Backbone interactions and secondary structures in phase separation of disordered proteins.

Intrinsically disordered proteins (IDPs) are one of the major drivers behind the formation and characteristics of biomolecular condensates. Due to their inherent flexibility, the backbones of IDPs are significantly exposed, rendering them highly influential and susceptible to biomolecular phase separation. In densely packed condensates, exposed backbones have a heightened capacity to interact with neighboring protein chains, which might lead to strong coupling between the secondary structures and phase separation and further modulate the subsequent transitions of the condensates, such as aging and fibrillization. In this mini-review, we provide an overview of backbone-mediated interactions and secondary structures within biomolecular condensates to underscore the importance of protein backbones in phase separation. We further focus on recent advances in experimental techniques and molecular dynamics simulation methods for probing and exploring the roles of backbone interactions and secondary structures in biomolecular phase separation involving IDPs.

Toward Accurate Simulation of Coupling between Protein Secondary Structure and Phase Separation.

Intrinsically disordered proteins (IDPs) frequently mediate phase separation that underlies the formation of a biomolecular condensate. Together with theory and experiment, efficient coarse-grained (CG) simulations have been instrumental in understanding the sequence-specific phase separation of IDPs. However, the widely used Cα-only models are limited in capturing the peptide nature of IDPs, particularly backbone-mediated interactions and effects of secondary structures, in phase separation. Here, we describe a hybrid resolution (HyRes) protein model toward a more accurate description of the backbone and transient secondary structures in phase separation. With an atomistic backbone and coarse-grained side chains, HyRes can semiquantitatively capture the residue helical propensity and overall chain dimension of monomeric IDPs. Using GY-23 as a model system, we show that HyRes is efficient enough for the direct simulation of spontaneous phase separation and, at the same time, appears accurate enough to resolve the effects of single His to Lys mutations. HyRes simulations also successfully predict increased ß-structure formation in the condensate, consistent with available experimental CD data. We further utilize HyRes to study the phase separation of TPD-43, where several disease-related mutants in the conserved region (CR) have been shown to affect residual helicities and modulate the phase separation propensity as measured by the saturation concentration. The simulations successfully recapitulate the effect of these mutants on the helicity and phase separation propensity of TDP-43 CR. Analyses reveal that the balance between backbone and side chain-mediated interactions, but not helicity itself, actually determines phase separation propensity. These results support that HyRes represents an effective protein model for molecular simulation of IDP phase separation and will help to elucidate the coupling between transient secondary structures and phase separation.

Accurate Simulation of Coupling between Protein Secondary Structure and Liquid-Liquid Phase Separation.

Intrinsically disordered proteins (IDPs) frequently mediate liquid-liquid phase separation (LLPS) that underlies the formation of membraneless organelles. Together with theory and experiment, efficient coarse-grained (CG) simulations have been instrumental in understanding sequence-specific phase separation of IDPs. However, the widely-used Cα-only models are severely limited in capturing the peptide nature of IDPs, including backbone-mediated interactions and effects of secondary structures, in LLPS. Here, we describe a hybrid resolution (HyRes) protein model for accurate description of the backbone and transient secondary structures in LLPS. With an atomistic backbone and coarse-grained side chains, HyRes accurately predicts the residue helical propensity and chain dimension of monomeric IDPs. Using GY-23 as a model system, we show that HyRes is efficient enough for direct simulation of spontaneous phase separation, and at the same time accurate enough to resolve the effects of single mutations. HyRes simulations also successfully predict increased beta-sheet formation in the condensate, consistent with available experimental data. We further utilize HyRes to study the phase separation of TPD-43, where several disease-related mutants in the conserved region (CR) have been shown to affect residual helicities and modulate LLPS propensity. The simulations successfully recapitulate the effect of these mutants on the helicity and LLPS propensity of TDP-43 CR. Analyses reveal that the balance between backbone and sidechain-mediated interactions, but not helicity itself, actually determines LLPS propensity. We believe that the HyRes model represents an important advance in the molecular simulation of LLPS and will help elucidate the coupling between IDP transient secondary structures and phase separation.

Self-Assembly of Peapod-like Micrometer Tubes from a Planet-Satellite-type Supramolecular Megamer.

This study presents interesting self-assembly of peapod-like micrometer tubes from a planet-satellite-type supramolecular megamer, which was constructed through the specific host-guest molecular recognition between azobenzene (AZO)-functionalized hyperbranched poly(ethyl-3-oxetanemethanol)-star-poly(ethylene oxide) (HSP-AZO) and ß-cyclodextrin(CD)-based hydrophilic hyperbranched polyglycerol (CD-g-HPG). A peapod-like structure with micrometer-sized tube as the pod and vesicles encapsulated inside as the peas was formed through sequential vesicle entosis, linear association, and fusion processes. Dissipative particle dynamics (DPD) simulations support the structural possibility of the supramolecular peapod formation and its mechanism. UV light irradiation could lead to the disassembly of the peapod-like structure. This study expands the family of supramolecular polymers and opens a new avenue to develop bioinspired complex hierarchical nanoarchitectures at the microscopic level.

One-Dimensional Helical Nanostructures from the Hierarchical Self-Assembly of an Achiral "Rod-Coil" Alternating Copolymer.

The self-assembly of alternating copolymers (ACPs) has attracted considerable interest due to their unique alternating nature. However, compared with block copolymers, their self-assembly behavior remains much less explored and their reported self-assembled structures are limited. Here, the formation of supramolecular helical structures by the self-assembly of an achiral rod-coil alternating copolymer named as poly(quarter(3-hexylthiophene)-alt-poly(ethylene glycol)) (P(Q3HT-alt-PEG)), is reported. The copolymer exhibits an interesting hierarchical self-assembly process, driven by the π-π stacking of the Q3HT segments and the solvophobic interaction of the alkyl chains in tetrahydrofuran (THF)-isopropanol mixed solvents. The copolymer first self-assembled into thin nanobelts with a uniform size, then grows to helical nanoribbons and eventually twisted into helical nanowires with an average diameter of 25 ± 9 nm and a mean pitch of 80 ± 10 nm. Dissipative particle dynamics (DPD) simulation supports the formation course of the helical nanowires. Furthermore, the addition of (S)-ethyl lactate and (R)-ethyl lactate in the self-assembly of P(Q3HT-alt-PEG) results in the formation of left-handed and right-handed chiral nanowires, respectively, demonstrating the tunability of the chirality of the helical wires. This study expands the library of ordered self-assembled structures of ACPs, and also brings a new strategy and mechanism to construct helical supramolecular structures.

Membrane-Bound Inward-Growth of Artificial Cytoskeletons and Their Selective Disassembly.

The cytoskeleton is one of the most important cellular components. Up to now, most of the reported artificial cytoskeletons are based on a gel-in-vesicle strategy. Herein, we report a membrane-bound inward-growth pathway to prepare cytoskeleton-like and radially aligned nanofibers grown from capsule membranes to get membrane-bound artificial cytoskeletons (MACs). The mechanism therein is disclosed through the direct observation of the intermediates in both dried and liquid states. Furthermore, the as-prepared MACs show a selective disassembly behavior in the presence of reductants: both capsule membranes and MACs can be disassembled or only MACs can be disassembled through the selective introduction of dynamic disulfide bonds (DS) into them and by the switch of ultraviolet (UV) irradiation. The present work provides a new hierarchical self-assembly way to construct artificial cytoskeletons with controlled compositions and orientations.

Coarse-Grained Model of Thiol-Epoxy-Based Alternating Copolymers in Explicit Solvents.

The cosolvent method has been widely used in the self-assembly of amphiphilic alternating copolymers (ACPs), but the role of good and selective solvents is rarely investigated. Here, we have developed a coarse-grained (CG) model for the widely studied thiol-epoxy-based amphiphilic ACPs and a three-bead CG model for tetrahydrofuran (THF) as the good solvent, which is compatible with the MARTINI water model. The accuracy of both the CG polymer and THF models was validated by reproducing the structural and thermodynamic properties obtained from experiments or atomistic simulation results. Density in bulk, the radius of gyration, and solvation free energy in water or THF showed a good agreement between CG and atomistic models. The CG models were further employed to explore the self-assembly of ACPs in THF/water mixtures with different compositions. Chain folding and liquid-liquid phase separation behaviors were found with increasing water fractions, which were the key steps of the self-assembly process. This work will provide a basic platform to explore the self-assembly of amphiphilic ACPs in solvent mixtures and to reveal the real role of different solvents in self-assembly.

Retention of deposited ammonium and nitrate and its impact on the global forest carbon sink.

The impacts of enhanced nitrogen (N) deposition on the global forest carbon (C) sink and other ecosystem services may depend on whether N is deposited in reduced (mainly as ammonium) or oxidized forms (mainly as nitrate) and the subsequent fate of each. However, the fates of the two key reactive N forms and their contributions to forest C sinks are unclear. Here, we analyze results from 13 ecosystem-scale paired 15N-labelling experiments in temperate, subtropical, and tropical forests. Results show that total ecosystem N retention is similar for ammonium and nitrate, but plants take up more labelled nitrate ([Formula: see text]%) ([Formula: see text]) than ammonium ([Formula: see text]%) while soils retain more ammonium ([Formula: see text]%) than nitrate ([Formula: see text]%). We estimate that the N deposition-induced C sink in forests in the 2010s  is [Formula: see text] Pg C yr-1, higher than previous estimates because of a larger role for oxidized N and greater rates of global N deposition.

pH-Controlled Stereoregular Polymerization of Poly(methyl methacrylate) in Vesicle Membranes.

Here, we report a pH-controlled stereoregular polymerization of methyl methacrylate (MMA) inside the membrane of H20-COOH hyperbranched polymer vesicles using a common radical polymerization process. The vesicle size decreases from 745 to 214 nm with an increase of solution pH from 2.60 to 7.26, and the isotacticity of the obtained polymethyl methacrylates (PMMAs) is accordingly elevated from 9 to 35%. The obtained isotactic-rich PMMAs show a lower glass transition temperature depending on the isotacticity than the commercial random PMMAs. A mechanism study according to the in situ Fourier transform infrared measurements indicates that the control of polymer isotacticity results from the monomer conformation confined effect inside the thin vesicle membranes. The present study provides a new method to realize the preparation of isotactic polymers with the characteristics of facile synthesis, pH controllability, and a green polymerization process in aqueous solution as well as under mild reaction conditions of ambient temperature and pressure.

Dynamics and multi-annual fate of atmospherically deposited nitrogen in montane tropical forests.

The effects of nitrogen (N) deposition on forests largely depend on its fate after entering the ecosystem. While several studies have addressed the forest fate of N deposition using 15 N tracers, the long-term fate and redistribution of deposited N in tropical forests remains unknown. Here, we applied 15 N tracers to examine the fates of deposited ammonium ( NH 4 + ) and nitrate ( NO 3 - ) separately over 3 years in a primary and a secondary tropical montane forest in southern China. Three months after 15 N tracer addition, over 60% of 15 N was retained in the forests studied. Total ecosystem retention did not change over the study period, but between 3 months and 3 years following deposition 15 N recovery in plants increased from 10% to 19% and 13% to 22% in the primary and secondary forests, respectively, while 15 N recovery in the organic soil declined from 16% to 2% and 9% to 2%. Mineral soil retained 50% and 35% of 15 N in the primary and secondary forests, with retention being stable over time. The total ecosystem retention of the two N forms did not differ significantly, but plants retained more 15 NO 3 - than 15 NH 4 + and the organic soil more 15 NH 4 + than NO 3 - . Mineral soil did not differ in 15 NH 4 + and 15 NO 3 - retention. Compared to temperate forests, proportionally more 15 N was distributed to mineral soil and plants in these tropical forests. Overall, our results suggest that atmospherically deposited NH 4 + and NO 3 - is rapidly lost in the short term (months) but thereafter securely retained within the ecosystem, with retained N becoming redistributed to plants and mineral soil from the organic soil. This long-term N retention may benefit tropical montane forest growth and enhance ecosystem carbon sequestration.

Asymmetric Vesicles Self-Assembled by Amphiphilic Sequence-Controlled Polymers.

The asymmetric distribution of lipids on the inner and outer membranes of a cell plays a pivotal role in the physiological and immunological activities of life. It has inspired the elaboration of synthetic asymmetric vesicles for the discovery of advanced materials and functions. The asymmetric vesicles were generally prepared by amphiphilic block copolymers. We herein report on the formation of asymmetric vesicles self-assembled by amphiphilic sequence-controlled polymers with two hydrophilic segments SU and TEO. We also developed an efficient fluorescence titration method with europium(III) ions (Eu3+) to determine the uneven distribution of SU and TEO. SU units are preferentially located on the outer membrane and TEO on the inner membrane of the resulting vesicles, which is facilitated by the electrostatic repulsion of SU and the U-shaped folding of the hydrophobic backbone of the resulting polymers. This work shows that sequence-controlled polymers with alternating monomer sequence provide a powerful toolbox for the elaboration of important yet challenging self-assembled structures for emerging functions and properties.

Computational design of Janus polymersomes with controllable fission from double emulsions.

Janus polymer vesicles (polymersomes) with biphasic membranes have special properties and potential applications in many fields. The big barrier for the preparation of Janus polymersomes lies in the difficulty of complete lateral microphase separation of polymers along the vesicle membrane due to the limited mobility. Herein, we present a systematic simulation study to provide a new strategy for the fabrication of Janus polymersomes based on water-in-oil-in-water double emulsions. Two incompatible block copolymers of AB and AC completely separate into two hemispheres of the polymersome driven by the dewetting of double emulsions, followed by the stabilization of the Janus structure with the block copolymers BC at the interface between AB and AC hemispheres. The simulation results demonstrate the formation of Janus polymersomes in a wide range of the incompatibility between blocks B and C. In addition, the morphologies of the Janus polymersomes can be readily regulated by changing the number of copolymers BC, the ratio of AB to AC, and the dewetting rate of organic solvents. Both the Janus and patchy polymersomes can be obtained through the adjustment of the dewetting rate. Besides, by introducing stimulus-cleavable copolymers of BC, the Janus polymersomes can perform controllable fission. Further comparison with similar experiments has also demonstrated the feasibility of our strategy. We believe the present work will be useful for the fabrication of polymersomes with controlled patches in a large quantity, and the stimulus-responsive fission process will also make the polymersomes promising in some applications like controlled drug delivery and cytomimetic membrane communication.

In silico study of structure and water dynamics in CNT/polyamide nanocomposite reverse osmosis membranes.

CNT-based reverse osmosis membranes have long been regarded as one of the most promising candidates for water desalination. However, it is a pity that there is no complete understanding of the exact role of CNTs in those nanocomposite membranes. To address this issue, three atomistic models of PA (pure polyamide membrane), PA-CNT1 (polyamide nanocomposite membrane with an embedded carbon nanotube oriented vertical to the membrane surface) and PA-CNT2 (polyamide nanocomposite with an embedded carbon nanotube oriented parallel to the membrane surface) were constructed respectively in this work. Then, equilibrium molecular dynamics (EMD) and non-equilibrium molecular dynamics (NEMD) simulations were conducted to investigate the structure and water dynamics in these three models. The EMD simulations revealed a better stacking of the PA matrix due to the addition of the CNT and this impact was more significant in PA-CNT1 than in PA-CNT2. Meanwhile, PA matrix near the mouth of the CNT was found to behave as an obstruction that hindered the exchange of water molecules inside and outside the CNT. In NEMD simulations, we found that water molecules were guided away from the CNT because of the better stacked surrounding PA matrix. The partially covered CNT might not help to increase water flux in PA-CNT1 while guided water molecules and the smaller polymer region afftected by the CNT contributed to a relatively high flux in PA-CNT2. The current work might serve as a comprehensive understanding of the role of CNTs in the reverse osmosis process.

In situ supramolecular polymerization-enhanced self-assembly of polymer vesicles for highly efficient photothermal therapy.

Vesicular photothermal therapy agents (PTAs) are highly desirable in photothermal therapy (PTT) for their excellent light-harvesting ability and versatile hollow compartments. However, up to now, the reported vesicular PTAs are generally self-assembled from small molecules like liposomes, and polymer vesicles have seldom been used as PTAs due to the unsatisfactory photothermal conversion efficiency resulting from the irregular packing of chromophores in the vesicle membranes. Here we report a nano-sized polymer vesicle from hyperbranched polyporphyrins with favorable photothermal stability and extraordinarily high photothermal efficiency (44.1%), showing great potential in imaging-guided PTT for tumors through in vitro and in vivo experiments. These excellent properties are attributed to the in situ supramolecular polymerization of porphyrin units inside the vesicle membrane into well-organized 1D monofilaments driven by π-π stacking. We believe the supramolecular polymerization-enhanced self-assembly process reported here will shed a new light on the design of supramolecular materials with new structures and functions.

High-χ alternating copolymers for accessing sub-5 nm domains via simulations.

The ever-growing semiconductor industry has encouraged the feature dimensions of nanolithography to reach the sub-10 nm length scale. It is highly necessary to find nanolithographic materials with high performance but ultra-small domains. We have designed a series of high-χ alternating copolymers (ACPs), in which the polar and apolar repeating units are four hydroxyl groups and alkyl chains, respectively. Careful coarse-grained molecular dynamics (CG-MD) simulations demonstrate that these ACPs can form a variety of mesophases, including lamellae, perforated lamellae, and hexagonally packed cylinders. All the domain periods of these mesophases are smaller than 5 nm, and the smallest domain is close to 1 nm. Most importantly, both the phase morphologies and domain periods are independent of the molecular weight (MW) and molecular weight distribution (MWD) when the degree of polymerization (N) exceeds the threshold value. Thus, using high-χ ACPs, ultranarrow domains can be realized with high MW for sufficient material performance, while the MWD-independence can ensure the uniformity of the domain sizes. We believe that these "high χ-high N" alternating copolymers are promising alternatives as new nanolithographic materials.

Solution self-assembly behavior of rod-alt-coil alternating copolymers via simulations.

Alternating copolymers (ACPs) have shown several attractive unique characteristics in solution self-assembly due to their special alternating structure. With the introduction of rod segments, much more complexity and multifunctionality can be achieved in the self-assembly of rod-alt-coil ACPs. Herein, we have performed a simulation study on the self-assembly of rod-alt-coil ACPs in dilute solution through dissipative particle dynamics (DPD) simulations. A morphological phase diagram was constructed as a function of rod and coil length, in which diverse assemblies were found, such as bicontinuous micelles and perforated membranes. Furthermore, the alignment of rod segments in the assemblies has been disclosed in detail. And, we deeply investigated the effects of rod length, coil length and π-π interaction strength on the self-assembly morphologies and rod alignment. With the increase of rod length, a disorder-order transition was observed, and π-π interactions could facilitate the orderly alignment of rods. Besides, our simulation results showed good agreement with available experiments. Furthermore, the unique characteristics in the self-assembly of rod-alt-coil alternating copolymers were discussed; in particular we found that the alternating molecular structures of ACPs could promote the orderly alignment of rod segments. We believe that the current work can provide a solid theoretical foundation for further experimental studies.

Molecular dynamics simulation studies of the structure and antifouling performance of a gradient polyamide membrane.

The polyamide (PA) layer on the surface of thin-film-composite reverse osmosis membranes is the core aspect of membrane-based desalination technology. In recent years, molecular dynamics simulations have been increasingly used to disclose the physicochemical properties of the PA layer. However, the currently reported all-atom PA layer models do not exhibit gradient variation of the structural properties of the layer, and they can only represent the innermost region of the PA layer. With the help of our recently developed universal toolkit "MembrFactory", this paper reports a modeling method that can be used to construct a gradient crosslinking model and surface grafting model for the PA layer. A fully atomistic model of the PA layer was constructed, in which the degree of crosslinking (DC) was changed gradiently along the thickness direction. The structure of the PA layer model and the transport dynamics of the water molecules within it were systematically investigated using equilibrium molecular dynamics simulations. We found that the DC is the lowest and the water molecules have the strongest self-diffusion ability in the interfacial region of the PA layer model. Meanwhile, the pore size is distributed widely in the region. Subsequently, we modified the surface of the PA layer model with PEG coatings, and their coverage ratio was around 75%. The radial distribution function analysis showed that water molecules prefer to coordinate with the oxygen atoms in PEG. Furthermore, two contaminant molecules, 1-ethyl-2-methyl benzene and n-decane, were selected to investigate the antifouling properties of the PEG-modified PA layer. By analysing the trajectories of the pollutants and calculating the potential of the mean force, we found that the antifouling performance of a PEG-modified PA layer is not only related to the hydrophobicity and the size of the pollutant, but is also related to the coverage ratio of the PEG layer.

Multigeometry Nanoparticles from the Orthogonal Self-Assembly of Block Alternating Copolymers via Simulation.

Multigeometry nanoparticles (MGNs) have a high level of complexity both in composition and structure, and they are prevailing in nature and have shown great potential for multifunctional nanomaterials and hierarchy self-assembly. Polymer self-assembly is a common way to construct MGNs. However, up to now, the self-assembly strategy and the polymer category to fabricate MGNs are quite limited, and it is still a big challenge to get MGNs in a controlled way. Herein, by employing dissipative particle dynamics simulation, we provide a new "covalent-bonding-forced orthogonal self-assembly" strategy for the preparation of MGNs through the self-assembly of block alternating copolymers. Without any additional cautious control, block alternating copolymers can directly self-assemble into various MGNs, except for two basic requirements: the critical molecular weight of each block and the incompatibility of different blocks. Any different simple geometries, like vesicles, cylinders, and spheres, can be combined at will to construct arbitrary customized MGNs by changing the blocks. We further explore the effect of polymer concentration and the volume ratio of different blocks, through which the sizes, components, and structures of the MGNs can be regulated simply. In addition, we extend this strategy to ternary systems to fabricate much more complicated nanoparticles with triple geometries. We believe the present work has provided a promising and simple strategy to efficiently construct MGNs with precisely controllable geometries.

Uptake Patterns of Glycine, Ammonium, and Nitrate Differ Among Four Common Tree Species of Northeast China.

Fundamental questions of how plant species within secondary forests and plantations in northeast China partition limited nitrogen (N) resource remain unclear. Here we conducted a 15N tracer greenhouse study to determine glycine, ammonium, and nitrate uptake by the seedlings of two coniferous species, Pinus koraiensis (Pinus) and Larix keampferi (Larix), and two broadleaf species, Quercus mongolica (Quercus) and Juglans mandshurica (Juglans), that are common in natural secondary forests in northeast China. Glycine contributed 43% to total N uptake of Pinus, but only 20, 11, and 21% to N uptake by Larix, Quercus, and Juglans, respectively (whole plant), whereas nitrate uptake was 24, 74, 88, and 68% of total uptake for these four species, respectively. Retention of glycine carbon versus nitrogen in Pinus roots indicated that 36% of glycine uptake was retained intact. Nitrate was preferentially used by Larix, Quercus, and Juglans, with nitrate uptake constituting 68∼88% of total N use by these three species. These results demonstrated that these dominant tree species in secondary forests in northeast China partitioned limited N resource by varying uptake of glycine, ammonium and nitrate, with all species, except Pinus, using nitrate that are most abundant within these soils. Such N use pattern may also provide potential underlying mechanisms for the higher retention of deposited nitrate than ammonium into aboveground biomass in these secondary forests.

MembrFactory: A Force Field and composition Double Independent Universal Tool for Constructing Polyamide Reverse Osmosis Membranes.

The topmost polyamide (PA) layer of the thin-film-composite reverse osmosis (RO) membrane is the most important part in the membrane-based RO technology. With the aid of molecular dynamics simulations, many PA layer-related features in the RO process can be revealed. With many novel types of PA RO membranes out of trimesoyl chloride/m-phenylenediamine monomers developed in the laboratory, a convenient model building tool for these PA layer systems is urgently needed to conduct the theoretical analysis. Here, we develop a new universal toolkit for constructing PA RO membranes, named as MembrFactory, which combines flexibility of force fields and membrane compositions. A key characteristic of our approach is the use of monomers as the starting state, and the final membrane model was obtained automatically by stepwise reaction between the functional groups on the monomers. The reliability of MembrFactory has been validated by constructing several common PA RO membranes. © 2019 Wiley Periodicals, Inc.