Biomolecular condensates form spatially inhomogeneous network fluids.

The functions of biomolecular condensates are thought to be influenced by their material properties, and these will be determined by the internal organization of molecules within condensates. However, structural characterizations of condensates are challenging, and rarely reported. Here, we deploy a combination of small angle neutron scattering, fluorescence recovery after photobleaching, and coarse-grained molecular dynamics simulations to provide structural descriptions of model condensates that are formed by macromolecules from nucleolar granular components (GCs). We show that these minimal facsimiles of GCs form condensates that are network fluids featuring spatial inhomogeneities across different length scales that reflect the contributions of distinct protein and peptide domains. The network-like inhomogeneous organization is characterized by a coexistence of liquid- and gas-like macromolecular densities that engenders bimodality of internal molecular dynamics. These insights suggest that condensates formed by multivalent proteins share features with network fluids formed by systems such as patchy or hairy colloids.

Intermolecular Interactions between Cysteine and Aromatic Amino Acids with a Phenyl Moiety in the DNA-Binding Domain of Heat Shock Factor 1 Regulate Thermal Stress-Induced Trimerization.

In this study, we investigated the trimerization mechanism and structure of heat shock factor 1 (HSF1) using western blotting, tryptophan (Trp) fluorescence spectroscopy, and molecular modeling. First, we examined the DNA-binding domains of human (Homo sapiens), goldfish (Carassius auratus), and walleye pollock (Gadus chalcogrammus) HSF1s by mutating key residues (36 and 103) that are thought to directly affect trimer formation. Human, goldfish, and walleye pollock HSF1s contain cysteine at residue 36 but cysteine (C), tyrosine (Y), and phenylalanine (F), respectively, at residue 103. The optimal trimerization temperatures for the wild-type HSF1s of each species were found to be 42, 37, and 20 °C, respectively. Interestingly, a mutation experiment revealed that trimerization occurred at 42 °C when residue 103 was cysteine, at 37 °C when it was tyrosine, and at 20 °C when it was phenylalanine, regardless of the species. In addition, it was confirmed that when residue 103 of the three species was mutated to alanine, trimerization did not occur. This suggests that in addition to trimerization via disulfide bond formation between the cysteine residues in human HSF1, trimerization can also occur via the formation of a different type of bond between cysteine and aromatic ring residues such as tyrosine and phenylalanine. We also confirmed that at least one cysteine is required for the trimerization of HSF1s, regardless of its position (residue 36 or 103). Additionally, it was shown that the trimer formation temperature is related to growth and survival in fish.

Thermodynamic modulation of gephyrin condensation by inhibitory synapse components.

Phase separation drives compartmentalization of intracellular contents into various biomolecular condensates. Individual condensate components are thought to differentially contribute to the organization and function of condensates. However, how intermolecular interactions among constituent biomolecules modulate the phase behaviors of multicomponent condensates remains unclear. Here, we used core components of the inhibitory postsynaptic density (iPSD) as a model system to quantitatively probe how the network of intra- and intermolecular interactions defines the composition and cellular distribution of biomolecular condensates. We found that oligomerization-driven phase separation of gephyrin, an iPSD-specific scaffold, is critically modulated by an intrinsically disordered linker region exhibiting minimal homotypic attractions. Other iPSD components, such as neurotransmitter receptors, differentially promote gephyrin condensation through distinct binding modes and affinities. We further demonstrated that the local accumulation of scaffold-binding proteins at the cell membrane promotes the nucleation of gephyrin condensates in neurons. These results suggest that in multicomponent systems, the extent of scaffold condensation can be fine-tuned by scaffold-binding factors, a potential regulatory mechanism for self-organized compartmentalization in cells.

pH Dependence of HSF1 trimerization is shaped by intramolecular interactions.

Heat shock factor 1 (HSF1) primarily regulates various cellular stress responses. Previous studies have shown that low pH within the physiological range directly activates HSF1 function in vitro. However, the detailed molecular mechanisms remain unclear. This study proposes a molecular mechanism based on the trimerization behavior of HSF1 at different pH values. Extensive mutagenesis of human and goldfish HSF1 revealed that the optimal pH for trimerization depended on the identity of residue 103. In particular, when residue 103 was occupied by tyrosine, a significant increase in the optimal pH was observed, regardless of the rest of the sequence. This behavior can be explained by the protonation state of the neighboring histidine residues, His101 and His110. Residue 103 plays a key role in trimerization by forming disulfide or non-covalent bonds with Cys36. If tyrosine resides at residue 103 in an acidic environment, its electrostatic interactions with positively charged histidine residues prevent effective trimerization. His101 and His110 are neutralized at a higher pH, which releases Tyr103 to interact with Cys36 and drives the effective trimerization of HSF1. This study showed that the protonation state of a histidine residue can regulate the intramolecular interactions, which consequently leads to a drastic change in the oligomerization behavior of the entire protein.

Biomolecular condensates form spatially inhomogeneous network fluids.

The functions of biomolecular condensates are thought to be influenced by their material properties, and these will be determined by the internal organization of molecules within condensates. However, structural characterizations of condensates are challenging, and rarely reported. Here, we deploy a combination of small angle neutron scattering, fluorescence recovery after photobleaching, and coarse-grained molecular dynamics simulations to provide structural descriptions of model condensates that are formed by macromolecules from nucleolar granular components (GCs). We show that these minimal facsimiles of GCs form condensates that are network fluids featuring spatial inhomogeneities across different length scales that reflect the contributions of distinct protein and peptide domains. The network-like inhomogeneous organization is characterized by a coexistence of liquid- and gas-like macromolecular densities that engenders bimodality of internal molecular dynamics. These insights suggest that condensates formed by multivalent proteins share features with network fluids formed by systems such as patchy or hairy colloids.

Biomolecular condensates form spatially inhomogeneous network fluids.

The functions of biomolecular condensates are thought to be influenced by their material properties, and these are in turn determined by the multiscale structural features within condensates. However, structural characterizations of condensates are challenging, and hence rarely reported. Here, we deploy a combination of small angle neutron scattering, fluorescence recovery after photobleaching, and bespoke coarse-grained molecular dynamics simulations to provide structural descriptions of model condensates that mimic nucleolar granular components (GCs). We show that facsimiles of GCs are network fluids featuring spatial inhomogeneities across hierarchies of length scales that reflect the contributions of distinct protein and peptide domains. The network-like inhomogeneous organization is characterized by a coexistence of liquid- and gas-like macromolecular densities that engenders bimodality of internal molecular dynamics. These insights, extracted from a combination of approaches, suggest that condensates formed by multivalent proteins share features with network fluids formed by associative systems such as patchy or hairy colloids.

Decoding the Roles of Amyloid-ß (1-42)'s Key Oligomerization Domains toward Designing Epitope-Specific Aggregation Inhibitors.

Fibrillar amyloid aggregates are the pathological hallmarks of multiple neurodegenerative diseases. The amyloid-ß (1-42) protein, in particular, is a major component of senile plaques in the brains of patients with Alzheimer's disease and a primary target for disease treatment. Determining the essential domains of amyloid-ß (1-42) that facilitate its oligomerization is critical for the development of aggregation inhibitors as potential therapeutic agents. In this study, we identified three key hydrophobic sites (17LVF19, 32IGL34, and 41IA42) on amyloid-ß (1-42) and investigated their involvement in the self-assembly process of the protein. Based on these findings, we designed candidate inhibitor peptides of amyloid-ß (1-42) aggregation. Using the designed peptides, we characterized the roles of the three hydrophobic regions during amyloid-ß (1-42) fibrillar aggregation and monitored the consequent effects on its aggregation property and structural conversion. Furthermore, we used an amyloid-ß (1-42) double point mutant (I41N/A42N) to examine the interactions between the two C-terminal end residues with the two hydrophobic regions and their roles in amyloid self-assembly. Our results indicate that interchain interactions in the central hydrophobic region (17LVF19) of amyloid-ß (1-42) are important for fibrillar aggregation, and its interaction with other domains is associated with the accessibility of the central hydrophobic region for initiating the oligomerization process. Our study provides mechanistic insights into the self-assembly of amyloid-ß (1-42) and highlights key structural domains that facilitate this process. Our results can be further applied toward improving the rational design of candidate amyloid-ß (1-42) aggregation inhibitors.

Probabilistic establishment of speckle-associated inter-chromosomal interactions.

Inter-chromosomal interactions play a crucial role in genome organization, yet the organizational principles remain elusive. Here, we introduce a novel computational method to systematically characterize inter-chromosomal interactions using in situ Hi-C results from various cell types. Our method successfully identifies two apparently hub-like inter-chromosomal contacts associated with nuclear speckles and nucleoli, respectively. Interestingly, we discover that nuclear speckle-associated inter-chromosomal interactions are highly cell-type invariant with a marked enrichment of cell-type common super-enhancers (CSEs). Validation using DNA Oligopaint fluorescence in situ hybridization (FISH) shows a strong but probabilistic interaction behavior between nuclear speckles and CSE-harboring genomic regions. Strikingly, we find that the likelihood of speckle-CSE associations can accurately predict two experimentally measured inter-chromosomal contacts from Hi-C and Oligopaint DNA FISH. Our probabilistic establishment model well describes the hub-like structure observed at the population level as a cumulative effect of summing individual stochastic chromatin-speckle interactions. Lastly, we observe that CSEs are highly co-occupied by MAZ binding and MAZ depletion leads to significant disorganization of speckle-associated inter-chromosomal contacts. Taken together, our results propose a simple organizational principle of inter-chromosomal interactions mediated by MAZ-occupied CSEs.

Conformational landscapes of artificial peptides predicted by various force fields: are we ready to simulate ß-amino acids?

With the introduction of artificial peptides as antimicrobial agents and organic catalysts, numerous efforts have been made to design foldamers with desirable structures and functions. Computational tools are a helpful proxy for revealing the dynamic structures at atomic resolution and understanding foldamer's complex structure-function relationships. However, the performance of conventional force fields in predicting the structures of artificial peptides has not been systematically evaluated. In this study, we critically assessed three popular force fields, AMBER ff14SB, CHARMM36m, and OPLS-AA/L, in predicting conformational propensities of a ß-peptide foldamer at monomer and hexamer levels. Simulation results were compared to those obtained from quantum chemistry calculations and experimental data. We also utilised replica exchange molecular dynamics simulations to investigate the energy landscape of each force field and assess the similarities and differences between force fields. We compared different solvent systems in the AMBER ff14SB and CHARMM36m frameworks and confirmed the unanimous role of hydrogen bonds in shaping energy landscapes. We anticipate that our data will pave the way for further improvements to force fields and for understanding the role of solvents in peptide folding, crystallisation, and engineering.

Thermodynamics of π-π Interactions of Benzene and Phenol in Water.

The π-π interaction is a major driving force that stabilizes protein assemblies during protein folding. Recent studies have additionally demonstrated its involvement in the liquid-liquid phase separation (LLPS) of intrinsically disordered proteins (IDPs). As the participating residues in IDPs are exposed to water, π-π interactions for LLPS must be modeled in water, as opposed to the interactions that are often established at the hydrophobic domains of folded proteins. Thus, we investigated the association of free energies of benzene and phenol dimers in water by integrating van der Waals (vdW)-corrected density functional theory (DFT) and DFT in classical explicit solvents (DFT-CES). By comparing the vdW-corrected DFT and DFT-CES results with high-level wavefunction calculations and experimental solvation free energies, respectively, we established the quantitative credibility of these approaches, enabling a reliable prediction of the benzene and phenol dimer association free energies in water. We discovered that solvation influences dimer association free energies, but not significantly when no direct hydrogen-bond-type interaction exists between two monomeric units, which can be explained by the enthalpy-entropy compensation. Our comprehensive computational study of the solvation effect on π-π interactions in water could help us understand the molecular-level driving mechanism underlying the IDP phase behaviors.

Recent trends in studies of biomolecular phase separation.

Biomolecular phase separation has recently attracted broad interest, due to its role in the spatiotemporal compartmentalization of living cells. It governs the formation, regulation, and dissociation of biomolecular condensates, which play multiple roles in vivo, from activating specific biochemical reactions to organizing chromatin. Interestingly, biomolecular phase separation seems to be a mainly passive process, which can be explained by relatively simple physical principles and reproduced in vitro with a minimal set of components. This Mini review focuses on our current understanding of the fundamental principles of biomolecular phase separation and the recent progress in the research on this topic. [BMB Reports 2022; 55(8): 363-369].

Kinetic Modulation of Amyloid-ß (1-42) Aggregation and Toxicity by Structure-Based Rational Design.

Several point mutations can modulate protein structure and dynamics, leading to different natures. Especially in the case of amyloidogenic proteins closely related to neurodegenerative diseases, structural changes originating from point mutations can affect fibrillation kinetics. Herein, we rationally designed mutant candidates to inhibit the fibrillation process of amyloid-ß with its point mutants through multistep in silico analyses. Our results showed that the designed mutants induced kinetic self-assembly suppression and reduced the toxicity of the aggregate. A multidisciplinary biophysical approach with small-angle X-ray scattering, ion mobility-mass spectrometry, mass spectrometry, and additional in silico experiments was performed to reveal the structural basis associated with the inhibition of fibril formation. The structure-based design of the mutants with suppressed self-assembly performed in this study could provide a different perspective for modulating amyloid aggregation based on the structural understanding of the intrinsically disordered proteins.

Current Understanding of Molecular Phase Separation in Chromosomes.

Biomolecular phase separation denotes the demixing of a specific set of intracellular components without membrane encapsulation. Recent studies have found that biomolecular phase separation is involved in a wide range of cellular processes. In particular, phase separation is involved in the formation and regulation of chromosome structures at various levels. Here, we review the current understanding of biomolecular phase separation related to chromosomes. First, we discuss the fundamental principles of phase separation and introduce several examples of nuclear/chromosomal biomolecular assemblies formed by phase separation. We also briefly explain the experimental and computational methods used to study phase separation in chromosomes. Finally, we discuss a recent phase separation model, termed bridging-induced phase separation (BIPS), which can explain the formation of local chromosome structures.

Microbially Guided Discovery and Biosynthesis of Biologically Active Natural Products.

The design of small molecules that inhibit disease-relevant proteins represents a longstanding challenge of medicinal chemistry. Here, we describe an approach for encoding this challenge-the inhibition of a human drug target-into a microbial host and using it to guide the discovery and biosynthesis of targeted, biologically active natural products. This approach identified two previously unknown terpenoid inhibitors of protein tyrosine phosphatase 1B (PTP1B), an elusive therapeutic target for the treatment of diabetes and cancer. Both inhibitors appear to target an allosteric site, which confers selectivity, and can inhibit PTP1B in living cells. A screen of 24 uncharacterized terpene synthases from a pool of 4464 genes uncovered additional hits, demonstrating a scalable discovery approach, and the incorporation of different PTPs into the microbial host yielded alternative PTP-specific detection systems. Findings illustrate the potential for using microbes to discover and build natural products that exhibit precisely defined biochemical activities yet possess unanticipated structures and/or binding sites.

Generalized models for bond percolation transitions of associative polymers.

Polymers with stickers-and-spacers architectures can drive phase-separation-aided bond percolation transitions. Here, we present a generalized mean-field model to enable the calculation of bond percolation thresholds for polymers with multiple types of stickers. Further, using graph-based Monte Carlo simulations we demonstrate how cooperativity in bond formation can give rise to reentrant phase behavior. When combined with recent advances for modeling phase separation, our approaches for calculating percolation lines could be useful for modeling hardening transitions for multivalent proteins.

Client proximity enhancement inside cellular membrane-less compartments governed by client-compartment interactions.

Membrane-less organelles or compartments are considered to be dynamic reaction centers for spatiotemporal control of diverse cellular processes in eukaryotic cells. Although their formation mechanisms have been steadily elucidated via the classical concept of liquid-liquid phase separation, biomolecular behaviors such as protein interactions inside these liquid compartments have been largely unexplored. Here we report quantitative measurements of changes in protein interactions for the proteins recruited into membrane-less compartments (termed client proteins) in living cells. Under a wide range of phase separation conditions, protein interaction signals were vastly increased only inside compartments, indicating greatly enhanced proximity between recruited client proteins. By employing an in vitro phase separation model, we discovered that the operational proximity of clients (measured from client-client interactions) could be over 16 times higher than the expected proximity from actual client concentrations inside compartments. We propose that two aspects should be considered when explaining client proximity enhancement by phase separation compartmentalization: (1) clients are selectively recruited into compartments, leading to concentration enrichment, and more importantly, (2) recruited clients are further localized around compartment-forming scaffold protein networks, which results in even higher client proximity.

Multivalent-Interaction-Driven Assembly of Discrete, Flexible, and Asymmetric Supramolecular Protein Nano-Prisms.

Current approaches to design monodisperse protein assemblies require rigid, tight, and symmetric interactions between oligomeric protein units. Herein, we introduce a new multivalent-interaction-driven assembly strategy that allows flexible, spaced, and asymmetric assembly between protein oligomers. We discovered that two polygonal protein oligomers (ranging from triangle to hexagon) dominantly form a discrete and stable two-layered protein prism nanostructure via multivalent interactions between fused binding pairs. We demonstrated that protein nano-prisms with long flexible peptide linkers (over 80 amino acids) between protein oligomer layers could be discretely formed. Oligomers with different structures could also be monodispersely assembled into two-layered but asymmetric protein nano-prisms. Furthermore, producing higher-order architectures with multiple oligomer layers, for example, 3-layered nano-prisms or nanotubes, was also feasible.

Physical Principles Underlying the Complex Biology of Intracellular Phase Transitions.

Many biomolecular condensates appear to form via spontaneous or driven processes that have the hallmarks of intracellular phase transitions. This suggests that a common underlying physical framework might govern the formation of functionally and compositionally unrelated biomolecular condensates. In this review, we summarize recent work that leverages a stickers-and-spacers framework adapted from the field of associative polymers for understanding how multivalent protein and RNA molecules drive phase transitions that give rise to biomolecular condensates. We discuss how the valence of stickers impacts the driving forces for condensate formation and elaborate on how stickers can be distinguished from spacers in different contexts. We touch on the impact of sticker- and spacer-mediated interactions on the rheological properties of condensates and show how the model can be mapped to known drivers of different types of biomolecular condensates.

LASSI: A lattice model for simulating phase transitions of multivalent proteins.

Many biomolecular condensates form via spontaneous phase transitions that are driven by multivalent proteins. These molecules are biological instantiations of associative polymers that conform to a so-called stickers-and-spacers architecture. The stickers are protein-protein or protein-RNA interaction motifs and / or domains that can form reversible, non-covalent crosslinks with one another. Spacers are interspersed between stickers and their preferential interactions with solvent molecules determine the cooperativity of phase transitions. Here, we report the development of an open source computational engine known as LASSI (LAttice simulation engine for Sticker and Spacer Interactions) that enables the calculation of full phase diagrams for multicomponent systems comprising of coarse-grained representations of multivalent proteins. LASSI is designed to enable computationally efficient phenomenological modeling of spontaneous phase transitions of multicomponent mixtures comprising of multivalent proteins and RNA molecules. We demonstrate the application of LASSI using simulations of linear and branched multivalent proteins. We show that dense phases are best described as droplet-spanning networks that are characterized by reversible physical crosslinks among multivalent proteins. We connect recent observations regarding correlations between apparent stoichiometry and dwell times of condensates to being proxies for the internal structural organization, specifically the convolution of internal density and extent of networking, within condensates. Finally, we demonstrate that the concept of saturation concentration thresholds does not apply to multicomponent systems where obligate heterotypic interactions drive phase transitions. This emerges from the ellipsoidal structures of phase diagrams for multicomponent systems and it has direct implications for the regulation of biomolecular condensates in vivo.

Covalently-assembled single-chain protein nanostructures with ultra-high stability.

Protein nanostructures with precisely defined geometries have many potential applications in catalysis, sensing, signal processing, and drug delivery. While many de novo protein nanostructures have been assembled via non-covalent intramolecular and intermolecular interactions, a largely unexplored strategy is to construct nanostructures by covalently linking multiple individually folded proteins through site-specific ligations. Here, we report the synthesis of single-chain protein nanostructures with triangular and square shapes made using multiple copies of a three-helix bundle protein and split intein chemistry. Coarse-grained simulations confirm the experimentally observed flexibility of these nanostructures, which is optimized to produce triangular structures with high regularity. These single-chain nanostructures also display ultra-high thermostability, resist denaturation by chaotropes and organic solvents, and have applicability as scaffolds for assembling materials with nanometer resolution. Our results show that site-specific covalent ligation can be used to assemble individually folded proteins into single-chain nanostructures with bespoke architectures and high stabilities.