Structure of bovine β-lactoglobulin–lactoferrin coacervates

Abstract

Lactoferrin (LF) and β-lactoglobulin (BLG) are among the protein pairs that exhibit heteroprotein coacervation, a unique and relatively unexamined type of liquid-liquid phase separation (LLPS). In prior work we found that LF and BLG undergo coacervation at highly constrained conditions of pH, ionic strength and protein stoichiometry. The molar stoichiometry in coacervate and supernatant is LF : BLG2 1 : 2 (where BLG2 represents the 38 kDa BLG dimer), suggesting that this is the primary unit of the coacervate. The precise balance of repulsive and attractive forces among these units, thought to stabilize the coacervate, is achieved only at limited conditions of pH and I. Our purpose here is to define the process by which such structural units form, and to elucidate the forces among them that lead to the long-range order found in equilibrium coacervates. We use confocal laser scanning microscopy (CLSM), small angle neutron scattering (SANS), and rheology to (1) define the uniformity of interprotein spacing within the coacervate phase, (2) verify structural unit dimensions and spacing, and (3) rationalize bulk fluid properties in terms of inter-unit forces. Electrostatic modeling is used in concert with SANS to develop a molecular model for the primary unit of the coacervate that accounts for bulk viscoelastic properties. Modeling suggests that the charge anisotropies of the two proteins stabilize the dipole-like LF(BLG2)2 primary unit, while assembly of these dipoles into higher order equilibrium structures governs the macroscopic properties of the coacervate.

Linear viscoelasticity of complex coacervates

“…In numerous examples, the elastic behavior dominates (i.e., the storage modulus (G') dominates) [8,61,137,138,171,175,[177][178][179][180], while in other coacervates the viscous or liquid-like behavior (i.e., the loss modulus (G") dominates) [95,103,171]. Meanwhile, there are other systems where a crossover is observed between the two regimes, with G" dominating at low frequencies, and G' dominating at higher frequencies [61,71,72,79,111,130,162,171].…”

Complex coacervate‐based materials for biomedicine

“…Coacervation‐based encapsulation has been widely used in food science, medicine, sensing, and in the development of nanoreactors . Encapsulation can be achieved by utilizing the cargo material as part of the coacervate matrix, as a result of specific interactions, or by preferential partitioning . In all of these cases, two major advantages of coacervation are (1) the ability to perform encapsulation in a purely aqueous environment and (2) the potential for dramatically enriching the molecule of interest in the macromolecule‐rich coacervate phase when compared with the original solution.…”

Complex Equilibria, Speciation, and Heteroprotein Coacervation of Lactoferrin and β-Lactoglobulin

“…1−4 Many of these proteins exhibit significant electrostatic and geometric anisotropy, 5 features in common with monoclonal antibodies that undergo LLPS. 6−9 HP complexes are in equilibrium with both the coacervate and free proteins, 3,10 but the molecular interactions that determine these equilibria are not well understood.…”