Nonspecific Hydrophobic Interactions Research Articles

Nonspecific Yet Selective Interactions Contribute to Small Molecule Condensate Binding.

Biomolecular condensates are essential in various cellular processes, and their misregulation has been demonstrated to underlie disease. Small molecules that modulate condensate stability and material properties offer promising therapeutic approaches, but mechanistic insights into their interactions with condensates remain largely lacking. We employ a multiscale approach to enable long-time, equilibrated all-atom simulations of various condensate-ligand systems. Systematic characterization of the ligand binding poses reveals that condensates can form diverse and heterogeneous chemical environments with one or multiple chains to bind small molecules. Unlike traditional protein-ligand interactions, these chemical environments are dominated by nonspecific hydrophobic interactions. Nevertheless, the chemical environments feature unique amino acid compositions and physicochemical properties that favor certain small molecules over others, resulting in varied ligand partitioning coefficients within condensates. Notably, different condensates share similar sets of chemical environments but at different populations. This population shift drives ligand selectivity toward specific condensates. Our approach can enhance the interpretation of experimental screening data and may assist in the rational design of small molecules targeting specific condensates.

Redox conditions influence the chemical composition of iron-associated organic carbon in boreal lake sediments: A synchrotron-based NEXAFS study

The global carbon and iron cycles are intimately linked as redox-sensitive iron oxides readily bind organic carbon in a variety of environmental settings, including marine and lacustrine sediments. While these iron-organic carbon complexes sequester vast quantities of organic carbon, the composition of the organic matter within them remains unknown for lacustrine environments. Here we present C K1s and Fe L3,2 edge Near Edge X-ray Absorption Fine Structure (NEXAFS) spectra of surface sediments and authigenic iron complexes from adjacent basins of a pristine boreal lake located in Québec, Canada, with contrasting oxygen exposure regimes. We demonstrate differences in organic carbon speciation in sediments from both basins, as well as co-localization of organic carbon and iron on a sub-micron scale in 100 nm thick samples. Differences in redox cycling across these two basins allow for a direct comparison of the effect of oscillating redox conditions on the composition of organic carbon sequestered by iron. Our results suggest that reactive organic molecules, which may be polysaccharides, were found preferentially associated with iron in the perennially oxic sediments compared to more phenol rich organics in the seasonally anoxic sediments, highlighting the importance of iron oxides in the protection and preservation of labile organic compounds. Traces of aliphatic carbon were observed in sediments from the anoxic basin, alongside carboxyl and aromatic functionalities. This carboxyl-rich aliphatic material could possibly interact with the sediment mineral matrix either through a ligand exchange mechanism between the mineral phases and the carboxyl functionalities, or via non-specific hydrophobic interactions involving the aliphatic moieties. Finally, our work also shows that OC:Fe ratios should be used with caution when inferring a binding mechanism between OC and iron oxides.

Non-specific yet selective interactions contribute to small molecule condensate partitioning behavior.

Biomolecular condensates are essential in various cellular processes, and their mis-regulation has been demonstrated to be underly disease. Small molecules that modulate condensate stability and material properties offer promising therapeutic approaches, but mechanistic insights into their interactions with condensates remain largely lacking. We employ a multiscale approach to enable long-time, equilibrated all-atom simulations of various condensate-ligand systems. Systematic characterization of the ligand binding poses reveals that condensates can form diverse and heterogeneous chemical environments with one or multiple chains to bind small molecules. Unlike traditional protein-ligand interactions, these chemical environments are dominated by non-specific hydrophobic interactions. Nevertheless, the chemical environments feature unique amino acid compositions and physicochemical properties that favor certain small molecules over others, resulting in varied ligand partitioning coefficients within condensates. Notably, different condensates share similar sets of chemical environments but at different populations. This population shift drives ligand selectivity towards specific condensates. Our approach can enhance the interpretation of experimental screening data and may assist in the rational design of small molecules targeting specific condensates.

A Zwitterionic Anti-Fouling Coating Promotes Electrochemical Biosensing in Complex Media

Biofoulants present in complex media, such as blood, plasma, saliva, etc., can cause an increase in the background of the signal or a reduction in signal-to-noise ratio, due to which most biosensors require a sample processing step. One of the major interactions for biofouling in biosensing platforms is non-specific hydrophobic interactions, which can be prevented using hydrophilic polymeric coatings [1], [2]. However, most hydrophilic polymers are not conductive enough to provide a strong electrochemical current signal, whereas the existing conductive polymers are often hydrophobic. To avoid this, zwitterionic polymers that are both ionically conductive [3] and hydrophilic [3] can be used as anti-fouling polymers for electrochemical biosensing. Additionally, these polymers are highly effective at preventing fouling by electrostatically-induced water structuring [4]. Herein, we report a zwitterionic polymer (bearing thiols, aldehydes and carboxylic acid groups) that forms a thin conductive and hydrophilic layer on the electrodes, while also housing biorecognition layers for capturing target analytes of interest.The polymer was synthesized by free radical copolymerization and coated on gold electrodes to form a thin coating (16 nm when dry), which upon hydration results in a hydrophilic interface (contact angle <15 degrees). Anti-fouling properties were validated by determining the adsorption of radiolabeled human serum albumin (HSA) protein (spiked in plasma) on polymer-coated electrodes. The polymer-coated electrodes showed a ~67% reduction in protein adsorption relative to bare gold electrodes. Additionally, no significant change in anodic current was observed when 1% HSA was incubated on polymer-coated electrodes for 1 hour; whereas an 83% decrease in anodic current was observed on bare-gold electrodes under the same conditions. The polymer-modified electrode was used in detecting redox-labeled DNA sequences with a LOD of 11 nM in buffer and unprocessed plasma. The coating was also utilized to detect COVID-19, where the prepared setup detected 104 cp/mL of SARS-CoV-2 pseudovirus spiked in 50% unprocessed saliva. Similar coatings created from poly(ethylene glycol) (PEG) were unresponsive toward SARS-CoV-2 spiked in 50% saliva [5]. The anti-fouling polymer coatings developed herein show enhanced performance compared to previously reported strategies, based on self-assembled monolayers, in detecting target analytes in complex media, demonstrating the potential for use in clinical diagnostics.

Exploring the Interactions between two Ligands, UCB-J and UCB-F, and Synaptic Vesicle Glycoprotein 2 Isoforms.

In silico modeling was applied to study the efficiency of two ligands, namely, UCB-J and UCB-F, to bind to isoforms of the synaptic vesicle glycoprotein 2 (SV2) that are involved in the regulation of synaptic function in the nerve terminals, with the ultimate goal to understand the selectivity of the interaction between UCB-J and UCB-F to different isoforms of SV2. Docking and large-scale molecular dynamics simulations were carried out to unravel various binding patterns, types of interactions, and binding free energies, covering hydrogen bonding and nonspecific hydrophobic interactions, water bridge, π-π, and cation-π interactions. The overall preference for bonding types of UCB-J and UCB-F with particular residues in the protein pockets can be disclosed in detail. A unique interaction fingerprint, namely, hydrogen bonding with additional cation-π interaction with the pyridine moiety of UCB-J, could be established as an explanation for its high selectivity over the SV2 isoform A (SV2A). Other molecular details, primarily referring to the presence of π-π interactions and hydrogen bonding, could also be analyzed as sources of selectivity of the UCB-F tracer for the three isoforms. The simulations provide atomic details to support future development of new selective tracers targeting synaptic vesicle glycoproteins and their associated diseases.

Synergistic effects of tea polyphenols and phosphorylation on the gelation behavior of egg white proteins

Egg white protein (EWP) is widely used in the food industry owing to its excellent gelation properties. This study investigated the synergistic effect of tea polyphenol (TP) and sodium tripolyphosphate (STP) or sodium pyrophosphate (TSPP) on the gelation properties of EWP. Results showed that TP and phosphates enhanced the surface electrostatic formula of EWP to improve the stability of complexes. Meanwhile, STP and EWP were ligand bound as OC–N–P, whereas TSPP was bonded to EWP as P–O–P. Increase in TP content decreased α-helical content, and shifts in the β-turns and random structures of EWP were observed. TSPP enhanced the crystallinity of EWP and promoted the formation of ordered structures. The dominant forces of EWP gel (EWG) under synergistic treatment were non-specific cross-linking, ionic bonding, and hydrophobic interactions. Scanning electron microscopy (SEM) results revealed that the gel three-dimensional network cross-linking microstructures were evident, and pore size analysis showed that the EWG formed a large pore structure to contain the water molecules produced by the combination of phosphate and EWP. Evaluation of the gelation properties revealed that the synergistic effect of TP and each phosphate prolonged the gelation time of EWP and decreased the rigidity of EWG, but increased the springiness and true stress of the EWG. Simultaneously, phosphorylated and TP-treated EWGs showed reduced sensitivity to reagents and salt ions. Overall, this study provides a scientific basis for the improvement and regulation of the gelation behavior of EWP.

Partitioning behavior and mechanism of polyhalogenated carbazoles in water and suspended particulate matter

Polyhalogenated carbazoles (PHCZs), known as new dioxin-like compounds, are a new class of emerging environmental contaminants that have received increasing attention in recent years due to their wide distribution and dioxin-like toxicity. This study investigated the partition characteristics and adsorption mechanisms of eight PHCZs in the aqueous phase and suspended particulate matter (SPM). The competitive impact of humic acid (HA) on the adsorption of PHCZs was revealed when the effects of various environmental conditions (HA, temperature, perturbation disturbance, and pH) behavior were explored. The key finding of this study is that SPM, which is its effective vector, could adsorb 70.7 % of ΣPHCZs. The equilibrium adsorption amount is ranked as 3,6-ICZ > 3,6-BCZ > 2,7-BCZ > 3,6-CCZ > 1,3,6,8-BCZ > 3-BCZ > 2-BCZ > CZ. The halogen species, the degree of halogenation, and the substitution position of the PHCZs influence the amount of adsorption, where the log Kow values, the steric effect (Es), and the density contribute the most to the amount of adsorption, and the specific adsorption mechanisms are van der Waals force, π-π, hydrogen bonding, and nonspecific hydrophobic interactions. The adsorption reaction of PHCZs by SPM is endothermic, and the amount of adsorption increases with increasing temperature, oscillation velocity, and decrease in pH. HA may reduce the adsorption sites of hydroxyl, carbonyl, amide groups, and π-π bonds on SPM for PHCZs, while the SPM@HA conjugates can provide new sites for the adsorption of PHCZs. According to the experimental findings of this study, SPM plays a significant role in contaminant transport. As a result, when conducting environmental investigations of PHCZs and even other hydrophobic contaminants, we must fully consider the level of contaminants present in SPM to reveal the ecological risks accurately.

COMBINED IN SILICO AND EXPERIMENTAL INVESTIGATIONS OF VITAMIN D3 ENCAPSULATION BY STARCH β-OLIGOSACCHARIDE

The relevance of the research lies in the need to develop methods for encapsulating fat-soluble vitamins with natural oligosaccharides to increase their water solubility and use in the production of functional foods. The production ofnanostructured encapsulated vitamins is a new innovative direction in the food industry. This article presents the results of theoretical and experimental studies on the production of a clathrate complex of fat-soluble vitamin D3 (ViD3, cholecalciferol) with β-cyclodextrin (β-CD). The interaction of fat-soluble ViD3 with β-oligosaccharide was carried out by microwave activation. Encapsulation of ViD3 in the β-CD: ViD3 clathrate complex led to a change in the aggregate state of the ViD3 oil solution. The resulting supramolecular water-soluble ViD3 inclusion complex was investigated in silico by molecular docking and molecular modeling methods. The synthesized β-CD: ViD3 complex belongs to host-guest inclusion compounds and has better solubility. The spectral properties of the β-CD: ViD3 inclusion complex are characterized by the data of IR Fourier spectroscopy and the results of thermographic measurements on a differential scanning calorimeter. The activation energy of the reaction of the thermo-oxidative destruction of the β-CD: ViD3 inclusion complexes is calculated, the kinetic parameters of the thermal destruction of clathrates are determined. It is shown that the decisive role in the formation of the clathrate complex belongs to non-specific (hydrophobic, dispersion and van der Waals) interactions. The results obtained are promising for further understanding of the mechanism of molecular encapsulation of ViD3 compounds.

How oxidized EGCG remodels α-synuclein fibrils into non-toxic aggregates: insights from computational simulations.

The misfolding and aggregation of the presynaptic protein α-synuclein (α-syn) is a pathological hallmark of Parkinson's disease (PD). Targeting α-syn has emerged as a promising therapeutic strategy for PD. Emerging in vitro evidence supports a dual action of epigallocatechin-3-gallate (EGCG) against amyloid neurotoxicity. EGCG can halt the formation of toxic aggregates by redirecting the amyloid fibril aggregation pathway toward non-toxic aggregates and remodeling the existing toxic fibrils into non-toxic aggregates. Moreover, EGCG oxidation can enhance fibril's remodeling by forming Schiff bases, leading to crosslinking of the fibril. However, this covalent modification is not required for amyloid remodeling, and establishing non-specific hydrophobic interactions with sidechains seems to be the main driver of amyloid remodeling by EGCG. Thioflavin (ThT) is a gold standard probe to detect amyloid fibrils in vitro, and oxidized EGCG competes with ThT for amyloid fibrils' binding sites. In this work, we performed docking and molecular dynamics (MD) simulations to gain insights into the intermolecular interactions of oxidized EGCG and ThT with a mature α-syn fibril. We find that oxidized EGCG moves within lysine-rich sites within the hydrophobic core of the α-syn fibril, forming aromatic and hydrogen-bonding (H-bond) interactions with different residues during the whole MD simulation time. In contrast, ThT, which does not remodel amyloid fibrils, was docked to the same sites but only via aromatic interactions. Our findings suggest that non-covalent interactions play a role in oxidized EGCG binding into the hydrophobic core, including H-bond and aromatic interactions with some residues in the amyloid remodeling processes. These interactions would ultimately lead to a disturbance of structural features as determinants for stabilizing this fibril into a compact and pathogenic Greek key topology.

Degradable Glycopolyester-like Nanoparticles by Radical Ring-Opening Polymerization.

A small library of degradable polyester-like glycopolymers was successfully prepared by the combination of radical ring-opening copolymerization of 2-methylene-1,3-dioxepane as a cyclic ketene acetal (CKA) with vinyl ether (VE) derivatives and a Pd-catalyzed thioglycoconjugation. The resulting thioglycopolymers were formulated into self-stabilized thioglyconanoparticles, which were stable up to 4 months and were enzymatically degraded. Nanoparticles and their degradation products exhibited a good cytocompatibility on two healthy cell lines. Interactions between thioglyconanoparticles and lectins were investigated and highlighted the presence of both specific carbohydrate/lectin interactions and nonspecific hydrophobic interactions. Fluorescent thioglyconanoparticles were also prepared either by encapsulation of Nile red or by the functionalization of the polymer backbone with rhodamine B. Such nanoparticles were used to prove the cell internalization of the thioglyconanoparticles by lung adenocarcinoma (A549) cells, which underlined the great potential of P(CKA-co-VE) copolymers for biomedical applications.

Development of PD3 and PD3-B for PDEδ inhibition to modulate KRAS activity

Despite extensive efforts over 40 years, few effective KRAS inhibitors have been developed to date, mainly due to the undruggable features of KRAS proteins. In addition to the direct approach to KRAS via covalent inhibition, modulation of the prenyl-binding protein PDEδ that binds with farnesylated KRAS has emerged as an alternative strategy to abrogate KRAS activity. For the verification of new therapeutic strategies, chemical probes with the dual functions of visualisation and pharmacological inhibition against oncogenic proteins are enormously valuable to understand cellular events related to cancer. Here, we report indolizino[3,2-c]quinoline (IQ)-based fluorescent probes (PD3 and PD3-B) for PDEδ inhibition. By using the unique fluorescent characteristics of the IQ scaffold, a fluorescence polarisation (FP)-based binding assay identified PD3 as the most effective PDEδ probe among the tested PD analogues, with a low K d value of 0.491 µM and long retention time in the binding site of PDEδ. In particular, a FP-based competition assay using deltarasin verified that PD3 occupies the farnesylation binding site of PDEδ, excluding the possibility that the FP signals resulted from non-specific hydrophobic interactions between the ligand and protein in the assay. We also designed and synthesised PD3-B (5), an affinity-based probe (ABP) from the PD3 structure, which enabled us to pull down PDEδ from bacterial lysates containing a large number of intrinsic bacterial proteins. Finally, KRAS relocalization was verified in PANC-1 cells by treatment with PD3, suggesting its potential as an effective probe to target PDEδ.

Physical properties of the cytoplasm modulate the rates of microtubule polymerization and depolymerization.

The cytoplasm is a crowded, visco-elastic environment whose physical properties change according to physiological or developmental states. How the physical properties of the cytoplasm impact cellular functions invivo remains poorly understood. Here, we probe the effects of cytoplasmic concentration on microtubules by applying osmotic shifts to fission yeast, moss, and mammalian cells. We show that the rates of both microtubule polymerization and depolymerization scale linearly and inversely with cytoplasmic concentration; an increase in cytoplasmic concentration decreases the rates of microtubule polymerization and depolymerization proportionally, whereas a decrease in cytoplasmic concentration leads to the opposite. Numerous lines of evidence indicate that these effects are due to changes in cytoplasmic viscosity rather than cellular stress responses or macromolecular crowding per se. We reconstituted these effects on microtubules invitro by tuning viscosity. Our findings indicate that, even in normal conditions, the viscosity of the cytoplasm modulates the reactions that underlie microtubule dynamic behaviors.

Effects of Cathepsins on Gel Strength and Water-Holding Capacity of Myofibrillar Protein Gels from Bighead Carp (Aristichthys nobilis) under a Hydroxyl Radical-Generation Oxidizing System.

This study investigates the effects of cathepsins on the gel strength and water-holding capacity (WHC) of myofibrillar protein gels from bighead carp (Aristichthys nobilis) under a hydroxyl radical-generation oxidizing system. The myofibrillar proteins were divided into control group (with cathepsins) and E64 group (without cathepsins). The changes of cathepsin B and cathepsin L activities, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), protein oxidation (total sulfhydryl and carbonyl contents), and chemical interactions (nonspecific association, ionic bonds, hydrogen bonds, hydrophobic interactions, and disulfides) of myofibrillar protein and gels, as well as the gel strength and WHC of two groups under 0–100 mM H2O2, were measured. The results indicated that mild oxidation (10 mM H2O2) made a better gel strength and WHC. Cathepsin B and L activities decreased with increasing H2O2 concentrations but their effects on myofibrillar protein degradation still existed during 0.1–50 mM H2O2, which was expressed by higher carbonyl contents and ionic bonds at 0.1 and 50 mM H2O2, higher total sulfhydryl contents at 0 mM H2O2, and a lower intensity of MHC and actin of the control group than the E64 group. Besides more protein degradation, cathepsin proteolysis also resulted in lower gel strength and WHC in control gels than E64 gels under mild oxidation, which could be explained by lower hydrophobic interaction and moderate disulfides bonds between gel protein molecules of control gels.

Motif-pattern dependence of biomolecular phase separation driven by specific interactions.

Eukaryotic cells partition a wide variety of important materials and processes into biomolecular condensates—phase-separated droplets that lack a membrane. In addition to nonspecific electrostatic or hydrophobic interactions, phase separation also depends on specific binding motifs that link together constituent molecules. Nevertheless, few rules have been established for how these ubiquitous specific, saturating, motif-motif interactions drive phase separation. By integrating Monte Carlo simulations of lattice-polymers with mean-field theory, we show that the sequence of heterotypic binding motifs strongly affects a polymer’s ability to phase separate, influencing both phase boundaries and condensate properties (e.g. viscosity and polymer diffusion). We find that sequences with large blocks of single motifs typically form more inter-polymer bonds, which promotes phase separation. Notably, the sequence of binding motifs influences phase separation primarily by determining the conformational entropy of self-bonding by single polymers. This contrasts with systems where the molecular architecture primarily affects the energy of the dense phase, providing a new entropy-based mechanism for the biological control of phase separation.

The effect of fibrin on rheological behavior, gelling properties and microstructure of myofibrillar proteins

The aim of this study was to evaluate water-holding capacity, texture, rheological properties, interactions and microstructure of myofibrillar proteins (MP) gel with the addition of fibrin during the heat-induced process. Compared with the control, whiteness, water-holding capacity and hardness of MP gel with the addition of 2% fibrin increased by 9.70%, 79.36%, 323.07%, respectively. Storage modulus and loss modulus values in 2% fibrin addition group were higher than those of the control. The conformation of random coil gradually transformed to β-sheet and β-turn with the increase of fibrin content; nonspecific association and hydrophobic interactions increased from 14.27 g L−1 and 9.00 g L−1 in the control to 28.96 g L−1 and 12.90 g L−1 in the addition of 2% fibrin, respectively, indicating that these interactions played a key role in the improvement of gelling and rheological properties of MP. Scanning electron microscopy revealed that the incorporation of fibrin facilitated the formation of composited gel with a more ordered and homogeneous gel network structure. These results indicated that fibrin enhanced the functionality of MP, which could be beneficial to the development of gel-type meat products.

Site-Specific Nonenzymatic Peptide S/O-Glutamylation Reveals the Extent of Substrate Promiscuity in Glutamate Elimination Domains.

Formation of dehydroalanine and dehydrobutyrine residues via tRNA-dependent dehydration of serine and threonine is a key post-translational modification in the biosynthesis of lanthipeptide and thiopeptide RiPPs. The dehydration process involves two reactions, wherein the O-glutamyl Ser/Thr intermediate, accessed by a dedicated enzyme utilizing Glu-tRNAGlu as the acyl donor, is recognized by the second enzyme, referred to as the glutamate elimination domain (ED), which catalyzes the eponymous reaction yielding a dehydroamino acid. Many details of ED catalysis remain unexplored because the scope of available substrates for testing is limited to those that the upstream enzymes can furnish. Here, we report two complementary strategies for direct, nonenzymatic access to diverse ED substrates. We establish that a thiol-thioester exchange reaction between a Cys-containing peptide and an α thioester of glutamic acid leads an S-glutamylated intermediate which can act as a substrate for EDs. Furthermore, we show that the native O-glutamylated substrates can be accessible from S-glutamylated peptides upon a site-specific S-to-O acyl transfer reaction. Combined with flexible in vitro translation utilized for rapid peptide production, these chemistries enabled us to dissect the substrate recognition requirements of three known EDs. Our results establish that EDs are uniquely promiscuous enzymes capable of acting on substrates with arbitrary amino acid sequences and performing retro-Michael reaction beyond the canonical glutamate elimination. To facilitate substrate recruitment, EDs apparently engage in nonspecific hydrophobic interactions with their substrates. Altogether, our results establish the substrate scope of EDs and provide clues to their catalysis.

Comparison of photoactivatable crosslinkers for in-gel immunoassays.

While fluorescence readout is a key detection modality for hydrogel-based immunoassays, background fluorescence due to autofluorescence or non-specific antibody interactions impairs the lower limit of detection of fluorescence immunoassays. Chemical modifications to the hydrogel structure impact autofluorescence and non-specific interactions. Benzophenone is a common photoactivatable molecule, and benzophenone methacrylamide (BPMA) has been used for cross-linking protein in polyacrylamide (PA) hydrogels. However, previous studies have suggested that the aromatic structure of benzophenone can contribute to increased autofluorescence and non-specific hydrophobic interactions with unbound fluorescent probes. Here, we synthesize diazirine methacrylamide (DZMA) as an alternative photoactivatable molecule to crosslink into PA hydrogels for in-gel protein capture for in-gel immunoassays. We hypothesize that the less hydrophobic structure of diazirine (based on previously reported predicted and experimental log P values) exhibits both reduced autofluorescence and non-specific hydrophobic interactions. We find that while equal concentrations of DZMA and BPMA result in lower protein target photocapture in the diazirine configuration, increasing the DZMA concentration up to 12 mM improves in-gel protein capture to be on par with previously reported and characterized 3 mM BPMA hydrogels. Furthermore, despite the higher concentration of diazirine, we observe negligible autofluorescence signal and a 50% reduction in immunoassay fluorescence background signal in diazirine gels compared to BPMA gels resulting in comparable signal-to-noise ratios (SNR) of the probed protein target. Finally, we test the utility of DZMA for single-cell immunoblotting in an open microfluidic device and find that protein migrates ∼1.3× faster in DZMA hydrogels than in BPMA hydrogels. However, in DZMA hydrogels we detect only 15% of the protein signal compared to BPMA hydrogels suggesting that the diazirine chemistry results in greater protein losses following electrophoretic separations. We establish that while diazirine has lower background fluorescence signal, which may potentially improve immunoassay performance, the lower capture efficiency of diazirine reduces its utility in open microfluidic systems susceptible to sample losses.

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.

Unexpected specificity within dynamic transcriptional protein–protein complexes

A key functional event in eukaryotic gene activation is the formation of dynamic protein-protein interaction networks between transcriptional activators and transcriptional coactivators. Seemingly incongruent with the tight regulation of transcription, many biochemical and biophysical studies suggest that activators use nonspecific hydrophobic and/or electrostatic interactions to bind to coactivators, with few if any specific contacts. Here a mechanistic dissection of a set of representative dynamic activator•coactivator complexes, comprised of the ETV/PEA3 family of activators and the coactivator Med25, reveals a different molecular recognition model. The data demonstrate that small sequence variations within an activator family significantly redistribute the conformational ensemble of the complex while not affecting overall affinity, and distal residues within the activator-not often considered as contributing to binding-play a key role in mediating conformational redistribution. The ETV/PEA3•Med25 ensembles are directed by specific contacts between the disordered activator and the Med25 interface, which is facilitated by structural shifts of the coactivator binding surface. Taken together, these data highlight the critical role coactivator plasticity plays in recognition of disordered activators and indicate that molecular recognition models of disordered proteins must consider the ability of the binding partners to mediate specificity.

3D RNA nanocage for encapsulation and shielding of hydrophobic biomolecules to improve the in vivo biodistribution.

Ribonucleic acid (RNA) nanotechnology platforms have the potential of harboring therapeutics for in vivo delivery in disease treatment. However, the nonspecific interaction between the harbored hydrophobic drugs and cells or other components before reaching the diseased site has been an obstacle in drug delivery. Here we report an encapsulation strategy to prevent such nonspecific hydrophobic interactions in vitro and in vivo based on a self-assembled three-dimensional (3D) RNA nanocage. By placing an RNA three-way junction (3WJ) in the cavity of the nanocage, the conjugated hydrophobic molecules were specifically positioned within the nanocage, preventing their exposure to the biological environment. The assembly of the nanocages was characterized by native polyacrylamide gel electrophoresis (PAGE), atomic force microscopy (AFM), and cryogenic electron microscopy (cryo-EM) imaging. The stealth effect of the nanocage for hydrophobic molecules in vitro was evaluated by gel electrophoresis, flow cytometry, and confocal microscopy. The in vivo sheathing effect of the nanocage for hydrophobic molecules was assessed by biodistribution profiling in mice. The RNA nanocages with hydrophobic biomolecules underwent faster clearance in liver and spleen in comparison to their counterparts. Therefore, this encapsulation strategy holds promise for in vivo delivery of hydrophobic drugs for disease treatment.