fret-dilution-assay-for-analyzing-dynamic-exchange-between-protein

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 ...

University of California, Santa Barbara

Disordered proteins often undergo a stimuli-responsive, disorder-to-order transition which facilitates dynamic processes that modulate the physiological activities and material properties of cells, such as strength, chemical composition, and reflectance. It remains challenging to gain rapid and spatiotemporal control over such disorder-to-order transitions, which limits the incorporation of these proteins into novel materials. The reflectin protein is a cationic, disordered protein whose assembly is responsible for dynamic color camouflage in cephalopods. Stimuli-responsive control of reflectin's assembly would enable the design of biophotonic materials with tunable color. Herein, a novel, multivalent azobenzene photoswitch is shown to be an effective and non-invasive strategy for co-assembling with reflectin molecules and reversibly controlling assembly size. Photoisomerization between the  and  ( and ) photoisomers promotes or reduces Coulombic interactions, respectively, with reflectin proteins to repeatedly cycle the sizes of the photoswitch-reflectin assemblies between 70 nm and 40 nm. The protein assemblies formed with the  and  isomers show differences in interaction stoichiometry and secondary structure, which indicate that photoisomerization modulates the photoswitch-protein interactions to change assembly size. Our results highlight the utility of photoswitchable interactions to control reflectin assembly and provide a tunable synthetic platform that can be adapted to the structure, assembly, and function of other disordered proteins.

Reversible and size-controlled assembly of reflectin proteins using a charged azobenzene photoswitch.

The intrinsically disordered reflectin proteins drive tunable reflectivity for dynamic camouflage and communication in the recently evolved Loliginidae family of squid. Previous work revealed that reflectin A1 forms discrete assemblies whose size is precisely predicted by protein net charge density and charge screening by the local anion concentration. Using dynamic light scattering, FRET, and confocal microscopy, we show that these assemblies, of which 95 to 99% of bulk protein in solution is partitioned into, are dynamic intermediates to liquid protein-dense condensates formed by liquid-liquid phase separation (LLPS). Increasing salt concentration drives this progression by anionic screening of the cationic protein's Coulombic repulsion, and by increasing the contribution of the hydrophobic effect which tips the balance between short-range attraction and long-range repulsion to drive protein assembly and ultimately LLPS. Measuring fluorescence recovery after photobleaching and droplet fusion dynamics, we demonstrate that reflectin diffusivity in condensates is tuned by protein net charge density. These results illuminate the physical processes governing reflectin A1 assembly and LLPS and demonstrate the potential for reflectin A1 condensate-based tunable biomaterials. They also compliment previous observations of liquid phase separation in the Bragg lamellae of activated iridocytes and suggest that LLPS behavior may serve a critical role in governing the tunable and reversible dehydration of the membrane-bounded Bragg lamellae and vesicles containing reflectin in biophotonically active cells.

Article	Year
Reversible and size-controlled assembly of reflectin proteins using a charged azobenzene photoswitch. Chemical science\| PubMed ID: 39183923	2024
Charge screening and hydrophobicity drive progressive assembly and liquid-liquid phase separation of reflectin protein. The Journal of biological chemistry\| PubMed ID: 39922493	2025

Reid Gordon

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