Artificial Chaperone Research Articles

Green Synthesis of Quinoline-Based Ionic Liquid.

The ever-growing menace of Antimicrobial Resistance (AMR) jeopardizes the potency of the prevailing antibiotics against the relentlessly sprouting infections spawned by bacteria, viruses, parasites as well as fungi, posing a great threat to human health and well-being. In this regard, several novel molecules have proved their mettle, with Ionic Liquids (ILs) being one of the most eco-friendly, non-volatile, and thermally stable alternatives to the existing antimicrobials, possessing high solvating potential as well as low vapor pressure. Moreover, the utilization of these entities in both stabilizing as well as destabilizing protein structures and enhancing enzymatic activity has further raised their potential in the biomedical industry. With this in view, we present the green synthesis and characterization of quinoline-based IL, owing to its immense antimicrobial potency, with low cytotoxicity and great artificial chaperone activity. Here, maneuvering the one-pot synthesis approach in solvent-free, greener reaction conditions not only ameliorated the reaction efficiency but also augmented the chemical yield. The purity of the synthesized IL was corroborated using 1H Nuclear Magnetic Resonance (NMR), 13C NMR, and Infrared (IR) spectroscopy. The biological potential of the synthesized compound is further validated by analyzing its Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) properties and authenticated using disc diffusion assay.

Engineered polymer nanoparticles as artificial chaperones facilitating the selective refolding of denatured enzymes

Molecular chaperones assist in protein refolding by selectively binding to proteins in their nonnative states. Despite progress in creating artificial chaperones, these designs often have a limited range of substrates they can work with. In this paper, we present molecularly imprinted flexible polymer nanoparticles (nanoMIPs) designed as customizable biomimetic chaperones. We used model proteins such as cytochrome c, laccase, and lipase to screen polymeric monomers and identify the most effective formulations, offering tunable charge and hydrophobic properties. Utilizing a dispersed phase imprinting approach, we employed magnetic beads modified with destabilized whole-protein as solid-phase templates. This process involves medium exchange facilitated by magnetic pulldowns, resulting in the synthesis of nanoMIPs featuring imprinted sites that effectively mimic chaperone cavities. These nanoMIPs were able to selectively refold denatured enzymes, achieving up to 86.7% recovery of their activity, significantly outperforming control samples. Mechanistic studies confirmed that nanoMIPs preferentially bind denatured rather than native enzymes, mimicking natural chaperone interactions. Multifaceted analyses support the functionality of nanoMIPs, which emulate the protective roles of chaperones by selectively engaging with denatured proteins to inhibit aggregation and facilitate refolding. This approach shows promise for widespread use in protein recovery within biocatalysis and biomedicine.

An artificial chaperone serves a dual role in regulating the assembly of peptides through phase separation

AbstractIn biological systems, molecular assembly primarily relies on the assistance of molecular chaperones. Inspired by nature, strategies like ‘chaperone‐assisted assembly’ and ‘catalyzed assembly’ have been proposed for the sophisticated control of molecular assembly. Nonetheless, significant challenges remain in the rational design of such systems, calling for a deep understanding of underlying principles. Herein, we demonstrate an artificial chaperone serves a dual role, that is catalyst in low dosages and inhibitor in high dosages, in regulating the supramolecular polymerization of peptides. Low dosages of carboxymethyl cellulose, as the chaperones, catalyze the assembly of Aβ16‐22 peptides into fibrils through multi‐step phase separation, while high dosages trap the peptides into coacervate intermediates and therefore inhibit the fibrillation. Consequently, the quantity of chaperones does not follow the intuition that ‘more is better’ for catalyzing assembly but instead has an optimal molar ratio. Investigation reveals that the interplay and evolution of electrostatic and hydrophobic interactions are the keys to achieving these processes. This study provides insights into the multifaceted roles artificial chaperones may play in a dosage‐dependent manner and enriches the toolkit for efficient and controllable construction of complex assembly systems.

Exploiting cyclodextrins as artificial chaperones to enhance enzyme protection through supramolecular engineering.

We report a method of enzyme stabilisation exploiting the artificial protein chaperone properties of β-cyclodextrin (β-CD) covalently embedded in an ultrathin organosilica layer. Putative interaction points of this artificial chaperone system with the surface of the selected enzyme were studied in silico using a protein energy landscape exploration simulation algorithm. We show that this enzyme shielding method allows for drastic enhancement of enzyme stability under thermal and chemical stress conditions, along with broadening the optimal temperature range of the biocatalyst. The presence of the β-CD macrocycle within the protective layer supports protein refolding after treatment with a surfactant.

Tuning Phase Transition of Molecular Self-Assembly by Artificial Chaperones through Aromatic-Aromatic Interactions.

The molecular chaperones are essential and play significant roles in controlling the protein phase transition and maintaining physiological homeostasis. However, manipulating phase transformation in biomimetic peptide self-assembly is still challenging. This work shows that an artificial chaperone modulates the energy landscape of supramolecular polymerization, thus controlling the phase transition of amyloid-like assemblies from crystals to hydrogels to solution. The absence of a chaperone allows the NapP to form crystals, while the presence of the chaperone biases the pathway to form nanofibrous hydrogels to soluble oligomers by adjusting the chaperone ratios. Mechanistic studies reveal that the aromatic-aromatic interaction is the key to trapping the molecules in a higher energy fold. Adding the chaperone relieves this restriction, lowers the energy barrier, and transforms the crystal into a hydrogel. This phase transformation can also be achieved in the macromolecular crowding environment, thus providing new insights into understanding molecular self-assembly in multiple component systems.

Nano-Osmolyte Conjugation: Tailoring the Osmolyte-Protein Interactions at the Nanoscale.

Osmolytes are small organic compounds accumulated at higher concentrations in the cell under various stress conditions like high temperature, high salt, high pressure, etc. Osmolytes mainly include four major classes of compounds including sugars, polyols, methylamines, and amino acids and their derivatives. In addition to their ability to maintain protein stability and folding, these osmolytes, also termed as chemical chaperones, can prevent protein misfolding and aggregation. Although being efficient protein folders and stabilizers, these osmolytes exhibit certain unavoidable limitations such as nearly molar concentrations of osmolytes being required for their effect, which is quite difficult to achieve inside a cell or in the extracellular matrix due to nonspecificity and limited permeability of the blood-brain barrier system and reduced bioavailability. These limitations can be overcome to a certain extent by using smart delivery platforms for the targeted delivery of osmolytes to the site of action. In this context, osmolyte-functionalized nanoparticles, termed nano-osmolytes, enhance the protein stabilization and chaperone efficiency of osmolytes up to 105 times in certain cases. For example, sugars, polyols, and amino acid functionalized based nano-osmolytes have shown tremendous potential in preventing protein aggregation. The enhanced potential of nano-osmolytes can be attributed to their high specificity at low concentrations, high tunability, amphiphilicity, multivalent complex formation, and efficient drug delivery system. Keeping in view the promising potential of nano-osmolytes conjugation in tailoring the osmolyte-protein interactions, as compared to their molecular forms, the present review summarizes the recent advancements of the nano-osmolytes that enhance the protein stability/folding efficiency and ability to act as artificial chaperones with increased potential to prevent protein misfolding disorders. Some of the potential nano-osmolyte aggregation inhibitors have been highlighted for large-scale screening with future applications in aggregation disorders. The synthesis of nano-osmolytes by numerous approaches and future perspectives are also highlighted.

Malachite green-derivatized cationic comb-type copolymer acts as a photoresponsive artificial chaperone

Molecular chaperones play vital roles in various physiological reactions by regulating the folding and assembly of biomacromolecules. We have demonstrated that cationic comb-type copolymers exhibit chaperone activity for anionic biomolecules including DNA and ionic peptide via the formation of soluble interpolyelectrolyte complexes. The development of smart artificial chaperones that can be spatiotemporally controlled by a remotely guided signal would expand the functions of artificial chaperones. Herein, to enable photocontrol of chaperone activity, a cationic comb-type copolymer bearing malachite green as a photoresponsive unit was designed. We first prepared a series of carboxylic acid derivatives of malachite green identified a derivative that could be quickly and quantitatively converted to the cationic form from the nonionic form by photoirradiation. This derivative was conjugated to the cationic comb-type copolymer, poly(allylamine)-graft-poly(ethylene glycol) through a condensation reaction. Upon photoirradiation, the copolymer bearing 9 mol% malachite green enhanced the membrane disruptive activity of acidic peptide E5 and induced morphological changes in liposomes. This demonstration of photoresponsive activation of chaperoning activity of a copolymer suggests that the installation of carboxyl derivatives of malachite green will impart photoresponsiveness to various materials including biopolymers.

Gold Nanoclusters Enhance the Efficacy of the Polymer-Based Chaperone in Restoring and Maintaining the Native Conformation of Human Islet Amyloid Polypeptide.

The misfolding and un-natural fibrillation of proteins/peptides are associated with many conformation diseases, such as human islet amyloid polypeptide (hIAPP) in type 2 diabetes (T2D). Inspired by molecular chaperones maintaining protein homeostasis in vivo, many polymer-based artificial chaperones were introduced to regulate protein/peptide folding and fibrillation. However, the pure polymer chaperones prefer to agglomerate into large-size micelles in the physiological environment and thus lose their chaperone functions, which greatly restricts the application of polymer-based chaperones. Here, we designed and prepared a core-shell artificial chaperone based on a dozen poly-(N-isopropylacrylamide-co-N-acryloyl-O-methylated-l-arginine) (PNAMR) anchored on a gold-nanocluster (AuNC) core. The introduction of the AuNC core significantly reduced the size and enhanced the efficacy and stability of polymer-based artificial chaperones. The PNAMR@AuNCs, with a diameter of 2.5 ± 0.5 nm, demonstrated exceptional ability in maintaining the natively unfolded conformation of protein away from the misfolding and the following fibrillation by directly binding to the natively unfolded monomolecular hIAPP and hence in preventing their conversion into toxic oligomers. More excitingly, the PNAMR@AuNCs were able to restore the natural unfolded conformation of hIAPP via dissolving the β-sheet-rich hIAPP fibrils. Considering the uniform molecular mechanism of protein misfolding and fibrillation in conformation disorders, this finding provides a generic therapeutic strategy for neurodegenerative diseases and other conformation diseases by using PNAMR@AuNC artificial chaperones to restore and maintain the native conformation of amyloid proteins.

Bifunctional cysteine gold nanoclusters for β-amyloid fibril inhibition and fluorescence imaging: a distinctive approach to manage Alzheimer's disease.

Alzheimer's disease (AD) is a progressive complex neurodegenerative disorder affecting millions of individuals worldwide. Currently, there is no effective treatment for AD. AD is characterized by the deposition of amyloid plaques/fibrils. One major strategy for managing this disease is by slowing the progression of AD using different drugs which could potentially limit free-radical formation, oxidative stress and lipid peroxidation and promote the survival of neurons exposed to β-amyloid. Inhibition of amyloid fibrillization and clearance of amyloid plaques/fibrils are essential for the prevention and treatment of AD. The thiophilic interaction between the side chain of an aromatic residue in a polypeptide and a sulphur atom of the compound can effectively inhibit amyloid fibril formation. In this work, we have synthesized cysteine-capped gold nanoclusters (Cy-AuNCs) which exhibit inherent red emission and can disintegrate amyloid fibrils through the aforementioned thiophilic interactions. Herein, we also used molecular docking to study the thiophilic interactions between the sulphur atom of Cy-AuNCs and the aromatic rings of the protein. Finally, the gold cluster was functionalized with a brain targeting molecule, Levodopa (AuCs-LD), to specifically target the brain and to facilitate passage through the blood brain barrier (BBB). Both Cy-AuNCs and AuCs-LD showed good biocompatibility and the inherent fluorescence properties of nanoclusters enabled real time imaging. The efficacy of the nanoclusters to disintegrate amyloid fibrils and their ability to cross the BBB were demonstrated both in vitro and in vivo in the BBB model and the AD animal model respectively. Our results imply that nanoparticle-based artificial molecular chaperones may offer a promising therapeutic approach for AD.

Amphiphilic dendrons as supramolecular holdase chaperones.

The aggregation of incompletely or incorrectly folded proteins is implicated in diseases including Alzheimer's, cataracts, and other maladies. Natural systems express protein chaperones to prevent or even reverse harmful protein aggregation. Synthetic chaperone-like systems have sought to mimic the action of their biological counterparts but typically require substantial optimization and high concentrations to be functional, or lack programmability that would enable the targeting of specific protein substrates. Here we report a series of amphiphilic dendrons that undergo assembly and inhibit the aggregation of fragment 16-22 amyloid β protein (Aβ16-22). We show that monodisperse dendrons with hydrophilic tetraethylene glycol chains and a hydrophobic core based on naphthyl and benzyl ethers undergo supramolecular assembly in aqueous solutions to form sphere-like particles. The solubility of these dendrons and their assemblies is tuned by varying the relative sizes of their hydrophilic and hydrophobic regions. Two water-soluble dendrons are discovered and shown, via fluorescence experiments with rhodamine 6G, to generate a hydrophobic environment. Furthermore, we demonstrate that sub-stoichiometric concentrations of these amphiphilic dendrons stabilize Aβ16-22 peptide with respect to aggregation, mimicking the activity of holdase chaperones. Our results highlight the potential of these amphiphilic molecules as the basis for a novel approach to artificial chaperones that may address many of the challenges associated with existing synthetic chaperone mimics.

Electronic fluctuation difference between trimethylamine N-oxide and tert-butyl alcohol in water

Although small organic molecules in cells have been considered important to control the functions of proteins, their electronic fluctuation and the intermolecular interaction, which is physicochemical origin of the molecular functions, under physiological conditions, i.e., dilute aqueous solutions (0.18 mol L−1), has never been clarified due to the lack of observation methods with both accuracy and efficiency. Herein, the time evolutions of the interactions in dilute aqueous trimethylamine N-oxide (TMAO) and tert-butyl alcohol (TBA) solutions were analyzed via ab initio molecular dynamics simulations accelerated with the fragment molecular theory. It has been known that TMAO and TBA have similar structures, but opposite physiological functions to stabilize and destabilize proteins. It was clarified that TMAO induced stable polarization and charge-transfer interactions with water molecules near the hydrophilic group, and water molecules were caught even near the CH3– group. Those should affect protein stabilization. Understanding the solution dynamics will contribute to artificial chaperone design in next generation medicine.

Polyelectrolytes for Enzyme Immobilization and the Regulation of Their Properties.

In this review, we considered aspects related to the application of polyelectrolytes, primarily synthetic polyanions and polycations, to immobilize enzymes and regulate their properties. We mainly focused on the description of works in which polyelectrolytes were used to create complex and unusual systems (self-regulated enzyme–polyelectrolyte complexes, artificial chaperones, polyelectrolyte brushes, layer-by-layer immobilization and others). These works represent the field of “smart polymers”, whilst the trivial use of charged polymers as carriers for adsorption or covalent immobilization of proteins is beyond the scope of this short review. In addition, we have included a section on the molecular modeling of interactions between proteins and polyelectrolytes, as modeling the binding of proteins with a strictly defined, and already known, spatial structure, to disordered polymeric molecules has its own unique characteristics.

An Arabidopsis thaliana arabinogalactan-protein (AGP31) and several cationic AGP fragments catalyse the boron bridging of rhamnogalacturonan-II

Rhamnogalacturonan-II (RG-II) is a complex pectic domain in plant primary cell walls. In vivo, most RG-II domains are covalently dimerised via borate diester bridges, essential for correct cell-wall assembly, but the dimerisation of pure RG-II monomers by boric acid in vitro is extremely slow. Cationic ‘chaperones’ can promote dimerisation, probably by overcoming the mutual repulsion between neighbouring anionic RG-II molecules. Highly effective artificial chaperones include Pb2+ and polyhistidine, but the proposed natural chaperones remained elusive. We have now tested cationic peptide fragments of several Arabidopsis thaliana arabinogalactan-proteins (AGPs) as candidates. Fragments of AGP17, 18, 19 and 31 were effective, typically at ∼25 µg/ml (9–19 µM), promoting the boron bridging of 16–20 µM monomeric RG-II at pH 4.8 in vitro. Native AGP31 glycoprotein was also effective, and hexahistidine was moderately so. All chaperones tested interacted reversibly with RG-II and were not consumed during the reaction; thus they acted catalytically, and may constitute the first reported boron-acting enzyme activity, an RG-II borate diesterase. Many of the peptide chaperones became less effective catalysts at higher concentration, which we interpret as due to the formation of RG-II–peptide complexes with a net positive charge, as mutually repulsive as negatively charged pure RG-II molecules. The four unique AGPs studied here may serve an enzymic role in the living plant cell, acting on RG-II within Golgi cisternae and/or in the apoplast after secretion. In this way, RG-II and specific AGPs may contribute to cell-wall assembly and hence plant cell expansion and development.

Regulation by Different Types of Chaperones of Amyloid Transformation of Proteins Involved in the Development of Neurodegenerative Diseases.

The review highlights various aspects of the influence of chaperones on amyloid proteins associated with the development of neurodegenerative diseases and includes studies conducted in our laboratory. Different sections of the article are devoted to the role of chaperones in the pathological transformation of alpha-synuclein and the prion protein. Information about the interaction of the chaperonins GroE and TRiC as well as polymer-based artificial chaperones with amyloidogenic proteins is summarized. Particular attention is paid to the effect of blocking chaperones by misfolded and amyloidogenic proteins. It was noted that the accumulation of functionally inactive chaperones blocked by misfolded proteins might cause the formation of amyloid aggregates and prevent the disassembly of fibrillar structures. Moreover, the blocking of chaperones by various forms of amyloid proteins might lead to pathological changes in the vital activity of cells due to the impaired folding of newly synthesized proteins and their subsequent processing. The final section of the article discusses both the little data on the role of gut microbiota in the propagation of synucleinopathies and prion diseases and the possible involvement of the bacterial chaperone GroE in these processes.

Active Regulation of Supramolecular Chirality through Integration of CdSe/CdS Nanorods for Strong and Tunable Circular Polarized Luminescence.

Building the cooperativity in artificial self-assembling systems will synergistically reshape their properties and expand their application spectrum. Here, we show how the cooperativity between achiral CdSe/CdS nanorods (NRs) and chiral perylene diimide (PDI)-based molecules is built upon their coassembly. We demonstrate that chirality transfer from chiral molecular assemblies to CdSe/CdS NRs is enabled by the encapsulation of NRs into PDI suprascrolls through chain-chain van der Waals interactions, which in turn gives rise to markedly enhanced circularly polarized luminescence of the nanocomposites. Additionally, the circularly polarized emissive bands of the nanocomposites could be finely tuned by engineering the emissive bands of NRs. More importantly, these nanocomposites are able to invert their chirality when NRs are self-assembled into chiral superlattices under a larger amount of NRs. Detailed mechanistic studies unveil that these jammed NRs assemblies hamper the folding of two-dimensional (2D) chiral nanosheets, which consequently suppress the interlayer excitonic coupling and implement the nanocomposites with inverted chirality. Our finding exemplifies a way to invert the chirality switching from folding to unfolding of 2D supramolecular nanosheets, akin to the chirality inversion in the helix-to-superhelix transition in conventional one-dimensional (1D) supramolecular systems. We also show that the strong van der Waals interactions from aliphatic chains are crucial in achieving such chirality transfer and inversion. Overall, we demonstrate that these achiral CdS/CdSe NRs could serve as artificial molecular chaperones to aid the unfolding of supramolecular nanosheets with controlled chirality.

Chemically Designed Nanoscale Materials for Controlling Cellular Processes.

Nanoparticles are widely used in various biomedical applications as drug delivery carriers, imaging probes, single-molecule tracking/detection probes, artificial chaperones for inhibiting protein aggregation, and photodynamic therapy materials. One key parameter of these applications is the ability of the nanoparticles to enter into the cell cytoplasm, target different subcellular compartments, and control intracellular processes. This is particularly the case because nanoparticles are designed to interact with subcellular components for the required biomedical performance. However, cells are protected from their surroundings by the cell membrane, which exerts strict control over entry of foreign materials. Thus, nanoparticles need to be designed appropriately so that they can readily cross the cell membrane, target subcellular compartments, and control intracellular processes.In the past few decades there have been great advancements in understanding the principles of cellular uptake of foreign materials. In particular, it has been shown that internalization of foreign materials (small molecules, macromolecules, nanoparticles) is size-dependent: endocytotic uptake of materials requires sizes greater than 10 nm, and materials with sizes of 10-100 nm usually enter into cells by energy-dependent endocytosis via biomembrane-coated vesicles. Direct access to the cytosol is limited to very specific conditions, and endosomal escape of material appears to be the most practical approach for intracellular processing.In this Account, we describe how cellular uptake and intracellular processing of nanoscale materials can be controlled by appropriate design of size and surface chemistry. We first describe the cell membrane structure and principles of cellular uptake of foreign materials followed by their subcellular trafficking. Next, we discuss the designed surface chemistry of a 5-50 nm particle that offers preferential lipid-raft/caveolae-mediated endocytosis over clathrin-mediated endocytosis with minimum endosomal/lysosomal trafficking or energy-independent direct cell membrane translocation (without endocytosis) followed by cytosolic delivery without endosomal/lysosomal trafficking. In particular, we emphasize that the zwitterionic-lipophilic surface property of a nanoparticle offers preferential interaction with the lipid raft region of the cell membrane followed by lipid raft uptake, whereas a lower number of affinity biomolecules (<25) on the nanoparticle surface offers caveolae/lipid-raft uptake, while an arginine/guanidinium-terminated surface along with a size of <10 nm offers direct cell membrane translocation. Finally, we discuss how nanoprobes can be designed by adapting these surface chemistry and size preference principles so that they can readily enter into the cell, label different subcellular compartments, and control intracellular processes such as trafficking kinetics, exocytosis, autophagy, amyloid aggregation, and clearance of toxic amyloid aggregates. The Account ends with a Conclusions and Outlook where we discuss a vision for the development of subcellular targeting nanodrugs and imaging nanoprobes by adapting to these surface chemistry principles.

Nanoclay based study on protein stability and aggregation and its implication in human health

Protein aggregation is the major cause of several acute amyloid diseases such as Parkinson's, Huntington's, Alzheimer's, Lysozyme Systemic amyloidosis, Diabetes-II etc. While these diseases have attracted much attention but the cure is still unavailable. In the present study, Human Serum Albumin (HSA) and Human Lysozyme (HL) were used as the model proteins to investigate their aggregations. Nanoclays are hydrous silicates found in clay fraction of soil and known as natural nanomaterials. They have long been used in several applications in health-related products. In the present paper, the different types of nanoclays (MMT K-10, MMT K-30, Halloysite, Bentonite) were used to inhibit the process of HSA and HL aggregation. Aggregation experiments were evaluated using several biophysical tools such as Turbidity measurements, Intrinsic fluorescence, 1-anilino-8-naphthalene sulfonate (ANS), Thioflavin T (Th T), congo red (CR) binding assays and Circular dichroism. Results demonstrated that all the nanoclays inhibit the DTT-induced aggregation. However, bentonite and MMT K-10 were progressively intense and potent as they slowed down nucleation stage which can be perceived using several biophysical techniques. Hence, nanoclays can be used as an artificial chaperone and might provide effective treatment against several protein aggregation related disorders.

Amyloid Targeting "Artificial Chaperone" Impairs Oligomer Mediated Neuronal Damage and Mitochondrial Dysfunction Associated with Alzheimer's Disease.

Alzheimer's disease (AD) is an irreversible memory disorder associated with multiple neuropathological events including amyloid aggregation that triggers oxidative stress and mitochondrial dysfunction in humans. Herein, a new artificial chaperone, benzimidazole functionalized polyfluorene (PFBZ) is reported to efficiently sequester toxic amyloid beta (Aβ) by binding at their 'amyloidogenic domain' (Aβ16-21) with unprecedented selectivity and prevent amyloid-mediated neuronal damage in a wild-type (WT) mouse model. An accurate dose of PFBZ chaperone successfully attenuated an amyloid triggered internal hemorrhage and pyknosis in the cerebral cortex of WT mice. The structural advantage of the polymer results in an efficient Cu(II) chelation arresting a redox cycle to prevent reactive oxygen species (ROS) generation and protect mitochondria from ROS mediated damage. This was further evidenced by caspase activation and mitochondrial membrane potential (MMP) biomarkers and was complemented by brain histology and electron microscopy data which revealed that the PFBZ chaperone provided a protective coating over the amyloid surface and resists from interacting with cell membrane and prevents inducing toxicity. This conjugated polymer artificial chaperone-based nanodrug showed exceptional properties such as its multipotent and highly biocompatible nature, the first of its kind with specific amyloid (Aβ16-21) targeting behavior, bioimaging, and BBB permeability with a potential to suppress amyloid triggered neurotoxicity implicated in numerous human disorders through a rare synergistic mechanism.

Thermoresponsive Gigaporous Microspheres Facilitate the Efficient Refolding of Recombinant Nitrilase Inclusion Bodies.

In order to assist the refolding of recombinant nitrilase inclusion bodies, a series of thermoresponsive media were prepared by grafting poly(N-isopropylacrylamide-co-butyl-methacrylate) [P(NIPAM-co-BMA)] brushes onto PS microspheres with various particles and pore sizes via an atom transfer radical polymerization (ATRP) method. The effects of particle sizes, pore sizes, and brush grafting amounts of thermoresponsive microspheres on nitrilase refolding were investigated preliminarily. The results showed that the PS-P(NIPAM-co-BMA) microspheres with the medium particle size (74 μm), gigapore size (320 nm), and high grafting amount (35.6 mg/m2) were the most effective candidates. The final nitrilase activity yield could be up to 84.5% with a high initial protein concentration (1 mg/mL) at 30 °C, which was 52.5% higher than that of a simple dilution refolding method at the initial protein concentration (0.1 mg/mL). After the refolding process, the PS-P(NIPAM-co-BMA) microspheres can be easily separated by self-precipitation, and the activity yield of nitrilase still reached 74.5% after being reused for five batches. These results indicated that the thermoresponsive gigaporous medium was an ideal alternative as an artificial chaperone.

Artificial Molecular Chaperone Systems for Proteins, Nucleic Acids, and Synthetic Molecules.

Molecular chaperones play critical roles in biological functions. They are closely involved in the maintenance of cell homeostasis, proper folding of proteins and nucleic acids, and inhibition of irreversible aggregation in denatured proteins. In addition to protein production, molecular chaperone function is widely recognized as important for peptide and protein drug delivery systems. Therefore, much effort has been made in recent decades to develop chaperone-mimetic molecules that have similar structures and biological functions to natural chaperones. These artificial molecular chaperone systems have been demonstrated to facilitate proper protein and nucleic acid folding, in addition to the formation of higher-order structures of synthetic molecules. Furthermore, the functions of these artificial systems show promising clinical applications in drug delivery and biomolecule detection. This topical review focuses on recent advances in the design, construction, characterization, and potential applications of different artificial molecular systems with distinct functional roles, such as the folding of water-soluble and membrane proteins, nucleic acids, and the self-assembly of synthetic molecules. Strategies used in the construction of some artificial molecule chaperone systems for proteins (such as pairs of amphiphilic molecules or self-assembled nanogels) and their applications as biomaterials are described. Specific examples from each design strategy are also highlighted to demonstrate the mechanisms, challenges, and limitations of the different artificial molecular systems. By highlighting the many new developments that have expanded the applications of the artificial chaperones beyond protein folding, this review aims to stimulate further studies on their design and applications.