FRET Dilution Assay for Analyzing Dynamic Exchange Between Protein Assemblies

As the molecular driver for tunable iridescence in cephalopods, the tunable phase behavior of reflectin A1 protein continues to be a focus of biomaterial engineering. Modulating salt concentration and protein net charge density of reflectin A1 drives the protein to form dynamic assemblies as intermediates to liquid-liquid phase separation. Reflectin assemblies, while limited in size by the extent of charge neutralization of the protein's cationic, Coulombic repulsion, are initially in dynamic exchange with monomers or oligomers from the surrounding solution. A novel fluorescence resonance energy transfer (FRET) dilution assay was used, in conjunction with dynamic light scattering (DLS) and protein concentration assays, to characterize the two-way flux of protein between reflectin A1 assemblies and a dilute phase as a function of assembly age. This FRET dilution assay distinguishes between one-way and two-way flux of protein into and out of protein assemblies and, therefore, can be applied during assembly formation. Differentiating between dynamic and kinetically arrested protein assemblies is crucial to understanding their biophysical origins, and this novel FRET dilution assay can be adapted to supplement biophysical investigations of other protein assemblies.