Engineering bio-brick protein scaffolds for organizing enzyme assemblies.

Enzyme scaffolding is an emerging approach for enhancing the catalytic efficiency of multi-enzymatic cascades by controlling their spatial organization and stoichiometry. This study introduces a novel family of engineered SCAffolding Bricks, named SCABs, utilizing the consensus tetratricopeptide repeat (CTPR) domain for organized multi-enzyme systems. Two SCAB systems are developed, one employing head-to-tail interactions with reversible covalent disulfide bonds, the other relying on non-covalent metal-driven assembly via engineered metal coordinating interfaces. Enzymes are directly fused to SCAB modules, triggering assembly in a non-reducing environment or by metal presence. A proof-of-concept with formate dehydrogenase (FDH) and L-alanine dehydrogenase (AlaDH) shows enhanced specific productivity by 3.6-fold compared to free enzymes, with the covalent stapling outperforming the metal-driven assembly. This enhancement likely stems from higher-order supramolecular assembly and improved NADH cofactor regeneration, resulting in more efficient cascades. This study underscores the potential of protein engineering to tailor scaffolds, leveraging supramolecular spatial-organizing tools, for more efficient enzymatic cascade reactions.

Diverse crystalline protein scaffolds through metal-dependent polymorphism.

As protein crystals are increasingly finding diverse applications as scaffolds, controlled crystal polymorphism presents a facile strategy to form crystalline assemblies with controllable porosity with minimal to no protein engineering. Polymorphs of consensus tetratricopeptide repeat proteins with varying porosity were obtained through co-crystallization with metal salts, exploiting the innate metal ion geometric requirements. A single structurally exposed negative amino acid cluster was responsible for metal coordination, despite the abundance of negatively charged residues. Density functional theory calculations showed that while most of the crystals were the most thermodynamically stable assemblies, some were kinetically trapped states. Thus, crystalline porosity diversity is achieved and controlled with metal coordination, opening a new scope in the application of proteins as biocompatible protein-metal-organic frameworks (POFs). In addition, metal-dependent polymorphic crystals allow direct comparison of metal coordination preferences.

High-throughput virtual search of small molecules for controlling the mechanical stability of human CD4.

Protein mechanical stability determines the function of a myriad of proteins, especially proteins from the extracellular matrix. Failure to maintain protein mechanical stability may result in diseases and disorders such as cancer, cardiomyopathies, or muscular dystrophy. Thus, developing mutation-free approaches to enhance and control the mechanical stability of proteins using pharmacology-based methods may have important implications in drug development and discovery. Here, we present the first approach that employs computational high-throughput virtual screening and molecular docking to search for small molecules in chemical libraries that function as mechano-regulators of the stability of human cluster of differentiation 4, receptor of HIV-1. Using single-molecule force spectroscopy, we prove that these small molecules can increase the mechanical stability of CD4D1D2 domains over 4-fold in addition to modifying the mechanical unfolding pathways. Our experiments demonstrate that chemical libraries are a source of mechanoactive molecules and that drug discovery approaches provide the foundation of a new type of molecular function, that is, mechano-regulation, paving the way toward mechanopharmacology.

Multiparametric modulation of magnetic transduction for biomolecular sensing in liquids.

The recent COVID19 pandemic has remarkably boosted the research on in vitro diagnosis assays to detect biomarkers in biological fluids. Specificity and sensitivity are mandatory for diagnostic kits aiming to reach clinical stages. Whilst the modulation of sensitivity can significantly improve the detection of biomarkers in liquids, this has been scarcely explored. Here, we report on the proof of concept and parametrization of a novel biosensing methodology based on the changes of AC magnetic hysteresis areas observed for magnetic nanoparticles following biomolecular recognition in liquids. Several parameters are shown to significantly modulate the transducing capacity of magnetic nanoparticles to detect analytes dispersed in saline buffer at concentrations of clinical relevance. Magnetic nanoparticles were bio-conjugated with an engineered recognition peptide as a receptor. Analytes are engineered tetratricopeptide binding domains fused to the fluorescent protein whose dimerization state allows mono- or divalent variants. Our results unveil that the number of receptors per particle, analyte valency and concentration, nanoparticle composition and concentration, and field conditions play a key role in the formation of assemblies driven by biomolecular recognition. Consequently, all these parameters modulate the nanoparticle transduction capacity. Our study provides essential insights into the potential of AC magnetometry for customizing biomarker detection in liquids.

Fibroblast-derived extracellular vesicles as trackable efficient transporters of an experimental nanodrug with fibrotic heart and lung targeting.

The discovery of extracellular vesicles (EVs) as efficient exogenous biotransporters of therapeutic agents into cells across biological membranes is an exciting emerging field. Especially the potential of EVs as targeted delivery systems for diseases with selective treatments, such as fibrosis, whose treatment causes side effects in other organs not involved in the disease. Methods: In this study, we collected embryonic fibroblast-derived EVs from two different centrifugation fractions, 10 K g and 100 K g fractions from a NIH-3T3 cell line loaded with an experimental drug. Mice with fibrotic hearts and lungs were obtained by administration of angiotensin II. We generated fluorescent EVs and bioluminescent drug to observe their accumulation by colocalization of their signals in fibrotic heart and lung. The biodistribution of the drug in various organs was obtained by detecting the Au present in the drug nanostructure. Results: The drug-loaded EVs successfully reduced fibrosis in pathological fibroblasts in vitro, and modified the biodistribution of the experimental drug, enabling it to reach the target organs in vivo. We described the pre-analytical characteristics of EVs related to physical variables, culture and harvesting conditions, crucial for their in vivo application as nanotransporters using a previously validated protein-based antifibrotic drug. The results showed the colocalization of EVs and the experimental drug in vivo and ex vivo and the efficient reduction of fibrosis in vitro. This work demonstrates that 10K-EVs and 100K-EVs derived from fibroblasts can act as effective biotransporters for targeted drug delivery to profibrotic fibroblasts, lungs, or heart. Conclusion: We observed that fibroblast-derived 10K-EVs and 100K-EVs are useful biotransporters encapsulating a new generation drug leading to a reduction of fibrosis in profibrotic fibroblasts in vitro. In addition, drug containing EVs were shown to reach fibrotic heart and lungs in vivo, enhancing free drug biodistribution.

In vitro Production of Hemin-Based Artificial Metalloenzymes.

Developing enzyme alternatives is pivotal to improving and enabling new processes in biotechnology and industry. Artificial metalloenzymes (ArMs) are combinations of protein scaffolds with metal elements, such as metal nanoclusters or metal-containing molecules with specific catalytic properties, which can be customized. Here, we engineered an ArM based on the consensus tetratricopeptide repeat (CTPR) scaffold by introducing a unique histidine residue to coordinate the hemin cofactor. Our results show that this engineered system exhibits robust peroxidase-like catalytic activity driven by the hemin. The expression of the scaffold and subsequent coordination of hemin was achieved by recombinant expression in bulk and through in vitro transcription and translation systems in water-in-oil drops. The ability to synthesize this system in emulsio paves the way to improve its properties by means of droplet microfluidic screenings, facilitating the exploration of the protein combinatorial space to discover improved or novel catalytic activities.

Engineering Proteins for PEDOT Dispersions: A New Horizon for Highly Mixed Ionic-Electronic Biocompatible Conducting Materials.

Poly (3,4-ethylenedioxythiophene) (PEDOT) doped with polystyrene sulfonate (PSS) is the most used conducting polymer from energy to biomedical applications. Despite its exceptional properties, there is a need for developing new materials that can improve some of its inherent limitations, e.g., biocompatibility. In this context, doping PEDOT is propose with a robust recombinant protein with tunable properties, the consensus tetratricopeptide repeated protein (CTPR). The doping consists of an oxidative polymerization, where the PEDOT chains are stabilized by the negative charges of the CTPR protein. CTPR proteins are evaluated with three different lengths (3, 10, and 20 identical CTPR units) and optimized varied synthetic conditions. These findings revealed higher doping rate and oxidized state of the PEDOT chains when doped with the smallest scaffold (CTPR3). These PEDOT:CTPR hybrids possess ionic and electronic conductivity. Notably, PEDOT:CTPR3 displayed an electronic conductivity of 0.016 S cm-1 , higher than any other reported protein-doped PEDOT. This result places PEDOT:CTPR3 at the level of PEDOT-biopolymer hybrids, and brings it closer in performance to PEDOT:PSS gold standard. Furthermore, PEDOT:CTPR3 dispersion is successfully optimized for inkjet printing, preserving its electroactivity properties after printing. This approach opens the door to the use of these novel hybrids for bioelectronics.

Nanozymes with versatile redox capabilities inspired in metalloenzymes.

Metalloenzymes represent exemplary systems in which an organic scaffold combines with a functional inorganic entity, resulting in excellent redox catalysts. Inspired by these natural hybrid biomolecules, biomolecular templates have garnered significant attention for the controlled synthesis of inorganic nanostructures. These nanostructures ultimately benefit from the protection and colloidal stabilization provided by the biomacromolecule. In this study, we have employed this strategy to prepare nanozymes with redox capabilities, utilizing the versatile catalytic profile of Pt-loaded nanomaterials. Thus, we have investigated protein-templated Pt-based nanoclusters of different sizes and compositions, which exhibit remarkable oxidase, catalase, and reductase-like activities. The interplay between the composition and catalytic activity highlighted the size of the nanocluster as the most prominent factor in determining the performance of the nanozymes. Additionally, we have demonstrated the use of protein-templated nanozymes as potential co-catalysts in combination with enzymes for coupled reactions, under both sequential and concurrent one-pot conditions. This study provides valuable insights into nanozyme design and its wide range of applications in the design of complex catalytic systems.

Statistical Analysis and Tokenization of Epitopes to Construct Artificial Neoepitope Libraries.

Epitopes are specific regions on an antigen's surface that the immune system recognizes. Epitopes are usually protein regions on foreign immune-stimulating entities such as viruses and bacteria, and in some cases, endogenous proteins may act as antigens. Identifying epitopes is crucial for accelerating the development of vaccines and immunotherapies. However, mapping epitopes in pathogen proteomes is challenging using conventional methods. Screening artificial neoepitope libraries against antibodies can overcome this issue. Here, we applied conventional sequence analysis and methods inspired in natural language processing to reveal specific sequence patterns in the linear epitopes deposited in the Immune Epitope Database (www.iedb.org) that can serve as building blocks for the design of universal epitope libraries. Our results reveal that amino acid frequency in annotated linear epitopes differs from that in the human proteome. Aromatic residues are overrepresented, while the presence of cysteines is practically null in epitopes. Byte pair encoding tokenization shows high frequencies of tryptophan in tokens of 5, 6, and 7 amino acids, corroborating the findings of the conventional sequence analysis. These results can be applied to reduce the diversity of linear epitope libraries by orders of magnitude.

Potential use of heat shock protein 90 as a biomarker for the diagnosis of human diseases.

INTRODUCTION: The heat shock protein 90 (Hsp90) is a protein involved in many different biological processes and especially in cell survival. Some of these functions require the participation of other biological molecules, so Hsp90 is a chaperone that takes part in many protein-protein interactions working as a critical signaling hub protein. As a member of the heat shock protein family, Hsp90 expression is regulated under certain environmental and/or stressful situations, therefore Hsp90 concentration can be monitored and linked to these effects. AREAS COVERED: This review discusses the Hsp90 expression in samples from individuals affected by different diseases (from infectious to cancer origin), and the biological consequences of these disorders, including the potential use of Hsp90 as a biomarker for the diagnosis of human diseases. EXPERT OPINION: The potential of Hsp90 as a biomarker disease has been demonstrated in several studies in relation to infectious diseases and especially cancer. However, further research in this field is still needed, mainly to validate in statistically significant clinical studies that the detection of Hsp90 protein allows the diagnosis of some cancers at an early stage and also that it can act as a biomarker for monitoring the efficacy of their therapies.

Cell-Free Production Systems in Droplet Microfluidics.

The use of cell-free production systems in droplet microfluidic devices has gained significant interest during the last decade. Encapsulating DNA replication, RNA transcription, and protein expression systems in water-in-oil drops allows for the interrogation of unique molecules and high-throughput screening of libraries of industrial and biomedical interest. Furthermore, the use of such systems in closed compartments enables the evaluation of various properties of novel synthetic or minimal cells. In this chapter, we review the latest advances in the usage of the cell-free macromolecule production toolbox in droplets, with a special emphasis on new on-chip technologies for the amplification, transcription, expression, screening, and directed evolution of biomolecules.

Engineered repeat proteins as scaffolds to assemble multi-enzyme systems for efficient cell-free biosynthesis.

Multi-enzymatic cascades with enzymes arranged in close-proximity through a protein scaffold can trigger a substrate channeling effect, allowing for efficient cofactor reuse with industrial potential. However, precise nanometric organization of enzymes challenges the design of scaffolds. In this study, we create a nanometrically organized multi-enzymatic system exploiting engineered Tetrapeptide Repeat Affinity Proteins (TRAPs) as scaffolding for biocatalysis. We genetically fuse TRAP domains and program them to selectively and orthogonally recognize peptide-tags fused to enzymes, which upon binding form spatially organized metabolomes. In addition, the scaffold encodes binding sites to selectively and reversibly sequester reaction intermediates like cofactors via electrostatic interactions, increasing their local concentration and, consequently, the catalytic efficiency. This concept is demonstrated for the biosynthesis of amino acids and amines using up to three enzymes. Scaffolded multi-enzyme systems present up to 5-fold higher specific productivity than the non-scaffolded ones. In-depth analysis suggests that channeling of NADH cofactor between the assembled enzymes enhances the overall cascade throughput and the product yield. Moreover, we immobilize this biomolecular scaffold on solid supports, creating reusable heterogeneous multi-functional biocatalysts for consecutive operational batch cycles. Our results demonstrate the potential of TRAP-scaffolding systems as spatial-organizing tools to increase the efficiency of cell-free biosynthetic pathways.

An Electroactive and Self-Assembling Bio-Ink, based on Protein-Stabilized Nanoclusters and Graphene, for the Manufacture of Fully Inkjet-Printed Paper-Based Analytical Devices.

Hundreds of new electrochemical sensors are reported in literature every year. However, only a few of them makes it to the market. Manufacturability, or rather the lack of it, is the parameter that dictates if new sensing technologies will remain forever in the laboratory in which they are conceived. Inkjet printing is a low-cost and versatile technique that can facilitate the transfer of nanomaterial-based sensors to the market. Herein, an electroactive and self-assembling inkjet-printable ink based on protein-nanomaterial composites and exfoliated graphene is reported. The consensus tetratricopeptide proteins (CTPRs), used to formulate this ink, are engineered to template and coordinate electroactive metallic nanoclusters (NCs), and to self-assemble upon drying, forming stable films. The authors demonstrate that, by incorporating graphene in the ink formulation, it is possible to dramatically improve the electrocatalytic properties of the ink, obtaining an efficient hybrid material for hydrogen peroxide (H2 O2 ) detection. Using this bio-ink, the authors manufactured disposable and environmentally sustainable electrochemical paper-based analytical devices (ePADs) to detect H2 O2 , outperforming commercial screen-printed platforms. Furthermore, it is demonstrated that oxidoreductase enzymes can be included in the formulation, to fully inkjet-print enzymatic amperometric biosensors ready to use.

Engineered flavoproteins as bioorthogonal photo-triggers for the activation of metal-based anticancer prodrugs.

A multifunctional hybrid constructed for controlling the delivery and activation of Pt anticancer agents in vitro is described herein. We employed consensus tetratricopeptide repeat protein (CTPR) for the covalent co-anchoring of riboflavin (photocatalyst) and a Pt(IV) prodrug complex. The Pt-loaded flavoprotein induced a 40% reduction in PANC-1 cell viability as a result of the photocatalytic formation of cisplatin.

Emerging Approaches to DNA Data Storage: Challenges and Prospects.

With the total amount of worldwide data skyrocketing, the global data storage demand is predicted to grow to 1.75 × 1014 GB by 2025. Traditional storage methods have difficulties keeping pace given that current storage media have a maximum density of 103 GB/mm3. As such, data production will far exceed the capacity of currently available storage methods. The costs of maintaining and transferring data, as well as the limited lifespans and significant data losses associated with current technologies also demand advanced solutions for information storage. Nature offers a powerful alternative through the storage of information that defines living organisms in unique orders of four bases (A, T, C, G) located in molecules called deoxyribonucleic acid (DNA). DNA molecules as information carriers have many advantages over traditional storage media. Their high storage density, potentially low maintenance cost, ease of synthesis, and chemical modification make them an ideal alternative for information storage. To this end, rapid progress has been made over the past decade by exploiting user-defined DNA materials to encode information. In this review, we discuss the most recent advances of DNA-based data storage with a major focus on the challenges that remain in this promising field, including the current intrinsic low speed in data writing and reading and the high cost per byte stored. Alternatively, data storage relying on DNA nanostructures (as opposed to DNA sequence) as well as on other combinations of nanomaterials and biomolecules are proposed with promising technological and economic advantages. In summarizing the advances that have been made and underlining the challenges that remain, we provide a roadmap for the ongoing research in this rapidly growing field, which will enable the development of technological solutions to the global demand for superior storage methodologies.

Engineered Protein-Driven Synthesis of Tunable Iron Oxide Nanoparticles as T1 and T2 Magnetic Resonance Imaging Contrast Agents.

Iron oxide nanoparticles (IONPs) have become one of the most promising nanomaterials for biomedical applications because of their biocompatibility and physicochemical properties. This study demonstrates the use of protein engineering as a novel approach to design scaffolds for the tunable synthesis of ultrasmall IONPs. Rationally designed proteins, containing different number of metal-coordination sites, were evaluated to control the size and the physicochemical and magnetic properties of a set of protein-stabilized IONPs (Prot-IONPs). Prot-IONPs, synthesized through an optimized coprecipitation approach, presented good T1 and T2 relaxivity values, stability, and biocompatibility, showing potential for magnetic resonance imaging (MRI) applications.

Intraparticle Kinetics Unveil Crowding and Enzyme Distribution Effects on the Performance of Cofactor-Dependent Heterogeneous Biocatalysts.

Multidimensional kinetic analysis of immobilized enzymes is essential to understand the enzyme functionality at the interface with solid materials. However, spatiotemporal kinetic characterization of heterogeneous biocatalysts on a microscopic level and under operando conditions has been rarely approached. As a case study, we selected self-sufficient heterogeneous biocatalysts where His-tagged cofactor-dependent enzymes (dehydrogenases, transaminases, and oxidases) are co-immobilized with their corresponding phosphorylated cofactors [nicotinamide adenine dinucleotide phosphate (NAD(P)H), pyridoxal phosphate (PLP), and flavin adenine dinucleotide (FAD)] on porous agarose microbeads coated with cationic polymers. These self-sufficient systems do not require the addition of exogenous cofactors to function, thus avoiding the extensive use of expensive cofactors. To comprehend the microscopic kinetics and thermodynamics of self-sufficient systems, we performed fluorescence recovery after photobleaching measurements, time-lapse fluorescence microscopy, and image analytics at both single-particle and intraparticle levels. These studies reveal a thermodynamic equilibrium that rules out the reversible interactions between the adsorbed phosphorylated cofactors and the polycations within the pores of the carriers, enabling the confined cofactors to access the active sites of the immobilized enzymes. Furthermore, this work unveils the relationship between the apparent Michaelis-Menten kinetic parameters and the enzyme density in the confined space, eliciting a negative effect of molecular crowding on the performance of some enzymes. Finally, we demonstrate that the intraparticle apparent enzyme kinetics are significantly affected by the enzyme spatial organization. Hence, multiscale characterization of immobilized enzymes serves as an instrumental tool to better understand the in operando functionality of enzymes within confined spaces.

Boosting the Photoluminescent Properties of Protein-Stabilized Gold Nanoclusters through Protein Engineering.

This work reports on the use of protein engineering as a versatile tool to rationally design metal-binding proteins for the synthesis of highly photoluminescent protein-stabilized gold nanoclusters (Prot-AuNCs). The use of a single repeat protein scaffold allowed the incorporation of a set of designed metal-binding sites to understand the effect of the metal-coordinating residues and the protein environment on the photoluminescent (PL) properties of gold nanoclusters (AuNCs). The resulting Prot-AuNCs, synthesized by two sustainable procedures, showed size-tunable color emission and outstanding PL properties. In a second stage, tryptophan (Trp) residues were introduced at specific positions to provide an electron-rich protein environment and favor energy transfer from Trps to AuNCs. This modification resulted in improved PL properties relevant for future applications in sensing, biological labeling, catalysis, and optics.

Engineered Repeat Protein Hybrids: The New Horizon for Biologic Medicines and Diagnostic Tools.

ConspectusThe last decades have witnessed unprecedented scientific breakthroughs in all the fields of knowledge, from basic sciences to translational research, resulting in the drastic improvement of the lifespan and overall quality of life. However, despite these great advances, the treatment and diagnosis of some diseases remain a challenge. Inspired by nature, scientists have been exploring biomolecules and their derivatives as novel therapeutic/diagnostic agents. Among biomolecules, proteins raise much interest due to their high versatility, biocompatibility, and biodegradability.Protein binders (binders) are proteins that bind other proteins, in certain cases, inhibiting or modulating their action. Given their therapeutic potential, binders are emerging as the next generation of biopharmaceuticals. The most well-known example of binders are antibodies, and inspired by them researchers have developed alternative binders using protein design approaches. Protein design can be based on naturally occurring proteins in which, by means of rational design or combinatorial approaches, new binding interfaces can be engineered to obtain specific functions or based on de novo proteins emerging from state-of-the-art computational methodologies.Among the novel designed proteins, a class of engineered repeat proteins, the consensus tetratricopeptide repeat (CTPR) proteins, stand out due to their stability and robustness. The CTPR unit is a helix-turn-helix motif constituted of 34 amino acids, of which only 8 are essential to ensure correct folding of the structure. The small number of conserved residues of CTPR proteins leaves plenty of freedom for functional mutations, making them a base scaffold that can be easily and reproducibly tailored to endow desired functions to the protein. For example, the introduction of metal-binding residues (e.g., histidines, cysteines) drives the coordination of metal ions and the subsequent formation of nanomaterials. Additionally, the CTPR unit can be conjugated with other peptides/proteins or repeated in tandem to encode larger CTPR proteins with superhelical structures. These properties allow for the design of both binder and nanomaterial-coordination modules as well as their combination within the same molecule, making the CTPR proteins, as we have demonstrated in several recent examples, the ideal platform to develop protein-nanomaterial hybrids. Generally, the fusion of two distinct materials exploits the best properties of each; however, in protein-nanomaterial hybrids, the fusion takes on a new dimension as new properties arise.These hybrids have ushered the use of protein-based nanomaterials as biopharmaceuticals beyond their original therapeutic scope and paved the way for their use as theranostic agents. Despite several reports of protein-stabilized nanomaterials found in the literature, these systems offer limited control in the synthesis and properties of the grown nanomaterials, as the protein acts just as a stabilizing agent with no significant functional contribution. Therefore, the rational design of protein-based nanomaterials as true theranostic agents is still incipient. In this context, CTPR proteins have emerged as promising scaffolds to hold simultaneously therapeutic and diagnostic functions through protein engineering, as it has been recently demonstrated in pioneering in vitro and in vivo examples.

Enhancing the Photocatalytic Conversion of Pt(IV) Substrates by Flavoprotein Engineering.

Our recent work demonstrates that certain flavoproteins can catalyze the redox activation of Pt(IV) prodrug complexes under light irradiation. Herein, we used site-directed mutagenesis on the mini singlet oxygen generator (mSOG) to modulate the photocatalytic activity of this flavoprotein toward two model Pt(IV) substrates. Among the prepared mutants, Q103V mSOG displayed enhanced catalytic efficiency as a result of its longer triplet excited-state lifetime. This study shows, for the first time, that protein engineering can improve the catalytic capacity of a protein toward metal-containing substrates.