 12.5 % respectively in the casein cluster. TEM observation was consistent with the results of spectra, SAXS and MD that calcium sequestration resulted in a more extended and loose structure, instead, EDTA chelation induced a more compact conformation of the casein cluster.