Steric Modulation of Protein‐Mediated Nanoparticle Assembly: Controlling Cluster Size, Polydispersity, and FRET Responses by Rebalancing Short‐ and Long‐Range Interactions

Abstract Understanding and manipulating protein‐nanoparticle interactions is of broad interest to fields ranging from nanomedicine to the biological fabrication of functional hierarchical materials. This study investigates how steric forces introduced by a pegylated derivative of superfolder green fluorescent protein (sfGFP) that is monofunctional for silica binding modulate the delicate interplay of long‐range (electrostatic and van der Waals) and short‐range (protein‐mediated) interactions in pH‐responsive silica nanoparticle (SiNP) assembly by bifunctional silica‐binding sfGFP. Increasing the length of the PEG segment and pre‐incubating SiNPs with increasing concentrations of pegylated proteins enables precise control over cluster size within the 800–1450 nm range with a sixfold decrease in polydispersity index to a remarkable 0.1 endpoint. Weakening short‐range attractive interactions via mutagenesis extends this control to clusters in the 50–250 nm range and reveals that the Förster resonance energy transfer (FRET) efficiency of clusters scales linearly with cluster diameter below 230 nm but increases only by 15% as clusters grow to 1450 nm. These findings enable the development of a system that provides an optical readout to dynamic changes in solution conditions enacted by a combination of pH adjustment and ion charge screening.