Current practices in the study of biomolecular condensates: a community comment

The realization that the cell is abundantly compartmentalized into biomolecular condensates has opened new opportunities for understanding the physics and chemistry underlying many cellular processes1, fundamentally changing the study of biology2. The term biomolecular condensate refers to non-stoichiometric assemblies that are composed of multiple types of macromolecules in cells, occur through phase transitions, and can be investigated by using concepts from soft matter physics3. As such, they are intimately related to aqueous two-phase systems4 and water-in-water emulsions5. Condensates possess tunable emergent properties such as interfaces, interfacial tension, viscoelasticity, network structure, dielectric permittivity, and sometimes interphase pH gradients and electric potentials6–14. They can form spontaneously in response to specific cellular conditions or to active processes, and cells appear to have mechanisms to control their size and location15–17. Importantly, in contrast to membrane-enclosed organelles such as mitochondria or peroxisomes, condensates do not require the presence of a surrounding membrane

Tunable multiphase dynamics of arginine and lysine liquid condensates

AbstractLiquid phase separation into two or more coexisting phases has emerged as a new paradigm for understanding subcellular organization, prebiotic life, and the origins of disease. The design principles underlying biomolecular phase separation have the potential to drive the development of novel liquid-based organelles and therapeutics, however, an understanding of how individual molecules contribute to emergent material properties, and approaches to directly manipulate phase dynamics are lacking. Here, using microrheology, we demonstrate that droplets of poly-arginine coassembled with mono/polynucleotides have approximately 100 fold greater viscosity than comparable lysine droplets, both of which can be finer tuned by polymer length. We find that these amino acid-level differences can drive the formation of coexisting immiscible phases with tunable formation kinetics and can be further exploited to trigger the controlled release of droplet components. Together, this work provides a novel mechanism for leveraging sequence-level components in order to regulate droplet dynamics and multiphase coexistence.</jats:p

The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics.

P granules and other RNA/protein bodies are membrane-less organelles that may assemble by intracellular phase separation, similar to the condensation of water vapor into droplets. However, the molecular driving forces and the nature of the condensed phases remain poorly understood. Here, we show that the Caenorhabditis elegans protein LAF-1, a DDX3 RNA helicase found in P granules, phase separates into P granule-like droplets in vitro. We adapt a microrheology technique to precisely measure the viscoelasticity of micrometer-sized LAF-1 droplets, revealing purely viscous properties highly tunable by salt and RNA concentration. RNA decreases viscosity and increases molecular dynamics within the droplet. Single molecule FRET assays suggest that this RNA fluidization results from highly dynamic RNA-protein interactions that emerge close to the droplet phase boundary. We demonstrate than an N-terminal, arginine/glycine rich, intrinsically disordered protein (IDP) domain of LAF-1 is necessary and sufficient for both phase separation and RNA-protein interactions. In vivo, RNAi knockdown of LAF-1 results in the dissolution of P granules in the early embryo, with an apparent submicromolar phase boundary comparable to that measured in vitro. Together, these findings demonstrate that LAF-1 is important for promoting P granule assembly and provide insight into the mechanism by which IDP-driven molecular interactions give rise to liquid phase organelles with tunable properties

Sequence‐Tunable Phase Behavior and Intrinsic Fluorescence in Dynamically Interacting Peptides

A conceptual framework towards understanding biological condensed phases is emerging, derived from biological, biomimetic, and synthetic sequences. However, de novo peptide condensate design remains a challenge due to an incomplete understanding of the structural and interactive complexity. We designed peptide modules based on a simple repeat motif composed of tripeptide spacers (GSG, SGS, GLG) interspersed with adhesive amino acids (R/H and Y). We show, using sequence editing and a combination of computation and experiment, that n→π* interactions in GLG backbones are a dominant factor in providing sufficient backbone structure, which in turn regulates the water interface, collectively promoting liquid droplet formation. Moreover, these R(GLG)Y and H(GLG)Y condensates unexpectedly display sequence-dependent emission that is a consequence of their non-covalent network interactions, and readily observable by confocal microscopy

The Conformational Ensembles of α-Synuclein and Tau: Combining Single-Molecule FRET and Simulations

AbstractIntrinsically disordered proteins (IDPs) are increasingly recognized for their important roles in a range of biological contexts, both in normal physiological function and in a variety of devastating human diseases. However, their structural characterization by traditional biophysical methods, for the purposes of understanding their function and dysfunction, has proved challenging. Here, we investigate the model IDPs α-Synuclein (αS) and tau, that are involved in major neurodegenerative conditions including Parkinson’s and Alzheimer’s diseases, using excluded volume Monte Carlo simulations constrained by pairwise distance distributions from single-molecule fluorescence measurements. Using this, to our knowledge, novel approach we find that a relatively small number of intermolecular distance constraints are sufficient to accurately determine the dimensions and polymer conformational statistics of αS and tau in solution. Moreover, this method can detect local changes in αS and tau conformations that correlate with enhanced aggregation. Constrained Monte Carlo simulations produce ensembles that are in excellent agreement both with experimental measurements on αS and tau and with all-atom, explicit solvent molecular dynamics simulations of αS, with much lower configurational sampling requirements and computational expense

Sequestration within peptide coacervates improves the fluorescence intensity, kinetics, and limits of detection of dye-based DNA biosensors

Peptide-based liquid-liquid phase separated domains, or coacervates, are a biomaterial gaining new interest due to their exciting potential in fields ranging from biosensing to drug delivery. In this study, we demonstrate that coacervates provide a simple and biocompatible medium to improve nucleic acid biosensors through the sequestration of both the biosensor and target strands within the coacervate, thereby increasing their local concentration. Using the well-established polyarginine (R9) – ATP coacervate system and an energy transfer-based DNA molecular beacon we observed three key improvements: i) a greater than 20-fold reduction of the limit of detection within coacervates when compared to control buffer solutions; ii) an increase in the kinetics, equilibrium was reached more than 4-times faster in coacervates; and iii) enhancement in the dye fluorescent quantum yields within the coacervates, resulting in greater signal-to-noise. The observed benefits translate into coacervates greatly improving bioassay functionality