The Inertness of the Lysozyme Template: Assessing the Structural Impact of Silicification Byproducts and Precursors

Bioinspired silica deposition with biomolecular templates offers a sustainable route to producing advanced inorganic materials. The templating agent is usually a polycation. Recent experimental evidence links the templating capability of polycationic polymers to self‐assembly, coacervation, or liquid–liquid phase separation processes that precede silica deposition. However, when a polycationic protein is used, there is no evidence of liquid–liquid phase separation and the behavior of folded proteins during the hydrolysis of silicon alkoxides (TMOS/TEOS) remains poorly understood. In this study, we investigate the stability and dispersity of lysozyme in the presence of the alkoxide hydrolysis byproducts (methanol and ethanol) and a nonreactive tetrahedral mimic of orthosilicic acid (pentaerythritol). Using Small‐Angle X‐ray Scattering (SAXS) and Nuclear Magnetic Resonance (NMR) spectroscopy, we demonstrate that lysozyme retains its fold and monomeric state in the presence of these species, with no evidence of aggregation or unfolding. Furthermore, we show that unlike disordered peptides, lysozyme does not require anions for structural organization, even if some self‐interaction in the presence of phosphate or other anions is found to occur. These findings support the previously proposed mechanistic model in which the folded protein provides a robust, preorganized electrostatic scaffold for silica deposition, independent of solvent‐induced phase transitions.