Biomolecular Phase Research Articles

Capturing single-molecule properties does not ensure accurate prediction of biomolecular phase diagrams

Capturing single-molecule properties does not ensure accurate prediction of biomolecular phase diagrams

Biomolecular phase separation research: Milestones, insights, and future trajectories

Biomolecular phase separation research: Milestones, insights, and future trajectories

Probing Noncovalent Interactions by Fast Magic-Angle Spinning NMR at 100 kHz and More.

Noncovalent interactions are the basis for a large number of chemical and biological molecular-recognition processes, such as those occurring in supramolecular chemistry, catalysis, solid-state reactions in mechanochemistry, protein folding, protein-nucleic acid binding, and biomolecular phase separation processes. In this perspective article, some recent developments in probing noncovalent interactions by proton-detected solid-state Nuclear Magnetic Resonance (NMR) spectroscopy at Magic-Angle Spinning (MAS) frequencies of 100 kHz and more are reviewed. The development of MAS rotors with decreasing outer diameters, combined with the development of superconducting magnets operating at high static magnetic-field strengths up to 28.2 T (1200 MHz proton Larmor frequency) improves resolution and sensitivity in proton-detected solid-state NMR, which is the fundamental requirement for shedding light on noncovalent interactions in solids. The examples reported in this article range from protein-nucleic acid binding in large ATP-fueled motor proteins to a hydrogen-π interaction in a calixarene-lanthanide complex.

Targeting ROS in cancer: rationale and strategies.

Reactive oxygen species (ROS) in biological systems are transient but essential molecules that are generated and eliminated by a complex set of delicately balanced molecular machineries. Disruption of redox homeostasis has been associated with various human diseases, especially cancer, in which increased ROS levels are thought to have a major role in tumour development and progression. As such, modulation of cellular redox status by targeting ROS and their regulatory machineries is considered a promising therapeutic strategy for cancer treatment. Recently, there has been major progress in this field, including the discovery of novel redox signalling pathways that affect the metabolism of tumour cells as well as immune cells in the tumour microenvironment, and the intriguing ROS regulation of biomolecular phase separation. Progress has also been made in exploring redox regulation in cancer stem cells, the role of ROS in determining cell fate and new anticancer agents that target ROS. This Review discusses these research developments and their implications for cancer therapy and drug discovery, as well as emerging concepts, paradoxes and future perspectives.

Sequence dependence of critical properties for two-letter chains.

Histogram-reweighting grand canonical Monte Carlo simulations are used to obtain the critical properties of lattice chains composed of solvophilic and solvophobic monomers. The model is a modification of one proposed by Larson et al. [J. Chem. Phys. 83, 2411 (1985)], lowering the "contrast" between beads of different types to prevent aggregation into finite-size micelles that would mask true phase separation between bulk high- and low-density phases. Oligomeric chains of lengths between 5 and 24 beads are studied. Mixed-field finite-size scaling methods are used to obtain the critical properties with typical relative accuracies of better than 10-4 for the critical temperature and 10-3 for the critical volume fraction. Diblock chains are found to have lower critical temperatures and volume fractions relative to the corresponding homopolymers. The addition of solvophilic blocks of increasing length to a fixed-length solvophobic segment results in a decrease of both the critical temperature and the critical volume fraction, with an eventual slow asymptotic approach to the long-chain limiting behavior. Moving a single solvophobic or solvophilic bead along a chain leads to a minimum or maximum in the critical temperature, with no change in the critical volume fraction. Chains of identical length and composition have a significant spread in their critical properties, depending on their precise sequence. The present study has implications for understanding biomolecular phase separation and for developing design rules for synthetic polymers with specific phase separation properties. It also provides data potentially useful for the further development of theoretical models for polymer and surfactant phase behavior.

Combinatorial entropy determines the early stages of nucleation

AbstractBiomolecular phase separation, a complex phenomenon within living systems, has garnered significant interest due to its diverse roles in cellular organization and function. Despite its importance, studying phase separation dynamics experimentally, particularly in the early stages, poses challenges. Our study investigates the dynamics of biomolecular phase separation using a graph‐based simulation module, particularly emphasizing its early stages. Through a simplified model, we dissect the influences of various factors on collective behavior, highlighting the crucial role of combinatorial entropy in percolation dynamics. This study offers valuable insights into the fundamental principles governing biomolecular phase separation, with implications for understanding cellular processes and disease mechanisms.

Interplay of condensation and chromatin binding underlies BRD4 targeting.

Nuclear compartments form via biomolecular phase separation, mediated through multivalent properties of biomolecules concentrated within condensates. Certain compartments are associated with specific chromatin regions, including transcriptional initiation condensates, which are composed of transcription factors and transcriptional machinery, and form at acetylated regions including enhancer and promoter loci. While protein self-interactions, especially within low-complexity and intrinsically disordered regions, are known to mediate condensation, the role of substrate-binding interactions in regulating the formation and function of biomolecular condensates is underexplored. Here, utilizing live-cell experiments in parallel with coarse-grained simulations, we investigate how chromatin interaction of the transcriptional activator BRD4 modulates its condensate formation. We find that both kinetic and thermodynamic properties of BRD4 condensation are affected by chromatin binding: nucleation rate is sensitive to BRD4-chromatin interactions, providing an explanation for the selective formation of BRD4 condensates at acetylated chromatin regions, and thermodynamically, multivalent acetylated chromatin sites provide a platform for BRD4 clustering below the concentration required for off-chromatin condensation. This provides a molecular and physical explanation of the relationship between nuclear condensates and epigenetically modified chromatin that results in their mutual spatiotemporal regulation, suggesting that epigenetic modulation is an important mechanism by which the cell targets transcriptional condensates to specific chromatin loci.

Biomolecular phase separation through theoretical and computational microscope

AbstractBiomolecular phase separation is a vital mechanism for orchestrating biomolecules within living cells. This crucial role has spurred an intense pursuit to comprehend the molecular underpinnings governing and regulating these processes. Computational methodologies offer a unique perspective, augmenting experimental techniques by providing detailed information that cannot be obtained otherwise. In this review, we briefly overview the theoretical and computational approaches to investigate biomolecular phase separation. As a short primer, we explain the factors driving and affecting phase separation of biomolecules, and then we delve into analytical and simulation methods used to study phase separation. We explain how analytical methods like the Flory–Huggins theory, random phase approximation, and graph‐based methods have been used to study phase behaviors of various proteins. We also discuss principles and applications of all‐atom simulations, coarse‐grained simulations, and field‐theoretical approaches. Additionally, we explore the recent advances in machine learning approach to predict phase separation of biomolecules.

Thermodynamics of temperature modulation in biomolecular phase separation

Thermodynamics of temperature modulation in biomolecular phase separation

Fluorogenic Hyaluronan Nanogels Track Individual Early Protein Aggregates Originated under Oxidative Stress.

Proteins are broadly versatile biochemical materials, whose functionality is tightly related to their folding state. Native folding can be lost to yield misfolded conformations, often leading to formation of protein oligomers, aggregates, and biomolecular phase condensates. The fluorogenic hyaluronan HA-RB, a nonsulfonated glycosaminoglycan with a combination of polyanionic character and of hydrophobic spots due to rhodamine B dyes, binds to early aggregates of the model protein cytoplasmic glyceraldehyde-3-phosphate dehydrogenase 1 from Arabidopsis thaliana (AtGAPC1) since the very onset of the oligomeric phase, making them brightly fluorescent. This initial step of aggregation has, until now, remained elusive with other fluorescence- or scattering-based techniques. The information gathered from nanotracking (via light-sheet fluorescence microscopy) and from FCS in a confocal microscope converges to highlight the ability of HA-RB to bind protein aggregates from the very early steps of aggregation and with high affinity. Altogether, this fluorescence-based approach allows one to monitor and track individual early AtGAPC1 aggregates in the size range from 10 to 100 nm with high time (∼10-2 s) and space (∼250 nm) resolution.

Modulation of biomolecular phase behavior by metal ions.

Liquid-liquid phase separation (LLPS) appears to be a newly appreciated aspect of the cellular organization of biomolecules that leads to the formation of membraneless organelles (MLOs). MLOs generate distinct microenvironments where particular biomolecules are highly concentrated compared to those in the surrounding environment. Their thermodynamically driven formation is reversible, and their liquid nature allows them to fuse with each other. Dysfunctional biomolecular condensation is associated with human diseases. Pathological states of MLOs may originate from the mutation of proteins or may be induced by other factors. In most aberrant MLOs, transient interactions are replaced by stronger and more rigid interactions, preventing their dissolution, and causing their uncontrolled growth and dysfunction. For these reasons, there is great interest in identifying factors that modulate LLPS. In this review, we discuss an enigmatic and mostly unexplored aspect of this process, namely, the regulatory effects of metal ions on the phase behavior of biomolecules.

Phase separation in cGAS-STING signaling.

Biomolecular condensates formed by phase separation are widespread and play critical roles in many physiological and pathological processes. cGAS-STING signaling functions to detect aberrant DNA signals to initiate anti-infection defense and antitumor immunity. At the same time, cGAS-STING signaling must be carefully regulated to maintain immune homeostasis. Interestingly, exciting recent studies have reported that biomolecular phase separation exists and plays important roles in different steps of cGAS-STING signaling, including cGAS condensates, STING condensates, and IRF3 condensates. In addition, several intracellular and extracellular factors have been proposed to modulate the condensates in cGAS-STING signaling. These studies reveal novel activation and regulation mechanisms of cGAS-STING signaling and provide new opportunities for drug discovery. Here, we summarize recent advances in the phase separation of cGAS-STING signaling and the development of potential drugs targeting these innate immune condensates.

Extreme dynamics in a biomolecular condensate.

Proteins and nucleic acids can phase-separate in the cell to form concentrated biomolecular condensates1-4. The functions of condensatesspan many length scales: they modulate interactions and chemical reactions at the molecular scale5, organize biochemical processes at the mesoscale6 and compartmentalize cells4. Understanding the underlying mechanisms of these processeswill require detailed knowledge of the rich dynamics across these scales7. The mesoscopic dynamics of biomolecular condensates have been extensively characterized8, but their behaviour at the molecular scale has remained more elusive. Here, as an example of biomolecular phase separation, we study complexcoacervates of two highly and oppositely charged disordered human proteins9. Their dense phase is 1,000 times more concentrated than the dilute phase, and the resulting percolated interaction network10 leads to a bulk viscosity 300 times greater than that of water. However, single-molecule spectroscopy optimized for measurements within individual droplets reveals that at the molecular scale, the disordered proteins remain exceedingly dynamic, with their chain configurations interconverting on submicrosecond timescales. Massive all-atom moleculardynamics simulations reproduce the experimental observations and explain this apparent discrepancy: the underlying interactions between individual charged side chains are short-lived and exchange on a pico- to nanosecond timescale. Our results indicate that, despite the high macroscopic viscosity of phase-separated systems, local biomolecular rearrangements required for efficient reactions at the molecular scale can remain rapid.

Biomolecular phase separation in stress granule assembly and virus infection.

Liquid-liquid phase separation (LLPS) has emerged as a crucial mechanism for cellular compartmentalization. One prominent example of this is the stress granule. Found in various types of cells, stress granule is a biomolecular condensate formed through phase separation. It comprises numerous RNA and RNA-binding proteins. Over the past decades, substantial knowledge has been gained about the composition and dynamics of stress granules. SGs can regulate various signaling pathways and have been associated with numerous human diseases, such as neurodegenerative diseases, cancer, and infectious diseases. The threat of viral infections continues to loom over society. Both DNA and RNA viruses depend on host cells for replication. Intriguingly, many stages of the viral life cycle are closely tied to RNA metabolism in human cells. The field of biomolecular condensates has rapidly advanced in recent times. In this context, we aim to summarize research on stress granules and their link to viral infections. Notably, stress granules triggered by viral infections behave differently from the canonical stress granules triggered by sodium arsenite (SA) and heat shock. Studying stress granules in the context of viral infections could offer a valuable platform to link viral replication processes and host anti-viral responses. A deeper understanding of these biological processes could pave the way for innovative interventions and treatments for viral infectious diseases. They could potentially bridge the gap between basic biological processes and interactions between viruses and their hosts.

Sensitive and selective polymer condensation at membrane surface driven by positive co-operativity

Biomolecular phase separation has emerged as an essential mechanism for cellular organization. How cells respond to environmental stimuli in a robust and sensitive manner to build functional condensates at the proper time and location is only starting to be understood. Recently, lipid membranes have been recognized as an important regulatory center for biomolecular condensation. However, how the interplay between the phase behaviors of cellular membranes and surface biopolymers may contribute to the regulation of surface condensation remains to be elucidated. Using simulations and a mean-field theoretical model, we show that two key factors are the membrane's tendency to phase-separate and the surface polymer's ability to reorganize local membrane composition. Surface condensate forms with high sensitivity and selectivity in response to features of biopolymer when positive co-operativity is established between coupled growth of the condensate and local lipid domains. This effect relating the degree of membrane-surface polymer co-operativity and condensate property regulation is shown to be robust by different ways of tuning the co-operativity, such as varying membrane protein obstacle concentration, lipid composition, and the affinity between lipid and polymer. The general physical principle emerged from the current analysis may have implications in other biological processes and beyond.

Characterizing protein interactions in biomolecular phase separation.

Protein interactions are ubiquitous to life as they enable essential functions such as protein synthesis, signalling cascades or sequestration of foreign antigens. Pairwise interactions, in particular, have been studied extensively for centuries leading to the development of various techniques that can characterise binding events reliably. Recently, phase separation of proteins into membraneless compartments within the cellular environment has emerged as novel context of protein interactions. Although, great progress has been made in the discovery and study of phase separating systems, the protein interactions underlying these macromolecular assemblies remain difficult to characterize given their transient, multivalent nature. Here we show novel approaches for characterizing protein interactions within phase separating systems involving the usage of microfluidic tools such as microfluidic diffusional sizing and construction of tie lines from confocal detection. In doing so, we discover DNA-induced phase separation of antibodies as well as the formation of nanoscale clusters in antibody-DNA mixtures. Furthermore, we characterize the mechanistic impact of an RNA aptamer on multicomponent FUS condensate stability and determine the interactions of salt excipients with the protein dense phase. Crucially, establishing such novel pathways for characterizing protein interactions in phase separating systems will be key to accelerate the discovery of molecules that amend or interfere with phase separation, hence, show the potential to alter disease courses.

A universal code for phase separation of prion-like low complexity domains: Insights from quantitative computer simulations.

Biomolecular phase separation is a critical mechanism that contributes to intracellular spatiotemporal organization via the formation of biomolecular condensates. In addition to their widespread implications in aggregation-related disorders, prion-like low complexity domains (PLCDs) have been shown to exhibit demixing behavior and their aberrant condensation has been linked to disease. Here, we investigate the relationship between amino acid sequence and phase behaviour of the PLCDs exploring an unprecedented set of 140 different protein sequences. To this end, we perform direct coexistence molecular dynamics simulations of our residue-resolution coarse-grained protein model, which we have shown accurately predicts quantitative phase diagrams. For variants where experimental phase behaviors have been characterized, we show that our predictions are in excellent agreement with the experimental results. Analysis of the full simulation set of 140 variants reveals that the identity and patterning of sticker residues (e.g., Y, F) are crucial to define the phase behaviour of PLCDs. Notably, aromatic and charged residues have context-dependent effects on the critical temperature of PLCD condensation. Regarding spacer residues (e.g., S, G, N, T), PLCDs condensates are remarkably stable in front of mutations of glycine and serine but are more sensitive to mutations of asparagine and glutamine. Remarkably, these results extend to the entire family of PLCDs tested and, therefore, reveal universal codes for PLCDs phase behavior. Furthermore, the findings here help shed light on the functions of PLCDs and aid in the design of modifications to counter their aberrant phase separation.

Effect of a membrane protein condensation phase transition on reaction-diffusion kinetics.

Reversible biomolecular phase transitions, such as liquid-liquid phase transition in cytosolic proteins, and phosphorylation-driven condensation in membrane-bound systems have been cited as underlying mechanisms for numerous important cellular processes such as signal transduction. For example, condensation of the T cell signaling protein LAT, and, separately, Epidermal Growth Factor Receptor has been shown to elongate the membrane dwell time of the Ras-GEF SOS, enabling it to overcome a series of slow, membrane dependent activation steps. However, evidence from in-vitro reconstitution experiments suggests that formation of a condensed phase may not solely be beneficial to membrane-associated enzyme kinetics. SOS is both a scaffolding component, and a downstream effector of signaling condensates upstream of the Ras/Erk pathway. In such systems, its substrate Ras needs to have access to SOS for nucleotide exchange to take place. Sequestration of SOS proteins within a networked condensate may inhibit the ability of Ras to encounter SOS by diffusion. To investigate, we reconstitute protein condensation phase transitions consisting of the phosphotyrosine tails of either LAT or EGFR on supported lipid bilayers, along with the crosslinking components Grb2 and the PR domain of SOS to induce condensation. Under these conditions, single molecule TIRF microscopy can be used to localize and track membrane diffusion and partitioning between the condensed domains and surrounding sparse phase.

Phase-separating pyrenoid proteins form complexes in the dilute phase

While most studies of biomolecular phase separation have focused on the condensed phase, relatively little is known about the dilute phase. Theory suggests that stable complexes form in the dilute phase of two-component phase-separating systems, impacting phase separation; however, these complexes have not been interrogated experimentally. We show that such complexes indeed exist, using an in vitro reconstitution system of a phase-separated organelle, the algal pyrenoid, consisting of purified proteins Rubisco and EPYC1. Applying fluorescence correlation spectroscopy (FCS) to measure diffusion coefficients, we found that complexes form in the dilute phase with or without condensates present. The majority of these complexes contain exactly one Rubisco molecule. Additionally, we developed a simple analytical model which recapitulates experimental findings and provides molecular insights into the dilute phase organization. Thus, our results demonstrate the existence of protein complexes in the dilute phase, which could play important roles in the stability, dynamics, and regulation of condensates.

Chemical control of phase separation in DNA solutions.

We designed a series of DNA sequences comprising a trinucleotide repeat segment and a small molecule-binding aptamer. Optimization of the DNA sequences and reaction conditions enabled chemical control of phase separation of DNA condensates. Our results demonstrate a new strategy to regulate biomolecular phase transition.