How High Concentrations of Proteins Stabilize the Amorphous State of Calcium Orthophosphate: A Solid-State Nuclear Magnetic Resonance (NMR) Study of the Casein Case

Abstract

ABSTRACT:Understanding how proteins stabilize Amorphous Calcium ortho-Phosphate (ACP) phases is of great importance in biology and for pharmaceutical or food applications. Until now, most of the former investigations about ACP-protein stability and equilibrium were performed in conditions where ACP colloidal nanoclusters are surrounded by low to moderate concentrations of peptides or proteins (15-30 g.L -1 ). As a result, the question of ACP-protein interactions in highly concentrated protein systems has clearly been overlooked, whereas it corresponds to actual industrial conditions such as drying or membrane filtration in the dairy industry for instance. In this study, the structure of an ACP phase is monitored in association with one model phosphorylated protein (casein) using solid state NMR (ssNMR) at two conditions of high protein concentration (300 and 400 g.L -1 ). At both concentrations and at 25°C, it is found that the caseins maintain the mineral phase in an amorphous form with no detectable influence on its structure or size. Interestingly, and in both cases, a significant amount of the non-phosphorylated side chains interacts with ACP through H-bonds. The number of these interacting side-chains is found to be higher at the highest casein concentration. At 45°C, which is a destabilizing temperature of ACP in protein-free conditions, the amorphous structure of the mineral phase is partially transformed at 300 g.L -1 while it remains almost intact at 400 g.L -1 . These results thus clearly indicate that rising the concentration of proteins favor protein-ACP interactions and stabilize the ACP clusters more efficiently.

Unravelling Conformational Aspects of Milk Protein Structure—Contributions from Nuclear Magnetic Resonance Studies

“…Solid-state NMR was also used to detect the presence of hydrogen bonding from non-phosphorylated parts of the caseins to amorphous calcium phosphate nanoclusters [94]. The protons of the side chains of Arg, Lys, Glu or Asp can be close in space with P atoms, with a distance of 3.4-4.4 Å, and hence the terminal groups of the side chains including R-COOH and R-NH 2 have direct interactions with the inorganic phosphate of CCP [94].…”

“…Solid-state NMR was also used to detect the presence of hydrogen bonding from non-phosphorylated parts of the caseins to amorphous calcium phosphate nanoclusters [ 94 ]. The protons of the side chains of Arg, Lys, Glu or Asp can be close in space with P atoms, with a distance of 3.4–4.4 Å, and hence the terminal groups of the side chains including R-COOH and R-NH 2 have direct interactions with the inorganic phosphate of CCP [ 94 ]. Similarly, Cross et al [ 29 ], observing the β-casein peptide (1–25) interactions with amorphous calcium phosphate using NMR and molecular modeling, suggested that the entire length of the peptide is involved in interactions with calcium phosphate clusters.…”

Potential of Casein as a Carrier for Biologically Active Agents

“…Many biofluids, including milk, contain phosphate and calcium in concentrations exceeding the limits of solubility that are stabilised by proteins [21]. Caseins fulfill this role through their natural ability to self-assemble into micelles in presence of calcium phosphate [61].…”

“…Most of them form thermodynamically stable complexes with amorphous calcium phosphate, which accounts for 6 % of the micelle [2, 5]. Amorphous calcium phosphate forms spherical nanoclusters with diameter of 3.5–5.0 nm, spaced ~18 nm apart [21]. Phosphate is bound to the protein by phosphoseryl residues [22].…”

Metadynamics Simulations of the pH-Dependent Adsorption of Phosphoserine and Citrate on Disordered Apatite Surfaces: What Interactions Govern the Molecular Binding?

“…Such NMR studies also encompass the binding of small molecules, such as Pser, citrate, and amino acids, 15,16,28,29,31,40,41 as well as ACP interacting with Pser, 14 or Pser-bearing casein. 11,12 Given current limitations of a detailed experimental probing of the surface binding at atomic resolution, computer modeling by atomistic molecular dynamics (MD) simulations offers a rich source of structural information of the organic/inorganic interface, 27,29,41−54 enabling the extraction of the precise binding sites (atoms of a given functional group), as well as the detailed surface-immobilized molecular conformation hereafter referred to as the binding mode of the molecule. Such simulations typically target a three-component system that involves one or several biomolecules in an aqueous solution in contact with a slab of HA.…”