Molecular insights into alginate β-lactoglobulin A multivalencies-The foundation for their amorphous aggregates and coacervation

For improved control of biomaterial property design, a better understanding of complex coacervation involving anionic polysaccharides and proteins is needed. Here, we address the initial steps in condensate formation of beta-lactoglobulin A (beta-LgA) with nine defined alginate oligosaccharides (AOSs) and describe their multivalent interactions in structural detail. Binding of AOSs containing four, five, or six uronic acid residues (UARs), either all mannuronate (M), all guluronate (G), or alternating M and G embodying the block structural components of alginates, was characterized by isothermal titration calorimetry, nuclear magnetic resonance spectroscopy (NMR), and molecular docking. beta-LgA was highly multivalent exhibiting binding stoichiometries decreasing from five to two AOSs with increasing degree of polymerization (DP) and similar affinities in the mid micromolar range. The different AOS binding sites on beta-LgA were identified by NMR chemical shift perturbation analyses and showed diverse compositions of charged, polar and hydrophobic residues. Distinct sites for the shorter AOSs merged to accommodate longer AOSs. The AOSs bound dynamically to beta-LgA, as concluded from saturation transfer difference and H-1-ligand-targeted NMR analyses. Molecular docking using Glide within the Schrodinger suite 2016-1 revealed the orientation of AOSs to only vary slightly at the preferred beta-LgA binding site resulting in similar XP glide scores. The multivalency coupled with highly dynamic AOS binding with lack of confined conformations in the beta-LgA complexes may help explain the first steps toward disordered beta-LgA alginate coacervate structures.

Automated summary

In order to improve the control of biomaterial property design, a better understanding of the complex coacervation between anionic polysaccharides and proteins is essential. This study investigated the initial steps of condensate formation between beta-lactoglobulin A (beta-LgA) and various alginate oligosaccharides (AOSs), providing structural details of their multivalent interactions. The results revealed that beta-LgA exhibited high multivalency and showed similar affinities for AOSs with increasing degree of polymerization. NMR analyses identified different binding sites for AOSs on beta-LgA, and molecular docking indicated that the AOSs have similar orientations at the preferred binding site. The study highlights the dynamic nature of the AOS binding and its role in the formation of disordered beta-LgA alginate coacervate structures.