A Biomimetic Multi-Component Subunit Vaccine via Ratiometric Loading of Hierarchical Hydrogels.

The development of subunit vaccines that mimic the molecular complexity of attenuated vaccines has been limited by the difficulty of intracellular co-delivery of multiple chemically diverse payloads at controllable concentrations. We report on hierarchical hydrogel depots employing simple poly(propylene sulfone) homopolymers to enable ratiometric loading of a protein antigen and four physicochemically distinct adjuvants in a hierarchical manner. The optimized vaccine consisted of immunostimulants either adsorbed to or encapsulated within nanogels, which were capable of noncovalent anchoring to subcutaneous tissues. These 5-component nanogel vaccines demonstrated enhanced humoral and cell-mediated immune responses compared to formulations with standard single adjuvant and antigen pairing. The use of a single simple homopolymer capable of rapid and stable loading and intracellular delivery of diverse molecular cargoes holds promise for facile development and optimization of scalable subunit vaccines and complex therapeutic formulations for a wide range of biomedical applications.

Proteomimetic Polymers Trigger Potent Antigen-Specific T Cell Responses to Limit Tumor Growth.

Elicitation of effective antitumor immunity following cancer vaccination requires the selective activation of distinct effector cell populations and pathways. Here we report a therapeutic approach for generating potent T cell responses using a modular vaccination platform technology capable of inducing directed immune activation, termed the Protein-like Polymer (PLP). PLPs demonstrate increased proteolytic resistance, high uptake by antigen-presenting cells (APCs), and enhanced payload-specific T cell responses. Key design parameters, namely payload linkage chemistry, degree of polymerization, and side chain composition, were varied to optimize vaccine formulations. Linking antigens to the polymer backbone using an intracellularly cleaved disulfide bond copolymerized with a diluent amount of oligo(ethylene glycol) (OEG) resulted in the highest payload-specific potentiation of antigen immunogenicity, enhancing dendritic cell (DC) activation and antigen-specific T cell responses. Vaccination with PLPs carrying either gp100, E7, or adpgk peptides significantly increased the survival of mice inoculated with B16F10, TC-1, or MC38 tumors, respectively, without the need for adjuvants. B16F10-bearing mice immunized with gp100-carrying PLPs showed increased antitumor CD8+ T cell immunity, suppressed tumor growth, and treatment synergy when paired with two distinct stimulator of interferon gene (STING) agonists. In a human papillomavirus-associated TC-1 model, combination therapy with PLP and 2'3'-cGAMP resulted in 40% of mice completely eliminating implanted tumors while also displaying curative protection from rechallenge, consistent with conferment of lasting immunological memory. Finally, PLPs can be stored long-term in a lyophilized state and are highly tunable, underscoring the unique properties of the platform for use as generalizable cancer vaccines.

Allosteric regulation in SARS-CoV-2 spike protein.

Allosteric regulation is common in protein-protein interactions and is thus promising in drug design. Previous experimental and simulation work supported the presence of allosteric regulation in the SARS-CoV-2 spike protein. Here the route of allosteric regulation in SARS-CoV-2 spike protein is examined by all-atom explicit solvent molecular dynamics simulations, contrastive machine learning, and the Ohm approach. It was found that peptide binding to the polybasic cleavage sites, especially the one at the first subunit of the trimeric spike protein, activates the fluctuation of the spike protein's backbone, which eventually propagates to the receptor-binding domain on the third subunit that binds to ACE2. Remarkably, the allosteric regulation routes starting from the polybasic cleavage sites share a high fraction (39-67%) of the critical amino acids with the routes starting from the nitrogen-terminal domains, suggesting the presence of an allosteric regulation network in the spike protein. Our study paves the way for the rational design of allosteric antibody inhibitors.

Controlled adsorption of multiple bioactive proteins enables targeted mast cell nanotherapy.

Protein adsorption onto nanomaterials often results in denaturation and loss of bioactivity. Controlling the adsorption process to maintain the protein structure and function has potential for a range of applications. Here we report that self-assembled poly(propylene sulfone) (PPSU) nanoparticles support the controlled formation of multicomponent enzyme and antibody coatings and maintain their bioactivity. Simulations indicate that hydrophobic patches on protein surfaces induce a site-specific dipole relaxation of PPSU assemblies to non-covalently anchor the proteins without disrupting the protein hydrogen bonding or structure. As a proof of concept, a nanotherapy employing multiple mast-cell-targeted antibodies for preventing anaphylaxis is demonstrated in a humanized mouse model. PPSU nanoparticles displaying an optimized ratio of co-adsorbed anti-Siglec-6 and anti-FcεRIα antibodies effectively inhibit mast cell activation and degranulation, preventing anaphylaxis. Protein immobilization on PPSU surfaces provides a simple and rapid platform for the development of targeted protein nanomedicines.

Inhibiting the Keap1/Nrf2 Protein-Protein Interaction with Protein-Like Polymers.

Successful and selective inhibition of the cytosolic protein-protein interaction (PPI) between nuclear factor erythroid 2-related factor 2 (Nrf2) and Kelch-like ECH-associating protein 1 (Keap1) can enhance the antioxidant response, with the potential for a therapeutic effect in a range of settings including in neurodegenerative disease (ND). Small molecule inhibitors have been developed, yet many have off-target effects, or are otherwise limited by poor cellular permeability. Peptide-based strategies have also been attempted to enhance specificity, yet face challenges due to susceptibility to degradation and lack of cellular penetration. Herein, these barriers are overcome utilizing a polymer-based proteomimetics. The protein-like polymer (PLP) consists of a synthetic, lipophilic polymer backbone displaying water soluble Keap1-binding peptides on each monomer unit forming a brush polymer architecture. The PLPs are capable of engaging Keap1 and displacing the cellular protective transcription factor Nrf2, which then translocates to the nucleus, activating the antioxidant response element (ARE). PLPs exhibit increased Keap1 binding affinity by several orders of magnitude compared to free peptides, maintain serum stability, are cell-penetrant, and selectively activate the ARE pathway in cells, including in primary cortical neuronal cultures. Keap1/Nrf2-inhibitory PLPs have the potential to impact the treatment of disease states associated with dysregulation of oxidative stress, such as NDs.

Microporous water with high gas solubilities.

Liquids with permanent microporosity can absorb larger quantities of gas molecules than conventional solvents1, providing new opportunities for liquid-phase gas storage, transport and reactivity. Current approaches to designing porous liquids rely on sterically bulky solvent molecules or surface ligands and, thus, are not amenable to many important solvents, including water2-4. Here we report a generalizable thermodynamic strategy to preserve permanent microporosity and impart high gas solubilities to liquid water. Specifically, we show how the external and internal surface chemistry of microporous zeolite and metal-organic framework (MOF) nanocrystals can be tailored to promote the formation of stable dispersions in water while maintaining dry networks of micropores that are accessible to gas molecules. As a result of their permanent microporosity, these aqueous fluids can concentrate gases, including oxygen (O2) and carbon dioxide (CO2), to much higher densities than are found in typical aqueous environments. When these fluids are oxygenated, record-high capacities of O2 can be delivered to hypoxic red blood cells, highlighting one potential application of this new class of microporous liquids for physiological gas transport.

Acid-Base Equilibrium and Dielectric Environment Regulate Charge in Supramolecular Nanofibers.

Peptide amphiphiles are a class of molecules that can self-assemble into a variety of supramolecular structures, including high-aspect-ratio nanofibers. It is challenging to model and predict the charges in these supramolecular nanofibers because the ionization state of the peptides are not fixed but liable to change due to the acid-base equilibrium that is coupled to the structural organization of the peptide amphiphile molecules. Here, we have developed a theoretical model to describe and predict the amount of charge found on self-assembled peptide amphiphiles as a function of pH and ion concentration. In particular, we computed the amount of charge of peptide amphiphiles nanofibers with the sequence C 16 - V 2 A 2 E 2. In our theoretical formulation, we consider charge regulation of the carboxylic acid groups, which involves the acid-base chemical equilibrium of the glutamic acid residues and the possibility of ion condensation. The charge regulation is coupled with the local dielectric environment by allowing for a varying dielectric constant that also includes a position-dependent electrostatic solvation energy for the charged species. We find that the charges on the glutamic acid residues of the peptide amphiphile nanofiber are much lower than the same functional group in aqueous solution. There is a strong coupling between the charging via the acid-base equilibrium and the local dielectric environment. Our model predicts a much lower degree of deprotonation for a position-dependent relative dielectric constant compared to a constant dielectric background. Furthermore, the shape and size of the electrostatic potential as well as the counterion distribution are quantitatively and qualitatively different. These results indicate that an accurate model of peptide amphiphile self-assembly must take into account charge regulation of acidic groups through acid-base equilibria and ion condensation, as well as coupling to the local dielectric environment.

Functional enzyme-polymer complexes.

SignificanceThe use of biological enzyme catalysts could have huge ramifications for chemical industries. However, these enzymes are often inactive in nonbiological conditions, such as high temperatures, present in industrial settings. Here, we show that the enzyme PETase (polyethylene terephthalate [PET]), with potential application in plastic recycling, is stabilized at elevated temperature through complexation with random copolymers. We demonstrate this through simulations and experiments on different types of substrates. Our simulations also provide strategies for designing more enzymatically active complexes by altering polymer composition and enzyme charge distribution.

Specific Ion Effects in Lanthanide-Amphiphile Structures at the Air-Water Interface and Their Implications for Selective Separation.

The use of surfactants to attract dissolved ions to water surfaces and interfaces is an essential step in both solvent-based and solvent-free separation processes. We have studied the interactions of lanthanide ions in the aqueous subphase with monolayers of dihexadecyl phosphate at air-water interfaces. With heavier lanthanides (atomic number Z ≥ 65) in the subphase, the floating layer can be compressed to an area/molecule of about half the molecular cross section, indicating bilayer formation. X-ray fluorescence and reflectivity data support this conclusion. In the presence of lighter lanthanides (Z < 65), only monolayers are observed. Subphase-concentration-dependent studies using Er3+ (heavier) and Nd3+ (lighter) lanthanides show a stepwise progression, with ions attaching to the monolayer only when the solution concentration is >3 × 10-7 M. Above ∼10-5 M, bilayers form but only in the presence of the heavier lanthanide. Grazing incidence X-ray diffraction shows evidence of lateral ion-ion correlations in the bilayer structure but not in monolayers. Explicit solvent all-atom molecular dynamics simulations confirm the elevated ion-ion correlation in the bilayer system. This bilayer structure isolates heavier lanthanides but not lighter lanthanides from an aqueous solution and is therefore a potential mechanism for the selective separation of heavier lanthanides.

Origin of Proteolytic Stability of Peptide-Brush Polymers as Globular Proteomimetics.

Peptide-brush polymers (PBPs), wherein every side-chain of the polymers is peptidic, represent a new class of proteomimetic with unusually high proteolytic resistance while maintaining bioactivity. Here, we sought to determine the origin of this behavior and to assess its generality via a combined theory and experimental approach. A series of PBPs with various polymer backbone structures were prepared and examined for their proteolytic stability and bioactivity. We discovered that an increase in the hydrophobicity of the polymer backbones is predictive of an elevation in proteolytic stability of the side-chain peptides. Computer simulations, together with small-angle X-ray scattering (SAXS) analysis, revealed globular morphologies for these polymers, in which pendant peptides condense around hydrophobic synthetic polymer backbones driven by the hydrophobic effect. As the hydrophobicity of the polymer backbones increases, the extent of solvent exposure of peptide cleavage sites decreases, reducing their accessibility to proteolytic enzymes. This study provides insight into the important factors driving PBP aqueous-phase structures to behave as globular, synthetic polymer-based proteomimetics.

Origins of Clustering of Metalate-Extractant Complexes in Liquid-Liquid Extraction.

Effective and energy-efficient separation of precious and rare metals is very important for a variety of advanced technologies. Liquid-liquid extraction (LLE) is a relatively less energy intensive separation technique, widely used in separation of lanthanides, actinides, and platinum group metals (PGMs). In LLE, the distribution of an ion between an aqueous phase and an organic phase is determined by enthalpic (coordination interactions) and entropic (fluid reorganization) contributions. The molecular scale details of these contributions are not well understood. Preferential extraction of an ion from the aqueous phase is usually correlated with the resulting fluid organization in the organic phase, as the longer-range organization increases with metal loading. However, it is difficult to determine the extent to which organic phase fluid organization causes, or is caused by, metal loading. In this study, we demonstrate that two systems with the same metal loading may impart very different organic phase organizations and investigate the underlying molecular scale mechanism. Small-angle X-ray scattering shows that the structure of a quaternary ammonium extractant solution in toluene is affected differently by the extraction of two metalates (octahedral PtCl62- and square-planar PdCl42-), although both are completely transferred into the organic phase. The aggregates formed by the metalate-extractant complexes (approximated as reverse micelles) exhibit a more long-range order (clustering) with PtCl62- compared to that with PdCl42-. Vibrational sum frequency generation spectroscopy and complementary atomistic molecular dynamics simulations on model Langmuir monolayers indicate that the two metalates affect the interfacial hydration structures differently. Furthermore, the interfacial hydration is correlated with water extraction into the organic phase. These results support a strong relationship between the organic phase organizational structure and the different local hydration present within the aggregates of metalate-extractant complexes, which is independent of metalate concentration.

Insights into the Enhanced Catalytic Activity of Cytochrome c When Encapsulated in a Metal-Organic Framework.

The encapsulation of enzymes within porous materials has shown great promise, not only in protecting the enzymes from denaturation under nonbiological environments, but also, in some cases, in facilitating their enzymatic reaction rates at favorable reaction conditions. While a number of hypotheses have been developed to explain this phenomenon, the detailed structural changes of the enzymes upon encapsulation within the porous material, which are closely related to their activity, remain largely elusive. Herein, the structural change of cytochrome c (Cyt c) upon encapsulation within a hierarchical metal-organic framework, NU-1000, is investigated through a combination of experimental and computational methods, such as electron paramagnetic resonance, solid-state ultraviolet-visible spectroscopy, and all-atom explicit solvent molecular dynamics simulations. The enhanced catalytic performance of Cyt c after being encapsulated within NU-1000 is supported by the physical and in silico observations of a change around the heme ferric active center.

Homopolymer self-assembly of poly(propylene sulfone) hydrogels via dynamic noncovalent sulfone-sulfone bonding.

Natural biomolecules such as peptides and DNA can dynamically self-organize into diverse hierarchical structures. Mimicry of this homopolymer self-assembly using synthetic systems has remained limited but would be advantageous for the design of adaptive bio/nanomaterials. Here, we report both experiments and simulations on the dynamic network self-assembly and subsequent collapse of the synthetic homopolymer poly(propylene sulfone). The assembly is directed by dynamic noncovalent sulfone-sulfone bonds that are susceptible to solvent polarity. The hydration history, specified by the stepwise increase in water ratio within lower polarity water-miscible solvents like dimethylsulfoxide, controls the homopolymer assembly into crystalline frameworks or uniform nanostructured hydrogels of spherical, vesicular, or cylindrical morphologies. These electrostatic hydrogels have a high affinity for a wide range of organic solutes, achieving >95% encapsulation efficiency for hydrophilic small molecules and biologics. This system validates sulfone-sulfone bonding for dynamic self-assembly, presenting a robust platform for controllable gelation, nanofabrication, and molecular encapsulation.

Enhanced Binding of SARS-CoV-2 Spike Protein to Receptor by Distal Polybasic Cleavage Sites.

The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein plays a crucial role in binding the human cell receptor ACE2 that is required for viral entry. Many studies have been conducted to target the structures of RBD-ACE2 binding and to design RBD-targeting vaccines and drugs. Nevertheless, mutations distal from the SARS-CoV-2 RBD also impact its transmissibility and antibody can target non-RBD regions, suggesting the incomplete role of the RBD region in the spike protein-ACE2 binding. Here, in order to elucidate distant binding mechanisms, we analyze complexes of ACE2 with the wild-type spike protein and with key mutants via large-scale all-atom explicit solvent molecular dynamics simulations. We find that though distributed approximately 10 nm away from the RBD, the SARS-CoV-2 polybasic cleavage sites enhance, via electrostatic interactions and hydration, the RBD-ACE2 binding affinity. A negatively charged tetrapeptide (GluGluLeuGlu) is then designed to neutralize the positively charged arginine on the polybasic cleavage sites. We find that the tetrapeptide GluGluLeuGlu binds to one of the three polybasic cleavage sites of the SARS-CoV-2 spike protein lessening by 34% the RBD-ACE2 binding strength. This significant binding energy reduction demonstrates the feasibility to neutralize RBD-ACE2 binding by targeting this specific polybasic cleavage site. Our work enhances understanding of the binding mechanism of SARS-CoV-2 to ACE2, which may aid the design of therapeutics for COVID-19 infection.

Protein Surface Printer for Exploring Protein Domains.

The surface of proteins is vital in determining protein functions. Herein, a program, Protein Surface Printer (PSP), is built that performs multiple functions in quantifying protein surface domains. Two proteins, PETase and cytochrome P450, are used to validate that the program supports atomistic simulations with different combinations of programs and force fields. A case study is conducted on the structural analysis of the spike proteins of SARS-CoV-2 and SARS-CoV and the human cell receptor ACE2. Although the surface domains of both spike proteins are highly similar, their receptor-binding domains (RBDs) and the O-linked glycan domains are structurally different. The O-linked glycan domain of SARS-CoV-2 is highly positively charged, which may promote binding to negatively charged human cells.

PDADMAC/PSS Oligoelectrolyte Multilayers: Internal Structure and Hydration Properties at Early Growth Stages from Atomistic Simulations.

We analyze the internal structure and hydration properties of poly(diallyl dimethyl ammonium chloride)/poly(styrene sulfonate sodium salt) oligoelectrolyte multilayers at early stages of their layer-by-layer growth process. Our study is based on large-scale molecular dynamics simulations with atomistic resolution that we presented recently [Sánchez et al., Soft Matter 2019, 15, 9437], in which we produced the first four deposition cycles of a multilayer obtained by alternate exposure of a flat silica substrate to aqueous electrolyte solutions of such polymers at 0.1M of NaCl. In contrast to any previous work, here we perform a local structural analysis that allows us to determine the dependence of the multilayer properties on the distance to the substrate. We prove that the large accumulation of water and ions next to the substrate observed in previous overall measurements actually decreases the degree of intrinsic charge compensation, but this remains as the main mechanism within the interface region. We show that the range of influence of the substrate reaches approximately 3 nm, whereas the structure of the outer region is rather independent from the position. This detailed characterization is essential for the development of accurate mesoscale models able to reach length and time scales of technological interest.

Single-chain heteropolymers transport protons selectively and rapidly.

Precise protein sequencing and folding are believed to generate the structure and chemical diversity of natural channels1,2, both of which are essential to synthetically achieve proton transport performance comparable to that seen in natural systems. Geometrically defined channels have been fabricated using peptides, DNAs, carbon nanotubes, sequence-defined polymers and organic frameworks3-13. However, none of these channels rivals the performance observed in their natural counterparts. Here we show that without forming an atomically structured channel, four-monomer-based random heteropolymers (RHPs)14 can mimic membrane proteins and exhibit selective proton transport across lipid bilayers at a rate similar to those of natural proton channels. Statistical control over the monomer distribution in an RHP leads to segmental heterogeneity in hydrophobicity, which facilitates the insertion of single RHPs into the lipid bilayers. It also results in bilayer-spanning segments containing polar monomers that promote the formation of hydrogen-bonded chains15,16 for proton transport. Our study demonstrates the importance of the adaptability that is enabled by statistical similarity among RHP chains and of the modularity provided by the chemical diversity of monomers, to achieve uniform behaviour in heterogeneous systems. Our results also validate statistical randomness as an unexplored approach to realize protein-like behaviour at the single-polymer-chain level in a predictable manner.

Atomistic simulation of PDADMAC/PSS oligoelectrolyte multilayers: overall comparison of tri- and tetra-layer systems.

By employing large-scale molecular dynamics simulations of atomistically resolved oligoelectrolytes in aqueous solutions, we study in detail the first four layer-by-layer deposition cycles of an oligoelectrolyte multilayer made of poly(diallyl dimethyl ammonium chloride)/poly(styrene sulfonate sodium salt) (PDADMAC/PSS). The multilayers are grown on a silica substrate in 0.1 M NaCl electrolyte solutions and the swollen structures are then subsequently exposed to varying added salt concentration. We investigated the microscopic properties of the films, analyzing in detail the differences between three- and four-layer systems. Our simulations provide insights into the early stages of growth of a multilayer, which are particularly challenging for experimental observations. We found rather strong complexation of the oligoelectrolytes, with fuzzy layering of the film structure. The main charge compensation mechanism is for all cases intrinsic, whereas extrinsic compensation is relatively enhanced for the layer of the last deposition cycle. In addition, we quantified other fundamental observables of these systems, such as the film thickness, water uptake, and overcharge fractions for each deposition layer.

Water follows polar and nonpolar protein surface domains.

The conformation of water around proteins is of paramount importance, as it determines protein interactions. Although the average water properties around the surface of proteins have been provided experimentally and computationally, protein surfaces are highly heterogeneous. Therefore, it is crucial to determine the correlations of water to the local distributions of polar and nonpolar protein surface domains to understand functions such as aggregation, mutations, and delivery. By using atomistic simulations, we investigate the orientation and dynamics of water molecules next to 4 types of protein surface domains: negatively charged, positively charged, and charge-neutral polar and nonpolar amino acids. The negatively charged amino acids orient around 98% of the neighboring water dipoles toward the protein surface, and such correlation persists up to around 16 Å from the protein surface. The positively charged amino acids orient around 94% of the nearest water dipoles against the protein surface, and the correlation persists up to around 12 Å. The charge-neutral polar and nonpolar amino acids are also orienting the water neighbors in a quantitatively weaker manner. A similar trend was observed in the residence time of the nearest water neighbors. These findings hold true for 3 technically important enzymes (PETase, cytochrome P450, and organophosphorus hydrolase). Our results demonstrate that the water-amino acid degree of correlation follows the same trend as the amino acid contribution in proteins solubility, namely, the negatively charged amino acids are the most beneficial for protein solubility, then the positively charged amino acids, and finally the charge-neutral amino acids.