Protein-based nanomaterials are gaining importance in biomedical and biosensor applications where tunability of the protein particle size is highly desirable. Rationally designed proteins and peptides offer control over molecular interactions between monomeric protein units to modulate their self-assembly and thus particle formation. Here, using an example enzyme-peptide system produced as a single construct by bacterial expression, we explore how solution conditions affect the formation and size of protein nanoparticles. We found two independent routes to particle formation, one facilitated by charge interactions between protein-peptide and peptide-peptide exemplified by pH change or the presence of NO3- or NH4+ and the second route via metal-ion coordination ( e.g., Mg2+) within peptides. We further demonstrate that the two independent factors of pH and Mg2+ ions can be combined to regulate nanoparticle size. Charge interactions between protein-peptide monomers play a key role in either promoting or suppressing protein assembly; the intermolecular contact points within protein-peptide monomers involved in nanoparticle formation were identified by chemical cross-linking mass spectrometry. Importantly, the protein nanoparticles retain their catalytic activities, suggesting that their native structures are unaffected. Once formed, protein nanoparticles remain stable over long periods of storage or with changed solution conditions. Nevertheless, formation of nanoparticles is also reversible-they can be disassembled by desalting the buffer to remove complexing agents ( e.g., Mg2+). This study defines the factors controlling formation of protein nanoparticles driven by self-assembly peptides and an understanding of complex ion-peptide interactions involved within, offering a convenient approach to tailor protein nanoparticles without changing amino acid sequence.
Keywords: cross-linking mass spectrometry; metal-ion interaction; noncovalent assembly; pH effect; peptide self-assembly; protein−protein interaction.