Kimbrough, H. | Search bioRxiv

bioRxiv2025

A tunable affinity fusion tag for protein self-assembly

The concentrations of individual proteins vary between cells, both developmentally and stochastically. The functional consequences of this variation remain largely unexplored due to limited experimental tools to manipulate the relationship of protein concentration to activity. Here, we introduce a genetically encoded tool based on a tunable amyloid that enables precise control of protein concentration thresholds in cells. By systematically screening dipeptide repeats, we identified poly-threonine alanine (poly-TA) as an ideal candidate due to its unique ability to form amyloid-like assemblies with a negligible nucleation barrier at arbitrarily chosen concentration thresholds. We demonstrate that the saturating concentration (Csat) of poly-TA can be finely tuned by adjusting the length of uninterrupted TA repeats, even while maintaining length and composition, providing a modular system for manipulating protein solubility. This tool offers a powerful approach to investigate the relationship between protein concentration, phase separation, and cellular function, with potential applications in cell-, developmental-, and synthetic biology.

cell biology↗

bioRxiv2025

A tool to dissect heterotypic determinants of homotypic protein phase behavior

Proteins commonly self-assemble to create liquid or solid condensates with diverse biological activities. The mechanisms of assembly are determined by each proteins sequence and cellular context. We previously developed distributed amphifluoric FRET (DAmFRET) to analyze sequence determinants of self-assembly in cells. Here, we extend DAmFRET by creating a nanobody (mEosNb) against the fluorescent protein mEos3 to physically tether and thereby recruit candidate modifier proteins to mEos3-fused query proteins. This accessorization allows us to rapidly screen for effects on the phase behavior of query proteins by modulating the expression level and valency of mEosNb-fused modifiers. We show that our system recapitulates known effects of multivalency on liquid-liquid phase separation and can discriminate between nucleation mechanisms of amyloid and amyloid-like assemblies. Our approach adds a new experimental dimension for interrogating the mechanisms of intracellular phase transitions. Lay summaryProtein self-assemblies are essential for cellular function, but can also contribute to disease. We develop a new tool to study how their formation is influenced by other cellular factors. This tool allows us to control the location and number of interactions between a protein of interest and other proteins that may influence it. Our results provide new insight into mechanisms of self-assembly and will aid research toward treating diseases associated with aberrant assembly.

biophysics↗