Molecular interactions underlying the phase separation of HP1α: role of phosphorylation, ligand and nucleic acid binding.

Heterochromatin protein 1α (HP1α) is a crucial element of chromatin organization. It has been proposed that HP1α functions through liquid-liquid phase separation (LLPS), which allows it to compact chromatin into transcriptionally repressed heterochromatin regions. In vitro, HP1α can undergo phase separation upon phosphorylation of its N-terminus extension (NTE) and/or through interactions with DNA and chromatin. Here, we combine computational and experimental approaches to elucidate the molecular interactions that drive these processes. In phosphorylation-driven LLPS, HP1α can exchange intradimer hinge-NTE interactions with interdimer contacts, which also leads to a structural change from a compacted to an extended HP1α dimer conformation. This process can be enhanced by the presence of positively charged HP1α peptide ligands and disrupted by the addition of negatively charged or neutral peptides. In DNA-driven LLPS, both positively and negatively charged peptide ligands can perturb phase separation. Our findings demonstrate the importance of electrostatic interactions in HP1α LLPS where binding partners can modulate the overall charge of the droplets and screen or enhance hinge region interactions through specific and non-specific effects. Our study illuminates the complex molecular framework that can fine-tune the properties of HP1α and that can contribute to heterochromatin regulation and function.

Developing Bonded Potentials for a Coarse-Grained Model of Intrinsically Disordered Proteins.

Recent advances in residue-level coarse-grained (CG) computational models have enabled molecular-level insights into biological condensates of intrinsically disordered proteins (IDPs), shedding light on the sequence determinants of their phase separation. The existing CG models that treat protein chains as flexible molecules connected via harmonic bonds cannot populate common secondary-structure elements. Here, we present a CG dihedral angle potential between four neighboring beads centered at Cα atoms to faithfully capture the transient helical structures of IDPs. In order to parameterize and validate our new model, we propose Cα-based helix assignment rules based on dihedral angles that succeed in reproducing the atomistic helicity results of a polyalanine peptide and folded proteins. We then introduce sequence-dependent dihedral angle potential parameters (εd) and use experimentally available helical propensities of naturally occurring 20 amino acids to find their optimal values. The single-chain helical propensities from the CG simulations for commonly studied prion-like IDPs are in excellent agreement with the NMR-based α-helix fraction, demonstrating that the new HPS-SS model can accurately produce structural features of IDPs. Furthermore, this model can be easily implemented for large-scale assembly simulations due to its simplicity.

A predictive coarse-grained model for position-specific effects of post-translational modifications.

Biomolecules undergo liquid-liquid phase separation (LLPS), resulting in the formation of multicomponent protein-RNA membraneless organelles in cells. However, the physiological and pathological role of post-translational modifications (PTMs) on the biophysics of phase behavior is only beginning to be probed. To study the effect of PTMs on LLPS in silico, we extend our transferable coarse-grained model of intrinsically disordered proteins to include phosphorylated and acetylated amino acids. Using the parameters for modified amino acids available for fixed-charge atomistic force fields, we parameterize the size and atomistic hydropathy of the coarse-grained-modified amino acid beads and, hence, the interactions between the modified and natural amino acids. We then elucidate how the number and position of phosphorylated and acetylated residues alter the protein's single-chain compactness and its propensity to phase separate. We show that both the number and the position of phosphorylated threonines/serines or acetylated lysines can serve as a molecular on/off switch for phase separation in the well-studied disordered regions of Fused in Sarcoma (FUS) and DDX3X, respectively. We also compare modified residues to their commonly used PTM mimics for their impact on chain properties. Importantly, we show that the model can predict and capture experimentally measured differences in the phase behavior for position-specific modifications, showing that the position of modifications can dictate phase separation. In sum, this model will be useful for studying LLPS of post-translationally modified intrinsically disordered proteins and predicting how modifications control phase behavior with position-specific resolution.

Alteration of Microstructure in Biopolymeric Hydrogels via Compositional Modification of Resilin-Like Polypeptides.

Heterogeneities in hydrogel scaffolds are known to impact the performance of cells in cell-laden materials constructs, and we have employed the phase separation of resilin-like polypeptides (RLPs) as a means to generate such materials. Here, we study the compositional features of resilin-like polypeptides (RLPs) that further enable our control of their liquid-liquid phase separation (LLPS) and how such control impacts the formation of microstructured hydrogels. The evaluation of the phase separation of RLPs in solutions of ammonium sulfate offers insights into the sequence-dependent LLPS of the RLP solutions, and atomistic simulations, along with 2D-nuclear Overhauser effect spectroscopy (NOESY) and correlated spectroscopy (COSY) 1H NMR, suggest specific amino acid interactions that may mediate this phase behavior. The acrylamide functionalization of RLPs enables their photo-cross-linking into hydrogels and also enhances the phase separation of the polypeptides. A heating-cooling protocol promotes the formation of stable emulsions that yield different microstructured morphologies with tunable rheological properties. These findings offer approaches for choosing RLP compositions with phase behaviors that can be easily tuned with differences in temperature to control the resulting morphology and mechanical behavior of the heterogeneous hydrogels in regimes useful for biological applications.

Molecular interactions contributing to FUS SYGQ LC-RGG phase separation and co-partitioning with RNA polymerase II heptads.

The RNA-binding protein FUS (Fused in Sarcoma) mediates phase separation in biomolecular condensates and functions in transcription by clustering with RNA polymerase II. Specific contact residues and interaction modes formed by FUS and the C-terminal heptad repeats of RNA polymerase II (CTD) have been suggested but not probed directly. Here we show how RGG domains contribute to phase separation with the FUS N-terminal low-complexity domain (SYGQ LC) and RNA polymerase II CTD. Using NMR spectroscopy and molecular simulations, we demonstrate that many residue types, not solely arginine-tyrosine pairs, form condensed-phase contacts via several interaction modes including, but not only sp2-π and cation-π interactions. In phases also containing RNA polymerase II CTD, many residue types form contacts, including both cation-π and hydrogen-bonding interactions formed by the conserved human CTD lysines. Hence, our data suggest a surprisingly broad array of residue types and modes explain co-phase separation of FUS and RNA polymerase II.

N-terminal acetylation modestly enhances phase separation and reduces aggregation of the low-complexity domain of RNA-binding protein fused in sarcoma.

The RNA-binding protein fused in sarcoma (FUS) assembles via liquid-liquid phase separation (LLPS) into functional RNA granules and aggregates in amyotrophic lateral sclerosis associated neuronal inclusions. Several studies have demonstrated that posttranslational modification (PTM) can significantly alter FUS phase separation and aggregation, particularly charge-altering phosphorylation of the nearly uncharged N-terminal low complexity domain of FUS (FUS LC). However, the occurrence and impact of N-terminal acetylation on FUS phase separation remains unexplored, even though N-terminal acetylation is the most common PTM in mammals and changes the charge at the N-terminus. First, we find that FUS is predominantly acetylated in two human cell types and stress conditions. Next, we show that recombinant FUS LC can be acetylated when co-expressed with the NatA complex in Escherichia coli. Using NMR spectroscopy, we find that N-terminal acetylated FUS LC (FUS LC Nt-Ac) does not notably alter monomeric FUS LC structure or motions. Despite no difference in structure, Nt-Ac-FUS LC phase separates more avidly than unmodified FUS LC. More importantly, N-terminal acetylation of FUS LC reduces aggregation. Our findings highlight the importance of N-terminal acetylation of proteins that undergo physiological LLPS and pathological aggregation.

Biomolecular Condensates: Sequence Determinants of Phase Separation, Microstructural Organization, Enzymatic Activity, and Material Properties.

This perspective article highlights recent progress and emerging challenges in understanding the formation and function of membraneless organelles (MLOs). A long-term goal in the MLO field is to identify the sequence-encoded rules that dictate the formation of compositionally controlled biomolecular condensates, which cells utilize to perform a wide variety of functions. The molecular organization of the different components within a condensate can vary significantly, ranging from a homogeneous mixture to core-shell droplet structures. We provide many examples to highlight the richness of the observed behavior and potential research directions for improving our mechanistic understanding. The tunable environment within condensates can, in principle, alter enzymatic activity significantly. We examine recent examples where this was demonstrated, including applications in synthetic biology. An important question about MLOs is the role of liquid-like material properties in biological function. We discuss the need for improved quantitative characterization tools and the development of sequence-structure-dynamics relationships.

Molecular Details of Protein Condensates Probed by Microsecond Long Atomistic Simulations.

The formation of membraneless organelles in cells commonly occurs via liquid-liquid phase separation (LLPS) and is in many cases driven by multivalent interactions between intrinsically disordered proteins (IDPs). Investigating the nature of these interactions, and their effect on dynamics within the condensed phase, is therefore of critical importance but very challenging for either simulation or experiment. Here, we study these interactions and their dynamics by pairing a novel multiscale simulation strategy with microsecond all-atom MD simulations of a condensed, IDP-rich phase. We simulate two IDPs this way, the low complexity domain of FUS and the N-terminal disordered domain of LAF-1, and find good agreement with experimental information about average density, water content, and residue-residue contacts. We go significantly beyond what is known from experiments by showing that ion partitioning within the condensed phase is largely driven by the charge distribution of the proteins and-in the cases considered-shows little evidence of preferential interactions of the ions with the proteins. Furthermore, we can probe the microscopic diffusive dynamics within the condensed phase, showing that water and ions are in dynamic equilibrium between dense and dilute phases, and their diffusion is reduced in the dense phase. Despite their high concentration in the condensate, the protein molecules also remain mobile, explaining the observed liquid-like properties of this phase. We finally show that IDP self-association is driven by a combination of nonspecific hydrophobic interactions as well as hydrogen bonds, salt bridges, and π-π and cation-π interactions. The simulation approach presented here allows the structural and dynamical properties of biomolecular condensates to be studied in microscopic detail and is generally applicable to single- and multicomponent systems of proteins and nucleic acids involved in LLPS.

Spectroscopy of Oxygen-Sensitive Material for Measuring Contact Lens Oxygen Transmissibility.

PURPOSE: Oxygen permeability or transmissibility is a crucial parameter for contact lenses to ensure that extended wear will not induce corneal hypoxia. This work tests a new method of using the oxidation of cysteamine, an oxygen-sensitive chemical, to quantify the oxygen transmissibility of current commercial contact lenses and contact lenses loaded with vitamin E. METHODS: 3D printing was used to modify eye drop bottles and quartz cuvettes to create systems that allowed insertion of a contact lens in between the cysteamine solution and air. Both systems were exposed to atmospheric conditions where the only path of entry for oxygen was through the contact lens. The entering oxygen reacted with cysteamine, and the rate of cysteamine oxidation was measured using UV-vis spectrophotometry. The rate was then stoichiometrically related to oxygen transmissibility. RESULTS: The eye drop method predicted transmissibility values within 9% of established, commercial values. The cuvette method predicted values within 10% of established values for silicone hydrogel lenses without any correction factor and within 11% for poly-hydroxyethyl-methacrylate lenses after correcting for oxygen entering the system. Incorporation of 20% (w/w) vitamin E into Acuvue® Oasys® lenses did not have a significant impact on the oxygen transmissibility. CONCLUSIONS: Both methods presented in this work can reliably measure oxygen transmissibility of contacts lenses or other materials. Further improvements in manufacturing could lead to improved accuracy and reliability, allowing wider use of this method for quantifying the oxygen transport in contact lenses.