An overview of solid-state NMR approaches that are readily applicable and potential methodological developments to investigate protein condensates and coacervates as well as to study protein phase separation and phase transitions are discussed.
Abstract
The formation of biomolecular condensates has been linked to a broad range of essential cellular processes, including immune signaling cascades, mRNA transport, and autophagy. Conversely, phase-separated condensates have also been associated with aberrant protein misfolding and subsequent aggregation in neurodegenerative disease-related processes. Protein phase separation and phase transitions involve the formation of heterogeneous and dynamic assemblies that can evolve into gel-like or semi-crystalline states, which are challenging to characterize using high-resolution structural biology techniques. Solid-state nuclear magnetic resonance (NMR) spectroscopy now offers a broad arsenal of methods to probe the structural and dynamic features of viscous condensates, elastic solids, coacervates and rigid protein assemblies. This review provides an overview of solid-state NMR approaches that are readily applicable and discusses potential methodological developments to investigate protein condensates and coacervates as well as to study protein phase separation and phase transitions.
Biomolecular condensates, formed by proteins, nucleic acids, and other macromolecules through liquid-liquid phase separation, enable molecular enrichment and reaction regulation within membraneless microenvironments. As their physical principles have become better understood, condensates are increasingly being incorporated into in vitro analytical systems. Compared with conventional homogeneous assays, condensate-based systems can enhance effective reaction concentrations, regulate molecular partitioning, accelerate reaction kinetics, and facilitate signal generation through enrichment, selective compartmentalization, and spatial confinement. This review discusses the mechanisms by which biomolecular condensates contribute to in vitro analysis, emphasizing component enrichment, selective partitioning, kinetic enhancement, and programmable phase behavior. We then discuss programmable condensate biosensing systems as emerging platform-level frameworks for molecular recognition, signal processing, and multimodal readout. Representative applications are summarized in nucleic acid and protein analysis, enzyme activity regulation, ion and small-molecule detection, and complex sample analysis. Finally, we highlight key challenges for practical implementation, including robust phase-behavior control in complex biological samples, predictive design, reproducibility, standardization, and biological benchmarking.
Biomolecular condensates represent unique microenvironments that organize intracellular biology and promote biochemical reactions. However, the biomolecular interactions driving condensate phase separation are often weak, transient, and heterogeneous. Investigating the structural biology and chemical properties of condensate interiors has therefore proven experimentally challenging, often requiring the use of perturbative probes. To overcome this challenge, we combine label-free optical scattering and vibrational spectroscopy approaches spanning ultraviolet, visible, mid-infrared, and terahertz wavelengths with deep-learning-based ensemble prediction of intrinsically disordered protein conformations. This suite of label-free approaches provides quantitative insights into protein-protein/protein-solvent interactions and the chemical properties of condensate interiors. Investigating the N-terminal domain of the RNA DEAD-box helicase 4 (DDX4), our experimental and computational results support a model of phase separation involving protein chain collapse, reduced dielectric, and water release. These molecular events are expected to enhance the strength of multivalent protein-protein interactions within condensates, creating a positive feedback loop important for condensate growth and phase separation.
Ethan A. Perets, Jacob A. Spies, Justin H. Cheong et al.· Biophysical Journal· 0 citations
Biomolecular condensates can undergo striking changes, such as transitioning from a liquid-like to a gel- or a solid-like aggregate due to changes in molecular interactions in response to changes in the biochemical environment. The question of how modified molecular interactions lead to such a transition in the material properties and spatial organization of condensates has not yet been elucidated. To address this question, we represent the biochemical environment as a triphasic mixture comprising a liquid-like protein-rich phase, a network-like protein-rich phase, and solvent. Owing to a change in the biochemical environment, protein molecules can reversibly switch between two conformational states. In a switched conformational state, the cross-linking domains of molecules are exposed which promote transient network formation in phase separated states. We develop a transient-network model and a continuum framework that couples phase separation, molecular switching, and dynamic cross-linking to predict condensate morphology and mechanics. The transient-network model predicts that a non-aging network behaves like a Maxwell fluid. When a network slowly ages via stabilization of cross-links, it shows Maxwell-like behavior and waiting time-dependent relaxation. However, a strongly aged network shows elastic recoil like characteristic of a Kelvin-Voigt solid. Our coupled continuum model demonstrates that the interplay of molecular switching and dynamic cross-linking in network formation shapes the spatial organization of condensate phases. In summary, this work demonstrates a mechanistic route explaining how conformational switching and molecular cross-linking regulate material properties and morphology of condensates.
The data support aqueous solvent interaction analysis as a broadly applicable, mechanistically grounded technology for protein characterization, drug–protein interaction studies, and structure-centric biomarker development, exemplified by the clinical translation of IsoPSA.
B. Zaslavsky, M. Stovsky, V. Uversky· International Journal of Mol...· 0 citations
Liquid-liquid phase separation (LLPS) under crowded conditions promotes the formation of protein-rich condensates that can undergo structural and physicochemical transformations over time. Understanding how these dynamic assemblies evolve toward more ordered states is essential for elucidating the mechanisms governing condensate maturation and for exploiting LLPS in the design of functional biomaterials. Here, we investigate the phase behaviour of β-lactoglobulin (BLG) under polyethylene glycol (PEG)-induced crowding conditions, following the evolution of protein-rich condensates from their formation and growth to the emergence of intermolecular β-sheet-rich solid assemblies. By combining environment-sensitive fluorescent probes with fluorescence lifetime, spectral-phasor, and fluorescence recovery after photobleaching (FRAP) microscopy, we monitored changes in the local molecular environment, protein mobility, and internal organization of individual condensates. These complementary approaches reveal the progressive emergence of structural heterogeneity and localized β-sheet-rich domains within individual condensates. Notably, these transformations occur within hours under mild, non-denaturing conditions and at relatively low protein concentrations, where BLG is generally regarded as a stable globular protein. Presented results provide a comprehensive picture of condensate maturation in this model system and demonstrate how multimodal fluorescence imaging enables the spatially resolved characterization of dynamic structural transitions in protein condensates, offering a valuable framework for investigating and engineering protein-based soft materials.
S. Anselmo, Federica Scollo, Giuseppe Sancataldo et al.· International Journal of Bio...· 0 citations
It is demonstrated that single-molecule mass measurements with mass photometry can capture RNA-protein interactions in phase-separated protein systems and can distinguish between charge neutralization, which drives coacervation, and complex formation, which mediates phase re-entry, making it a highly complementary tool for the study of RNA-mediated phase separation.
Axel Leppert, Jesper Shiapan, Irena Papageorgiou et al.· bioRxiv· 0 citations
We use cookies to run the site and, with your consent, for analytics and to show ads.
See our Cookie Policy.
