Kodirov, R. | bioRxiv Science

bioRxiv2025

Label-Free Fluorescence Microscopy Reveals Multiphase Organization in Biomolecular Condensates

Phase transitions of proteins and nucleic acids (NA) leading to the formation of biomolecular condensates have been linked to various biological functions. Given the growing number of proteins/NA predicted to undergo liquid-liquid phase separation (LLPS), efficient tools to investigate this behavior are critical to advancing our understanding of biomolecular condensate function. The current standard used to study LLPS involves techniques that utilize exogenous fluorophore labels. The labeling process is often costly and time-consuming and comes with associated complexity that arises from unknown interactions from the bulky fluorescent tags. These aspects limit high throughput analysis of protein/NA phase separation based on external fluorophore labeling. Here, we report the discovery that intrinsic fluorescence, well into the visible spectrum, arises as an emergent property of biomolecular condensates. Leveraging this intrinsic fluorescence, we study condensate formation, directly measure their internal dynamics via Fluorescence Recovery after Photobleaching (FRAP), and examine the 3D morphology and transitions to various multiphase architectures. Through this approach, we find that a variety of G-quadruplex DNA readily form droplets with histone H1 and display dynamic exchange. In addition, we directly demonstrate that the 3D morphology, core-shell architecture, and sub-compartmentalization of condensate droplets are tunable via the charge ratio of components in solution and NA hybridization. Our method utilizes an inherent property of condensates, thus is broadly applicable to any phase-separated systems and can advance our understanding of biological phase transition.

biophysics↗

bioRxiv2023

Protein unfolding thermodynamics predict multicomponent phase behavior

An increasing number of proteins are known to undergo liquid-liquid phase separation (LLPS), with or without nucleic acids or partner proteins, forming dense liquid-like phases termed biomolecular condensates. This physical phenomenon has been implicated in the existence of cellular membraneless organelles as well as in the formation of pathological protein aggregates in several human diseases. While common structural features of proteins with a propensity to undergo LLPS have been well documented, currently there is no thermodynamic framework capable of predicting the phase behavior of native proteins. Here, we show that two fundamental thermodynamic properties associated with the unfolding of a native protein, change in heat capacity ({Delta}Cunfold) and change in Gibbs free energy ({Delta}Gunfold), are sufficient to predict the formation of multicomponent biomolecular condensates. We find that proteins with small{Delta} Cunfold and{Delta} Gunfold values, which indicate a native state that is thermodynamically similar to the fully unfolded state, promote LLPS. In contrast, proteins with large{Delta} Cunfold and{Delta} Gunfold values promote aggregation. We also demonstrate that the stability of the liquid-like condensate can be predicted from the proximity of a proteins thermodynamic variable values to the phase boundary. This work elucidates a deep connection between single-protein thermodynamics and multicomponent phase behavior, and provides an avenue for predicting pathological droplet-aggregate transition. Significance StatementBiomolecular phase transitions, such as LLPS of proteins and nucleic acids, is emerging as an important concept in understanding the link between dysregulation of membraneless compartmentalization in cells and several human diseases. Currently, analysis of structural features such as intrinsic disorder and charged residue content is the "rule-of-thumb" to predict a proteins propensity to undergo LLPS. There are several instances where this qualitative approach fails, potentially due to not accounting for a proteins native structure. We demonstrate an empirical correlation between the unfolding thermodynamics of native proteins and their phase behavior, which enables a quantitative prediction of multicomponent LLPS. This novel approach, based on single-protein thermodynamics, has the potential to quantitatively predict pathological phase transitions of proteins in degenerative human diseases.

biophysics↗