Spatial structure and composition of polysaccharide-protein complexes from Small Angle Neutron Scattering

We use Small Angle Neutron Scattering (SANS), with an original analysis method, to obtain both the characteristic sizes and the inner composition of lysozyme-pectin complexes depending on the charge density. Lysozyme is a globular protein and pectin a natural anionic semiflexible polysaccharide with a degree of methylation (DM) 0, 43 and 74. For our experimental conditions (buffer ionic strength I = 2.5 10-2 mol/L and pH between 3 and 7), the electrostatic charge of lysozyme is always positive (from 8 to 17 depending on pH). The pectin charge per elementary chain segment is negative and can be varied from almost zero to one through the change of DM and pH. The weight molar ratio of lysozyme on pectin monomers is kept constant. The ratio of negative charge content per volume to positive charge content per volume, -/+, is varied between 10 and 0.007. On a local scale, for all charged pectins, a correlation peak appears at 0.2 {\AA}-1 due to proteins clustering inside the complexes. On a large scale, the complexes appear as formed of spherical globules with a well defined radius of 10 to 50 nm, containing a few thousands proteins. The volume fraction Phi of organic matter within the globules derived from SANS absolute cross-sections is around 0.1. The protein stacking, which occurs inside the globules, is enhanced when pectin is more charged, due to pH or DM. The linear charge density of the pectin determines the size of the globules for pectin chains of comparable molecular weights whether it is controlled by the pH or the DM. The radius of the globules varies between 10 nm and 50 nm. In conclusion the structure is driven by electrostatic interactions and not by hydrophobic interactions. The molecular weight also has a large influence on the structure of the complexes since long chains tend to form larger globules. This maybe one reason why DM and pH are not completely equivalent in our system since DM 0 has a short mass, but this may not be the only one. For very low pectin charge (-/+ = 0.07), globules do not appear and the scattering signals a gel-like structure. We did not observe any beads-on-a-string structure.

💡 Research Summary

In this work the authors employed Small‑Angle Neutron Scattering (SANS) together with a novel data‑analysis protocol to obtain quantitative information on both the size and the internal composition of lysozyme‑pectin complexes over a wide range of charge conditions. Lysozyme, a globular protein that remains positively charged (8–17 elementary charges) under the experimental pH (3–7), was mixed with pectin, a natural anionic semiflexible polysaccharide whose degree of methylation (DM) was varied among 0 %, 43 % and 74 %. By adjusting DM and pH, the linear charge density of the pectin chain could be tuned from almost neutral to one negative charge per elementary segment, thereby spanning a volume‑charge ratio (‑/+) from 10 down to 0.007 while keeping the lysozyme‑to‑pectin monomer weight ratio constant.

The SANS profiles revealed two distinct structural regimes. For systems where the negative‑to‑positive charge ratio exceeded ≈0.07, the scattering displayed a pronounced correlation peak at q ≈ 0.2 Å⁻¹, indicating that lysozyme molecules cluster inside the complexes with a characteristic inter‑protein distance of roughly 3 nm. At lower q, the data were well described by spherical “globules” with radii ranging from 10 nm to 50 nm. Absolute intensity calibration allowed the authors to estimate the organic volume fraction (Φ) inside the globules at about 0.1, implying that the globules are relatively dilute, soft aggregates rather than dense particles. Each globule contains on the order of a few thousand lysozyme molecules, and the number of proteins per globule increases with the overall polymer concentration.

A systematic dependence on charge density emerged. When pectin carried a higher negative charge (low DM or low pH), the correlation peak became more intense, reflecting stronger protein stacking, while the globule radius decreased. This suggests that a higher linear charge density promotes more efficient electrostatic neutralisation, leading to smaller, more tightly packed aggregates. Conversely, when the charge imbalance was reduced (‑/+ < 0.07), the globular signature vanished and the scattering adopted a q⁻¹–q⁻² power‑law characteristic of a gel‑like network. In this regime the system no longer forms discrete particles; instead, pectin chains interpenetrate and create a percolated matrix in which lysozyme is homogeneously dispersed.

Molecular weight (or chain length) also proved decisive. For comparable charge densities, pectins with higher DM (and thus higher molecular weight) generated larger globules than low‑DM, short‑chain pectins. The authors interpret this as a consequence of longer chains providing more contour length to accommodate lysozyme, thereby allowing the formation of larger aggregates. Consequently, DM and pH are not strictly interchangeable: DM = 0 % pectin, despite having a high charge density, possesses a short chain length and therefore yields smaller globules than a high‑DM, long‑chain counterpart with a similar charge density.

Importantly, the authors conclude that electrostatic interactions dominate the self‑assembly process. No evidence was found for “beads‑on‑a‑string” morphologies or for significant contributions from hydrophobic forces or specific hydrogen‑bonding patterns. The observed structures can be rationalised by a simple picture: lysozyme binds to negatively charged pectin segments until local charge neutrality is reached, then the neutralised patches aggregate into spherical domains. The size of these domains is set by the balance between the linear charge density of the polysaccharide (which controls how many proteins can be accommodated per unit length) and the overall chain length (which determines how many such domains can be linked together).

These findings have practical implications for the design of protein‑polysaccharide materials in food science, pharmaceutical delivery, and biomaterials engineering. By tuning pH, degree of methylation, and polymer molecular weight, one can deliberately steer the system toward discrete nano‑ or microscale particles, dense protein‑rich gels, or intermediate soft colloids, thereby tailoring rheological properties, stability, and release characteristics for specific applications.