Self-assembly Of Proteins Research Articles

Elucidating the Supramolecular Interaction of Positively Supercharged Fluorescent Protein with Anionic Phthalocyanines.

Developing bioinspired materials to convert sunlight into electricity efficiently is paramount for sustainable energy production. Fluorescent proteins are promising candidates as photoactive materials due to their high fluorescence quantum yield and absorption extinction coefficients in aqueous media. However, developing artificial bioinspired photosynthetic systems requires a detailed understanding of molecular interactions and energy transfer mechanisms in the required operating conditions. Here, the supramolecular self-assembly and photophysical properties of fluorescent proteins complexed with organic dyes are investigated in aqueous media. Supercharged mGreenLantern protein, mutated to have a charge of +22, is complexed together with anionic zinc phthalocyanines having 4 or 16 carboxylate groups. The structural characterization reveals a strong electrostatic interaction between the moieties, accompanied by partial conformational distortion of the protein structure, yet without compromising the mGreenLantern chromophore integrity as suggested by the lack of emission features related to the neutral form of the chromophore. The self-assembled biohybrid shows a total quenching of protein fluorescence, in favor of an energy transfer process from the protein to the phthalocyanine, as demonstrated by fluorescence lifetime and ultrafast transient absorption measurements. These results provide insight into the rich photophysics of fluorescent protein-dye complexes, anticipating their applicability as water-based photoactive materials.

Multifunctional Self-Assembled Bio-Interfacial Layers for High-Performance Zinc Metal Anodes.

Rechargeable aqueous zinc-ion (Zn-ion) batteries are widely regarded as important candidates for next-generation energy storage systems for low-cost renewable energy storage. However, the development of Zn-ion batteries is currently facing significant challenges due to uncontrollable Zn dendrite growth and severe parasitic reactions on Zn metal anodes. Herein, we report an effective strategy to improve the performance of aqueous Zn-ion batteries by leveraging the self-assembly of bovine serum albumin (BSA) into a bilayer configuration on Zn metal anodes. BSA's hydrophilic and hydrophobic fragments form unique and intelligent ion channels, which regulate the migration of Zn ions and facilitate their desolvation process, significantly diminishing parasitic reactions on Zn anodes and leading to a uniform Zn deposition along the Zn (002) plane. Notably, the Zn||Zn symmetric cell with BSA as the electrolyte additive demonstrated a stable cycling performance for up to 2400 hours at a high current density of 10 mA cm-2. This work demonstrates the pivotal role of self-assembled protein bilayer structures in improving the durability of Zn anodes in aqueous Zn-ion batteries.

Rapid Coaggregation of Proteins Without Sequence Similarity: Possible Role of Conformational Complementarity.

Despite extensive research on the sequence-determined self-assembly of both pathogenic and nonpathogenic proteins, the question of how the sequence identity would influence the coassembly or cross-seeding of diverse proteins without distinct sequence similarity remains largely unanswered. Here, we demonstrate that the rapid coaggregation of proteins with negligible sequence similarity is fundamentally governed by preferred heteromeric interactions between their partially unfolded states via the gain of additional charge complementarity and hydrophobic interactions. The partial loss of intramolecular interactions and concurrent gain of non-native intrinsically disordered regions with sticky groups become crucial for both aggressive heteromeric primary nucleation and secondary nucleation events. The results signify the direct relevance of sequence-independent conformational cross-talk between diverse proteins to the foundational events required for the growth of biological multiprotein amyloid deposits.

Biosynthesis of plant-derived triterpenoid asiatic acid in Saccharomyces cerevisiae cell factories

Asiatic acid, a bioactive component of Centella asiatica (L.) Urban, exhibits plentiful valuable pharmacological properties. Herein, we engineered Saccharomyces cerevisiae to produce asiatic acid. Initially, asiatic acid was synthesized by expressing the Centella asiatica cytochrome P450 monooxygenases CYP714E19 and CYP716C11 in a Saccharomyces cerevisiae strain optimized for ursolic acid production. The engineered strain yielded 0.42 ± 0.01 mg/L and 0.067 ± 0.0013 mg/g dry cell weight (DCW) of asiatic acid. Subsequently, a suitable cytochrome P450 reductase was screened, and key enzymes were overexpressed to effectively convert ursolic acid to asiatic acid. Strengthening heme biosynthesis, promoting endoplasmic reticulum (ER) expansion, and enhancing the cofactor supply were implemented to improve P450 catalytic activity. Additionally, a PDZ-PDZlig-mediated protein self-assembly strategy was used to improve the efficiency of the CYP714E19 and CYP716C11 catalytic cascade. Finally, the highest production was achieved (30.09 ± 0.15 mg/L, 4.09 ± 0.01 mg/g DCW) in microbial cell factories. This work establishes a foundation for efficient production of asiatic acid.

Development of two-dimensional amyloid fibril/carboxymethyl cellulose hybrid membranes for effective adsorption of hexavalent chromium

Amyloid fibrils (AFs) are protein aggregates with highly ordered fibrillar structures and can be formed via self-assembly of proteins under appropriate conditions. Owing to the unique properties of AFs, e.g., high surface-to-volume ratio and versatile functional groups, they offer abundant binding sites for the efficient adsorption of hexavalent chromium (Cr(VI)). This study was aimed at examining the performance of two-dimensional AF-based hybrid membranes for adsorbing Cr(VI). First, AF/carboxymethyl cellulose (AF/CMC) hybrid membranes were synthesized via chemical crosslinking coupled with phase inversion, followed by investigating the synthesis mechanism using Fourier transform infrared spectroscopy. Our results revealed that the surface microstructures and mechanical properties of the hybrid membranes could be affected by the AF:CMC mass ratio of the membrane. The porosity and ζ-potential of hybrid membranes were found to be dependent on both pH value and membrane composition. Analyses of the kinetics and thermodynamic behavior of Cr(VI) adsorption on the AF/CMC hybrid membranes revealed that the initial adsorption rate and maximum adsorption capacity were determined to be ∼149.20 mg g–1 h–1 and ∼311.11 mg g–1, respectively. The Cr(VI) removal percentage of hybrid membrane with high CMC content was found to remain at >75 % after five successive adsorption-desorption cycles. Finally, the mechanism and interactions involved in the adsorption of Cr(VI) on the hybrid membrane were further investigated via thermodynamic analysis and X-ray photoelectron spectroscopy. This study provides an excellent example of harnessing AF-based membranes in water remediation, highlighting the potential of AF-based hybrid materials for wastewater treatment applications.

Heteromeric amyloid filaments of ANXA11 and TDP-43 in FTLD-TDP type C

Neurodegenerative diseases are characterized by the abnormal filamentous assembly of specific proteins in the central nervous system1. Human genetic studies have established a causal role for protein assembly in neurodegeneration2. However, the underlying molecular mechanisms remain largely unknown, which is limiting progress in developing clinical tools for these diseases. Recent advances in cryo-electron microscopy have enabled the structures of the protein filaments to be determined from the brains of patients1. All neurodegenerative diseases studied to date have been characterized by the self-assembly of proteins in homomeric amyloid filaments, including that of TAR DNA-binding protein 43 (TDP-43) in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) types A and B3,4. Here we used cryo-electron microscopy to determine filament structures from the brains of individuals with FTLD-TDP type C, one of the most common forms of sporadic FTLD-TDP. Unexpectedly, the structures revealed that a second protein, annexin A11 (ANXA11), co-assembles with TDP-43 in heteromeric amyloid filaments. The ordered filament fold is formed by TDP-43 residues G282/G284–N345 and ANXA11 residues L39–Y74 from their respective low-complexity domains. Regions of TDP-43 and ANXA11 that were previously implicated in protein–protein interactions form an extensive hydrophobic interface at the centre of the filament fold. Immunoblots of the filaments revealed that the majority of ANXA11 exists as an approximately 22 kDa N-terminal fragment lacking the annexin core domain. Immunohistochemistry of brain sections showed the colocalization of ANXA11 and TDP-43 in inclusions, redefining the histopathology of FTLD-TDP type C. This work establishes a central role for ANXA11 in FTLD-TDP type C. The unprecedented formation of heteromeric amyloid filaments in the human brain revises our understanding of amyloid assembly and may be of significance for the pathogenesis of neurodegenerative diseases.

Current research status of virus-like particle vaccine

Virus-like particles (VLPs) are self-assembled protein nanoparticles with repetitive antigen epitopes, which can stimulate immune response and do not contain viral genetic materials. VLPs has important research value and application potential in vaccine development, targeted drug delivery and bioengineering materials. In this review, the mechanism of VLPs vaccine induced immune responses is discussed. The existing VLPs expression systems are summarized. The research progress of VLPs vaccine in prevention and treatment of virus infection are summarized. This review provides general reference and guidance for the design and development of antiviral VLPs vaccine.

Collagen-targeted protein nanomicelles for the imaging of non-alcoholic steatohepatitis

In vivo molecular imaging tools hold immense potential to drive transformative breakthroughs by enabling researchers to visualize cellular and molecular interactions in real-time and/or at high resolution. These advancements will facilitate a deeper understanding of fundamental biological processes and their dysregulation in disease states. Here, we develop and characterize a self-assembling protein nanomicelle called collagen type I binding – thermoresponsive assembled protein (Col1-TRAP) that binds tightly to type I collagen in vitro with nanomolar affinity. For ex vivo visualization, Col1-TRAP is labeled with a near-infrared fluorescent dye (NIR-Col1-TRAP). Both Col1-TRAP and NIR-Col1-TRAP display approximately a 3.8-fold greater binding to type I collagen compared to TRAP when measured by surface plasmon resonance (SPR). We present a proof-of-concept study using NIR-Col1-TRAP to detect fibrotic type I collagen deposition ex vivo in the livers of mice with non-alcoholic steatohepatitis (NASH). We show that NIR-Col1-TRAP demonstrates significantly decreased plasma recirculation time as well as increased liver accumulation in the NASH mice compared to mice without disease over 4 hours. As a result, NIR-Col1-TRAP shows potential as an imaging probe for NASH with in vivo targeting performance after injection in mice. Statement of significanceDirect molecular imaging of fibrosis in NASH patients enables the diagnosis and monitoring of disease progression with greater specificity and resolution than do elastography-based methods or blood tests. In addition, protein-based imaging probes are more advantageous than alternatives due to their biodegradability and scalable biosynthesis. With the aid of computational modeling, we have designed a self-assembled protein micelle that binds to fibrillar and monomeric collagen in vitro. After the protein was labeled with near-infrared fluorescent dye, we injected the compound into mice fed on a NASH diet. NIR-Col1-TRAP clears from the serum faster in these mice compared to control mice, and accumulates significantly more in fibrotic livers.This work advances the development of targeted protein probes for in vivo fibrosis imaging.

Pore Engineering as a General Strategy to Improve Protein-Based Enzyme Nanoreactor Performance.

Enzyme nanoreactors are nanoscale compartments consisting of encapsulated enzymes and a selectively permeable barrier. Sequestration and colocalization of enzymes can increase catalytic activity, stability, and longevity, highly desirable features for many biotechnological and biomedical applications of enzyme catalysts. One promising strategy to construct enzyme nanoreactors is to repurpose protein nanocages found in nature. However, protein-based enzyme nanoreactors often exhibit decreased catalytic activity, partially caused by a mismatch of protein shell selectivity and the substrate requirements of encapsulated enzymes. No broadly applicable and modular protein-based nanoreactor platform is currently available. Here, we introduce a pore-engineered universal enzyme nanoreactor platform based on encapsulins-microbial self-assembling protein nanocompartments with programmable and selective enzyme packaging capabilities. We structurally characterize our protein shell designs via cryo-electron microscopy and highlight their polymorphic nature. Through fluorescence polarization assays, we show their improved molecular flux behavior and highlight their expanded substrate range via a number of proof-of-concept enzyme nanoreactor designs. This work lays the foundation for utilizing our encapsulin-based nanoreactor platform for diverse future biotechnological and biomedical applications.

Chemical regulation of Tau oligomers in phase separation in Alzheimer’s disease

A huge number of patients suffering from certain neurodegenerative disorder, which are collectively called as tauopathies, may exhibit pathological tau protein aggregates in their brains. This category of diseases includes Alzheimer's disease (AD). In AD, post translational modifications such as phos phorylations, glycosylations, truncations, and subsequent aggregation into oligomers, paired helical filaments (PHFs), and neurofibrillary tangles (NFTs) are closely associated with cognitive decline and neurodegeneration. As a result, tau oligomers have emerged as the primary toxic species in AD and tauo pathies. Tau oligomers are soluble, self-assembled tau proteins that are formed prior to fibrils and have been demonstrated to play a pivotal role in neuronal cell death and the induction of neurodegeneration in animal models. In this succinct review, we collate and summarize literature pertaining to tau oligomer formation and its role in Alzheimer's disease. Secondly, we explore the crucial role of zinc ions (Zn²⁺) in tau aggregation, as studies suggest that zinc induces reversible tau oligomerization and can lead to tau hyper phosphorylation. The concentration of zinc is critical, as excessive levels can promote harmful tau aggregation, while normal levels are essential for physiological functions. We also examine natural and chemical compounds that can modulate tau aggregation, and lastly, we discuss how Tau protein can undergo liquid-liquid phase separation (LLPS) in neurons, forming droplets that can later develop into toxic oligomers, which are the primary hallmark of AD. We mention some molecules, such as proteins, nucleic acids, and metal ions, that influence tau LLPS and aggregation.

Modification of rice protein and its components: Enhanced fibrils formation and improved foaming properties

Plant-derived proteins like rice protein are considered an ideal source for yielding amyloid fibrils due to sustainability and affordability. However, the inherent low solubility of rice protein often restricts its self-assembly capability under common fibrillization protocols. Hence, this study comprehensively investigated the effect of modification on the self-assembly behavior, protein structure and foaming properties of rice protein and its components. The modification facilitated the transformation of α-helix to β-sheet, promoting fibrils formation. All modified proteins, except rice protein, exhibited significantly increased zeta potential values. AFM results revealed that the modified rice protein and globulin fibrils exhibited entangled aggregation behavior, while the glutelin fibril was notably extended, with several micrometers in length. Additionally, modification promoted the aggregation of albumin and prolamin particles to form short and straight fibrils. Amino acid analysis indicated that the content difference of amino acids with different characteristics and the change of aromatic amino acid interaction caused by modification may lead to different self-assembly behavior of the same protein. Furthermore, the foaming capability and stability of most protein fibrils were significantly improved by the modification. These findings provide valuable insights into the regulation of plant-derived protein fibrillation through modification.

A Single-Component Multilayered Self-Assembling Protein Nanoparticle Vaccine Based on Extracellular Domains of Matrix Protein 2 against Both Influenza A and B.

The development of an effective and broadly protective influenza vaccine against circulating and emerging strains remains elusive. In this study, we evaluated a potentially universal influenza vaccine based on single-component self-assembling protein nanoparticles (1c-SApNPs) presenting the conserved matrix protein 2 ectodomain (M2e) from influenza A and B viruses (IAV and IBV, respectively). We previously designed a tandem antigen comprising three IAV M2e domains of human, avian/swine, and human/swine origins (termed M2ex3). The M2ex3-presenting 1c-SApNPs conferred complete protection in mice against sequential lethal challenges with H1N1 and H3N2. To broaden this protection to cover IBVs, we designed a series of antigens incorporating different arrangements of three IAV M2e domains and three copies of IBV M2e. Tandem repeats of IAV and IBV (termed influenza A-B) M2e arrayed on the I3-01v9a 60-mer 1c-SApNP, when formulated with an oil-in-water emulsion adjuvant, generated greater M2e-specific immunogenicity and protective efficacy than the soluble influenza A-B M2e trimer, indicated by higher survival rates and reduced weight loss post-challenge. Importantly, one of the influenza A-B M2e SApNP constructs elicited 100% protection against a lethal influenza A/Puerto Rico/8/1934 (H1N1) challenge in mice and 70% protection against a lethal influenza B/Florida/4/2006 (Yamagata lineage) challenge, the latter of which has not been reported in the literature to date. Our study thus provides a promising M2e-based single-component universal vaccine candidate against the two major types of influenza virus circulating in humans.

Self-Assembled Protein Micelles for Encapsulation of Docosahexaenoic Acid (DHA): The Improvement of Bioaccessibility and Lipid-Lowering Activity

Self-Assembled Protein Micelles for Encapsulation of Docosahexaenoic Acid (DHA): The Improvement of Bioaccessibility and Lipid-Lowering Activity

Integrative in vivo analysis of the ethanolamine utilization bacterial microcompartment in Escherichia coli.

Bacterial microcompartments (BMCs) are self-assembling protein megacomplexes that encapsulate metabolic pathways. Although approximately 20% of sequenced bacterial genomes contain operons encoding putative BMCs, few have been thoroughly characterized, nor any in the most studied Escherichia coli strains. We used an interdisciplinary approach to gain deep molecular and functional insights into the ethanolamine utilization (Eut) BMC system encoded by the eut operon in E. coli K-12. The eut genotype was linked with the ethanolamine utilization phenotype using deletion and overexpression mutants. The subcellular dynamics and morphology of the E. coli Eut BMCs were characterized in cellula by fluorescence microscopy and electron (cryo)microscopy. The minimal proteome reorganization required for ethanolamine utilization and the in vivo stoichiometric composition of the Eut BMC were determined by quantitative proteomics. Finally, the first flux map connecting the Eut BMC with central metabolism in cellula was obtained by genome-scale modeling and 13C-fluxomics. Our results reveal that contrary to previous suggestions, ethanolamine serves both as a nitrogen and a carbon source in E. coli K-12, while also contributing to significant metabolic overflow. Overall, this study provides a quantitative molecular and functional understanding of the BMCs involved in ethanolamine assimilation by E. coli.IMPORTANCEThe properties of bacterial microcompartments make them an ideal tool for building orthogonal network structures with minimal interactions with native metabolic and regulatory networks. However, this requires an understanding of how BMCs work natively. In this study, we combined genetic manipulation, multi-omics, modeling, and microscopy to address this issue for Eut BMCs. We show that the Eut BMC in Escherichia coli turns ethanolamine into usable carbon and nitrogen substrates to sustain growth. These results improve our understanding of compartmentalization in a widely used bacterial chassis.

Strategies and Applications for Supramolecular Protein Self-Assembly.

Supramolecular chemistry achieves higher-order molecular self-assembly through non-covalent interactions. Utilizing supramolecular methods to explore the polymorphism of proteins, the building blocks of life, from a "bottom-up" perspective is essential for constructing diverse and functional biomaterials. In recent years, significant progress has been achieved in the design strategies and functional applications of supramolecular protein self-assembly, becoming a focal point for researchers. This paper reviews classical supramolecular strategies driving protein self-assembly, including electrostatic interactions, metal coordination, hydrogen bonding, hydrophobic interactions, host-guest interactions, and other mechanisms. We discuss how these supramolecular interactions regulate protein assembly processes and highlight protein supramolecular assemblies' unique structural and functional advantages in constructing artificial photosynthetic systems, protein hydrogels, bio-delivery systems, and other functional materials. The enormous potential and significance of supramolecular protein materials are elucidated. Finally, the challenges in preparing and applying protein supramolecular assemblies are summarized, and future development directions are projected.

Catalytic Nanomaterials by Conjugation of an Artificial Heme-Peroxidase to Amyloid Fibrils.

The self-assembly of proteins and peptides into fibrillar amyloid aggregates is a highly promising route to define the next generation of functional nanomaterials. Amyloid fibrils, traditionally associated with neurodegenerative diseases, offer exceptional conformational and chemical stability and mechanical properties, and resistance to degradation. Here, we report the development of catalytic amyloid nanomaterials through the conjugation of a miniaturized artificial peroxidase (FeMC6*a) to a self-assembling amyloidogenic peptide derived from human transthyretin, TTR(105-115), whose sequence is YTIAALLSPYS. Our synthetic approach relies on fast and selective click ligation upon proper modification of both the peptide and FeMC6*a, leading to TTRLys108@FeMC6*a. Mixing unmodified TTR(105-115) with TTRLys108@FeMC6*a allowed the generation of enzyme-loaded amyloid fibrils, namely, FeMC6*a@fibrils. Catalytic studies, performed in aqueous solution at nearly neutral pH, using ABTS as a model substrate and H2O2 as the oxidizing agent revealed that the enzyme retains its catalytic activity. Moreover, the activity was found to depend on the TTRLys108@FeMC6*a/unmodified TTR(105-115) peptide ratio. In particular, those with the 2:100 ratio showed the highest activity in terms of initial rates and substrate conversion among the screened nanoconjugates and compared to the freely diffusing enzyme. Finally, the newly developed nanomaterials were integrated into a flow system based on a polyvinylidene difluoride membrane filter. Within this flow-reactor, multiple reaction cycles were performed, showcasing the reusability and stability of the catalytic amyloids over extended periods, thus offering significantly improved characteristics compared to the isolated FeMC6*a in the application to a number of practical scenarios.

Biocompatible protein-based self-assembled nanocomposite as efficient drug nanocarrier and antibacterial agent

To fulfill diverse application-specific purposes, functional nanoparticles encapsulated into protein-based self-assemblies show promising advantages includes easy preparation, high biocompatibility and multiple properties. In this study, the model protein of BSA used for multi-component self-assembly, including hydrophobic Fe3O4 nanoparticles, nano-ZnO and curcumin, was constructed through hydrophobic interactions. The obtained composites of BSA-Fe3O4&ZnO and BSA-Fe3O4&ZnO&curcumin self-assemblies were tested for antibacterial and drug delivery, respectively. It was found that the multicomponents loaded into protein assembly confer a simple yet robust synthetic method resulting in the produced nanocomposites with high stability and desirable biocompatibility. Notably, the protein self-assemblies successfully increased the antibacterial efficacy of hydrophobic ZnO nanoparticles, and promote the intracellular delivery of poorly soluble curcumin to exert significant killing effects against cancer cells. Therefore, the constructed multi-component contained protein self-assembly that enables hydrophobic interactions of compositional nanoparticles holds promising potentials for the versatile uses in biomedical aspects.

Sound-mediated nucleation and growth of amyloid fibrils

Mechanical energy, specifically in the form of ultrasound, can induce pressure variations and temperature fluctuations when applied to an aqueous media. These conditions can both positively and negatively affect protein complexes, consequently altering their stability, folding patterns, and self-assembling behavior. Despite much scientific progress, our current understanding of the effects of ultrasound on the self-assembly of amyloidogenic proteins remains limited. In the present study, we demonstrate that when the amplitude of the delivered ultrasonic energy is sufficiently low, it can induce refolding of specific motifs in protein monomers, which is sufficient for primary nucleation; this has been revealed by MD. These ultrasound-induced structural changes are initiated by pressure perturbations and are accelerated by a temperature factor. Furthermore, the prolonged action of low-amplitude ultrasound enables the elongation of amyloid protein nanofibrils directly from natively folded monomeric lysozyme protein, in a controlled manner, until it reaches a critical length. Using solution X-ray scattering, we determined that nanofibrillar assemblies, formed either under the action of sound or from natively fibrillated lysozyme, share identical structural characteristics. Thus, these results provide insights into the effects of ultrasound on fibrillar protein self-assembly and lay the foundation for the potential use of sound energy in protein chemistry.

Interfacially Self-Assembled Mutifunctional Protein Thin Films for Accelerated Wound Healing.

The design of mutifunctional protein films for large-area spatially ordered arrays of functional components holds great promise in the field of biomedical applications. Herein, interfacial electrostatic self-assembly was employed to construct a large-scale protein thin film by inducing electrostatic interactions between three bovine serum albumin (BSA)-coated nanoclusters and cetyltrimethylammonium bromide (CTAB), leading to their spontaneous organization and uniform distribution at the oil-water interface. This protein film demonstrated excellent multienzyme functions, high antibacterial activity, and pH-responsive drug release capability. Therefore, it can accelerate the wound closure process through a synergistic effect that includes reducing local blood glucose levels, regulating cellular oxidative stress, eradicating bacteria, and promoting cell proliferation.

Architecting Multicompartmentalized, Giant Vesicles with Recombinant Fusion Proteins.

We present a straightforward strategy for constructing giant, multicompartmentalized vesicles using recombinant fusion proteins. Our method leverages the self-assembly of globule-zipper-elastin-like polypeptide fusion protein complexes in aqueous conditions, eliminating the need for organic solvents and chemical conjugation. By employing the thin-film rehydration method, we have successfully encapsulated a diverse range of bioactive macromolecules and engineered organelle-like compartments─ranging from soluble proteins and coacervate droplets to vesicles─within these protein-assembled giant vesicles. This approach also facilitates the integration of water-soluble block copolymers, enhancing the structural stability and functional versatility of the vesicles. Our results suggest that these multicompartment giant protein vesicles not only mimic the complex architecture of living cells but also support biochemically distinct reactions regulated by functionally folded proteins, providing a robust model for studying cellular processes and designing microreactor systems. This work highlights the transformative potential of self-assembling recombinant fusion proteins in artificial cell design.