In-Cell Engineering of Protein Crystals into Hybrid Solid Catalysts for Artificial Photosynthesis.

The emergence of protein-based crystalline materials offers promising opportunities in enzyme immobilization. However, the current systems used for encapsulation of protein crystals are limited to either exogenous small molecules or monomeric proteins. In this work, polyhedra crystals were used to simultaneously encapsulate the foreign enzymes FDH and the organic photocatalyst eosin Y. These hybrid protein crystals are prepared easily by cocrystallization within a cell without a requirement for complex purification processes because they spontaneously form 1 µm scale solid particles. After immobilization within protein crystals, the recombinant FDH is recyclable and thermally stable and maintains 94.4% activity compared to the free enzyme. In addition, the incorporation of eosin Y endows the solid catalyst with CO2-formate conversion activity based on a cascade reaction. This work indicates that engineering protein crystals by both in vivo and in vitro strategies will provide robust and environmentally friendly solid catalysts for artificial photosynthesis.

Single-Molecule Observation of Selenoenzyme Intermediates in a Semisynthetic Seleno-α-Hemolysin Nanoreactor.

The development of monitoring methods to capture short-lived intermediates is crucial for kinetic mechanism validation of enzymatic reaction steps. In this work, a semisynthetic selenoenzyme nanoreactor was constructed by introducing the unnatural amino acid (Sec) into the lumen of the α-hemolysin (αHL) nanopore. This nanoreactor not only created a highly confined space to trap the enzyme-substrate complex for a highly efficient antioxidant activity but also provided a single channel to characterize a series of selenoenzyme intermediates in the whole catalytic cycle through electrochemical analysis. In particular, the unstable intermediate of SeOH can be clearly detected by the characteristic blocking current. The duration time corresponding to the lifetime of each intermediate that stayed within the nanopore was also determined. This label-free approach showed a high detection sensitivity and temporal-spatial resolution to scrutinize a continuous enzymatic process, which would facilitate uncovering the mysteries of selenoenzyme catalysis at the single-molecule level.

Selenacrown Macrocycle in Aqueous Medium: Synthesis, Redox-Responsive Self-Assembly, and Enhanced Disulfide Formation Reaction.

Organic selenides are famous for their coordination and catalytic functions in the organic phase, albeit challenging for aqueous medium. Herein, the combination of a hydrophilic body of crown ether and substitution of one oxygen atom with a selenium one provides a new type of design route for organic selenide entities with charming functions in aqueous solution. The selenacrown ether C9Se presented here intrinsically shows an amphiphile-like property. Its nanosphere structure in water readily expands the catalysis of organic selenide to aqueous substrates in thiol/disulfide conversion.

Allosteric Switching of Calmodulin in a Mycobacterium smegmatis porin†A (MspA) Nanopore-Trap.

Recent developments concerning large protein nanopores suggest a new approach to structure profiling of native folded proteins. In this work, the large vestibule of Mycobacterium smegmatis porin A (MspA) and calmodulin (CaM), a Ca2+ -binding protein, were used in the direct observation of the protein structure. Three conformers, including the Ca2+ -free, Ca2+ -bound, and target peptide-bound states of CaM, were unambiguously distinguished. A disease related mutant, CaM D129G was also discriminated by MspA, revealing how a single amino acid replacement can interfere with the Ca2+ -binding capacity of the whole protein. The binding capacity and aggregation effect of CaM induced by different ions (Mg2+ /Sr2+ /Ba2+ /Ca2+ /Pb2+ /Tb3+ ) were also investigated and the stability of MspA in extreme conditions was evaluated. This work demonstrates the most systematic single-molecule investigation of different allosteric conformers of CaM, acknowledging the high sensing resolution offered by the MspA nanopore trap.

Biological Macrocycle: Supramolecular Hydrophobic Guest Transport System Based on Nanodiscs with Photodynamic Activity.

A biogenic macrocycle-based guest loading system has been developed by the self-assembly of membrane scaffold protein and phospholipids. The resulting 10 nm level transport system can increase the solubility of hydrophobic photodynamic agent hypocrellin B in aqueous medium and exhibited a cellular internalization capacity with substantial photodynamic activity.

Supramolecular Gel Based on Crown-Ether-Appended Dynamic Covalent Macrocycles.

A new type of dynamic covalent macrocycle with self-promoted supramolecular gelation behavior is developed. Under oxidative conditions, the dithiol compound containing a diamide alkyl linker with an odd number (7) of carbon chain and an appended crown ether shows a remarkable gelation ability in acetonitrile, without any template molecules. Due to the existence of crown ethers and disulfide bonds, the obtained gel shows a multiple stimuli-responsiveness behavior. The mechanical properties and reversibility of the gel are investigated. Computational modeling suggests that the peripheral chain for diamide hydrogen bonding is responsible for the gelation process.

Self-assembly behaviors of perylene- and naphthalene-crown macrocycle conjugates in aqueous medium.

The synthesis of conjugates of perylene diimide (PDI) and naphthalene diimide (NDI) modified with two benzo-21-crown-7 ethers (B21C7) are herein described. Their self-assembly behavior in various solvents was investigated particularly in aqueous medium, due to the recently discovered hydrophilic properties of B21C7 crown macrocycle. An unexpected fluorescence quenching phenomenon was observed in the PDI-B21C7 macrocycle conjugate in chloroform. The detailed UV-vis absorption and fluorescence spectra of these PDI/NDI derivatives in different solvents as well as their morphologies were investigated.

Catalysts Encapsulated in Molecular Machines.

Smart catalysts offer the control of chemical processes and sequences of transformations, and catalysts with unique catalytic behavior can afford chiral products or promote successive polymerization. To meet advanced demands, the key to constructing smart catalysts is to incorporate traditional catalytic functional groups with trigger-induced factors. Molecular machines with dynamic properties and particular topological structures have typical stimulus-responsive features. In recent years, scientists have made efforts to utilize molecular machines (molecular switches, rotaxanes, motors, etc.) as scaffolds to develop smart catalysts. This Minireview focuses on the achievements of developing catalysts encapsulated in molecular machines and their remarkable specialties. This strategy is believed to provide more potential applications in switchable reactions, asymmetric synthesis, and processive catalysis.

Robust, flexible, and bioadhesive free-standing films for the co-delivery of antibiotics and growth factors.

Free-standing polymer films that adhere strongly to tissue and can codeliver multiple therapeutic agents in a controlled manner are useful as medical plasters. In this study, a bilayer polymer film comprising a drug reservoir layer and a supporting layer is fabricated by spin-coating poly(lactic-co-glycolic acid) (PLGA) on top of a layer-by-layer assembled film of poly(ß-amino esters) (PAE), alginate sodium (ALG), and recombinant human basic fibroblast growth factor (bFGF). Apart from bFGF, the bilayer film can also load antibiotic drug ceftriaxone sodium (CTX) by a postdiffusion process. The PLGA supporting layer facilitates the direct peeling of the bilayer film from substrate to produce a robust and flexible free-standing film with excellent adhesion onto the human skin and porcine liver. The excellent adhesion of the bilayer film originates from the ALG component in the drug reservoir layer. CTX is quickly released by easily breaking its electrostatic interaction with the drug reservoir layer, whereas the sustained release of bFGF is due to the slow degradation of PAE component in the drug reservoir layer. Wounds can be synergetically treated by fast release of CTX to effectively eradicate invasive bacteria and by sustained release of bFGF to accelerate wound healing. Our results serve as a basis for designing multifunctional free-standing films with combination therapy for biomedical applications.

Reversible Ca(2+) switch of an engineered allosteric antioxidant selenoenzyme.

A Ca(2+) -responsive artificial selenoenzyme was constructed by computational design and engineering of recoverin with the active center of glutathione peroxidase (GPx). By combining the recognition capacity for the glutathione (GSH) substrate and the steric orientation of the catalytic selenium moiety, the engineered selenium-containing recoverin exhibits high GPx activity for the catalyzed reduction of H2 O2 by glutathione (GSH). Moreover, the engineered selenoenzyme can be switched on/off by Ca(2+) -induced allosterism of the protein recoverin. This artificial selenoenzyme also displays excellent antioxidant ability when it was evaluated using a mitochondrial oxidative damage model, showing great potential for controlled catalysis in biomedical applications.

A method for in situ self-assembly of the catalytic peptide in enzymatic compartments of glucan particles.

Drawing inspiration from cellular compartmentalization, enzymatic compartments play a pivotal role in bringing enzymes and substrates into confined environments, offering heightened catalytic efficiency and prolonged enzyme lifespan. Previously, we engineered bioinspired enzymatic compartments, denoted as TPE-Q18H@GPs, achieved through the spatiotemporally controllable self-assembly of the catalytic peptide TPE-Q18H within hollow porous glucan particles (GPs). This design strategy allows substrates and products to freely traverse, while retaining enzymatic aggregations. The confined environment led to the formation of catalytic nanofibers, resulting in enhanced substrate binding affinity and a more than two-fold increase in the second-order kinetic constant (kcat/Km) compared to TPE-Q18H nanofibers in a dispersed system. In this work, we will introduce how to synthesize the above-mentioned enzymatic compartments using salt-responsive catalytic peptides and GPs.

A redox-responsive macrocycle based on the crown ether C7Te for enhanced bacterial inhibition.

Due to increasing bacterial resistance to disinfectants, there is an urgent need for new therapeutic agents and strategies to effectively inhibit bacteria. Accordingly, we have designed and synthesized a novel crown ether known as C7Te, and its oxidized form C7TeO. These compounds have demonstrated antibacterial effectiveness against Gram-negative E. coli (BL21). Notably, C7Te has the capability to enhance the inhibition of E. coli and the prevention of biofilm formation by H2O2 through a redox response. It can also effectively disrupt preformed E. coli biofilms by penetrating biofilm barriers effectively. Additionally, computer modeling of the bacterial cell membrane using nanodiscs composed of phospholipids and encircled amphipathic proteins with helical belts has revealed that C7Te can insert into and interact with phospholipids and proteins. This interaction results in the disruption of the bacterial cell membrane leading to bacterial cell death. The utilization of redox-responsive crown ethers to augment the antibacterial capabilities of H2O2-based disinfectants represents a novel approach to supramolecular bacterial inhibition.

Developing Isomeric Peptides for Mimicking the Sequence-Activity Landscapes of Enzyme Evolution.

Enzymes catalyze almost all material conversion processes within living organisms, yet their natural evolution remains unobserved. Short peptides, derived from proteins and featuring active sites, have emerged as promising building blocks for constructing bioactive supramolecular materials that mimic native proteins through self-assembly. Herein, we employ histidine-containing isomeric tetrapeptides KHFF, HKFF, KFHF, HFKF, FKHF, and FHKF to craft supramolecular self-assemblies, aiming to explore the sequence-activity landscapes of enzyme evolution. Our investigations reveal the profound impact of peptide sequence variations on both assembly behavior and catalytic activity as hydrolytic simulation enzymes. During self-assembly, a delicate balance of multiple intermolecular interactions, particularly hydrogen bonding and aromatic-aromatic interactions, influences nanostructure formation, yielding various morphologies (e.g., nanofibers, nanospheres, and nanodiscs). Furthermore, the analysis of the structure-activity relationship demonstrates a strong correlation between the distribution of the His active site on the nanostructures and the formation of the catalytic microenvironment. This investigation of the sequence-structure-activity paradigm reflects how natural enzymes enhance catalytic activity by adjusting the primary structure during evolution, promoting fundamental research related to enzyme evolutionary processes.

Janus liposozyme for the modulation of redox and immune homeostasis in infected diabetic wounds.

Diabetic foot ulcers often become infected, leading to treatment complications and increased risk of loss of limb. Therapeutics to manage infection and simultaneously promote healing are needed. Here we report on the development of a Janus liposozyme that treats infections and promotes wound closure and re-epithelialization. The Janus liposozyme consists of liposome-like selenoenzymes for reactive oxygen species (ROS) scavenging to restore tissue redox and immune homeostasis. The liposozymes are used to encapsulate photosensitizers for photodynamic therapy of infections. We demonstrate application in methicillin-resistant Staphylococcus aureus-infected diabetic wounds showing high ROS levels for antibacterial function from the photosensitizer and nanozyme ROS scavenging from the liposozyme to restore redox and immune homeostasis. We demonstrate that the liposozyme can directly regulate macrophage polarization and induce a pro-regenerative response. By employing single-cell RNA sequencing, T cell-deficient Rag1-/- mice and skin-infiltrated immune cell analysis, we further reveal that IL-17-producing γδ T cells are critical for mediating M1/M2 macrophage transition. Manipulating the local immune homeostasis using the liposozyme is shown to be effective for skin wound repair and tissue regeneration in mice and mini pigs.

Feasible pickup from intact ossicular chain with floating piezoelectric microphone.

OBJECTIVES: Many microphones have been developed to meet with the implantable requirement of totally implantable cochlear implant (TICI). However, a biocompatible one without destroying the intactness of the ossicular chain still remains under investigation. Such an implantable floating piezoelectric microphone (FPM) has been manufactured and shows an efficient electroacoustic performance in vitro test at our lab. We examined whether it pick up sensitively from the intact ossicular chain and postulated whether it be an optimal implantable one. METHODS: Animal controlled experiment: five adult cats (eight ears) were sacrificed as the model to test the electroacoustic performance of the FPM. Three groups were studied: (1) the experiment group (on malleus): the FPM glued onto the handle of the malleus of the intact ossicular chains; (2) negative control group (in vivo): the FPM only hung into the tympanic cavity; (3) positive control group (Hy-M30): a HiFi commercial microphone placed close to the site of the experiment ear. The testing speaker played pure tones orderly ranged from 0.25 to 8.0 kHz. The FPM inside the ear and the HiFi microphone simultaneously picked up acoustic vibration which recorded as .wav files to analyze. RESULTS: The FPM transformed acoustic vibration sensitively and flatly as did the in vitro test across the frequencies above 2.0 kHz, whereas inefficiently below 1.0 kHz for its overloading mass. Although the HiFi microphone presented more efficiently than the FPM did, there was no significant difference at 3.0 kHz and 8.0 kHz. CONCLUSIONS: It is feasible to develop such an implantable FPM for future TICIs and TIHAs system on condition that the improvement of Micro Electromechanical System and piezoelectric ceramic material technology would be applied to reduce its weight and minimize its size.

Structure, thermal stability and electrical properties of reduced graphene/poly(vinylidene fluoride) nanocomposite films.

The reduced graphene/poly(vinylidene fluoride) nanocomposite films were prepared by the solution casting-thermal reduction process using graphene oxide (GO) and poly(vinylidene fluoride) (PVDF) resin. With the presence of reduced graphene (RG) nano sheets in the nanocomposite, the structure of PVDF is transformed from alpha to beta phase, and the beta phase fraction and its crystallinity are largely affected by the RG content. The PVDF thermal stability is improved by the RG introduction, with about 15 degrees C increase of the half-life of PVDF decomposition temperature. The RG/PVDF nanocomposites show a better electrical conductivity than that for the GO/PVDF nanocomposites. At a low RG content (0.8 wt.%), the dielectric constant of RG/PVDF nanocomposite film with a very low loss tangent is dramatically increased from about 6 to 23. The mechanisms for the thermal stability and electrical property improvements are discussed.

Rational design of allosteric switchable catalysts.

Allosteric regulation, in many cases, involves switching the activities of natural enzymes, which further affects the enzymatic network and cell signaling in the living systems. The research on the construction of allosteric switchable catalysts has attracted broad interests, aiming to control the progress and asymmetry of catalytic reactions, expand the chemical biology toolbox, substitute unstable natural enzymes in the biological detection and biosensors, and fabricate the biomimetic cascade reactions. Thus, in this review, we summarize the recent outstanding works in switchable catalysts based on the allosterism of single molecules, supramolecular complexes, and self-assemblies. The concept of allosterism was extended from natural proteins to polymers, organic molecules, and supramolecular systems. In terms of the difference between these building scaffolds, a variety of design methods that tailor biological and synthetic molecules into controllable catalysts were introduced with emphasis.

Bioinspired enzymatic compartments constructed by spatiotemporally confined in situ self-assembly of catalytic peptide.

Enzymatic compartments, inspired by cell compartmentalization, which bring enzymes and substrates together in confined environments, are of particular interest in ensuring the enhanced catalytic efficiency and increased lifetime of encapsulated enzymes. Herein, we constructed bioinspired enzymatic compartments (TPE-Q18H@GPs) with semi-permeability by spatiotemporally controllable self-assembly of catalytic peptide TPE-Q18H in hollow porous glucan particles (GPs), allowing substrates and products to pass in/out freely, while enzymatic aggregations were retained. Due to the enrichment of substrates and synergistic effect of catalytic nanofibers formed in the confined environment, the enzymatic compartments exhibited stronger substrate binding affinity and over two-fold enhancement of second-order kinetic constant (kcat/Km) compared to TPE-Q18H nanofibers in disperse system. Moreover, GPs enabled the compartments sufficient stability against perturbation conditions, such as high temperature and degradation. This work opens an intriguing avenue to construct enzymatic compartments using porous biomass materials and has fundamental implications for constructing artificial organelles and even artificial cells.

Macrocycles in Bioinspired Catalysis: From Molecules to Materials.

Macrocyclic compounds have been studied extensively as the host molecules in supramolecular chemistry. Their structural characteristics make macrocycles desirable in the field of molecular recognition, which is the key to high catalytic efficiencies of natural enzymes. Therefore, macrocycles are ideal building blocks for the design of bioinspired catalysts. This mini review highlights recent advances ranging from single-molecule to metal-organic framework materials, exhibiting multilevel macrocycle catalysts with unique catalytic centers and substrate-binding affinities.

Guanidinium-Responsive Crown Ether-Based Macrocyclic Amphiphile in Aqueous Medium.

Supramolecular assemblies based on oligo(ethylene glycol) (OEG) building blocks are well-known for their neutral chemical property and thermal-responsive behavior. Here, the cyclic "CLOSED" and linear "OPEN" typologies of OEGs led to dramatic difference in the sensitivity to guanidinium-containing species. From thermodynamic studies, the association constant (Ka) between the "CLOSED" form amphiphile and guanidinium salt was determined to be 28.7 M-1, whereas there was no detectable binding affinity for the "OPEN" form. Therefore, considering ion specificity, the present results establish that crown ether derivatives with "CLOSED" and "OPEN" topologies provide an easy-to-access model pair with designed ion-recognition sites and special functional moieties and geometries (like the binding pockets of enzymes or ion channels in cellular members) that allow the manipulation of the intercrossed relationship between supramolecular solutes, waters, and guanidinium salts. These supramolecular forces in aqueous solution offered an alternative strategy to fabricate thermal-responsive systems in ionic medium.