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Bacterial adhesins are cell‐surface proteins that anchor to the cell wall of the host. The first stage of infection involves the specific attachment to fibrinogen (Fg), a protein found in human blood. This attachment allows bacteria to colonize tissues causing diseases such as endocarditis. The study of this family of proteins is hence essential to develop new strategies to fight bacterial infections. In the case of the Gram‐positive bacterium Staphylococcus aureus, there exists a class of adhesins known as microbial surface components recognizing adhesive matrix molecules (MSCRAMMs). Here, we focus on one of them, the clumping factor A (ClfA), which has been found to bind Fg through the dock‐lock‐latch mechanism. Interestingly, it has recently been discovered that MSCRAMM proteins employ a catch‐bond to withstand forces exceeding 2 nN, making this type of interaction as mechanically strong as a covalent bond. However, it is not known whether this strength is an evolved feature characteristic of the bacterial protein or is typical only of the interaction with its partner. Here, we combine single‐molecule force spectroscopy, biophysical binding assays, and molecular simulations to study the intrinsic mechanical strength of ClfA. We find that despite the extremely high forces required to break its interactions with Fg, ClfA is not by itself particularly strong. Integrating the results from both theory and experiments we dissect contributions to the mechanical stability of this protein. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In this study, the goal is to fabricate robust and highly efficient peroxidase‐like nanozymes that can ultimately be assembled into films for their easy reuse in catalytic cycles. Nanozymes are designed by mimicking the strategy adopted by metalloproteins to accommodate metal cofactors within their protein structure. The engineered consensus tetratricopeptide repeat (CTPR) protein module is selected as the scaffold to guide the growth and the stabilization of a library of in situ synthesized metal nanoclusters. A deep investigation of the interplay between the composition and function of the nanozymes reveals the impact of the protein templates and nanocluster composition on the peroxidase‐like activity of the hybrids. Moreover, among a total of 24 hybrids, a top‐performing nanozyme results from the growth of Au/Pt bimetallic nanoclusters on a CTPR protein with engineered histidine coordination sites. These nanozymes exhibit improved thermostability and resistance to hydrogen peroxide compared to natural peroxidases like horseradish peroxidase. Finally, it shows the easy fabrication of nanozyme composite films guided throughout the intrinsic self‐assembling properties of the CTPR scaffold. These heterogeneous solid materials are reused in several reaction cycles without significant loss of the catalytic performance, proving these protein‐templated nanozymes as an advantageous alternative to natural enzymes. [ABSTRACT FROM AUTHOR]

Biophosphors with fluorescent proteins (FPs) are promising candidates to replace rare‐earth color down‐converting filters for white light‐emitting diodes (LEDs). There is, however, a lack of deep‐red FPs meeting high photostabilities, photoluminescence quantum yields (ϕ), and throughput expression yields. Herein, a new approach for the design of highly emissive and stable deep‐red biophosphors combining an artificial FP (Lactococcal multidrug resistance Regulator (LmrR) as protein host and an archetypal red‐emitting squaraine (S) as guest) with a polymer network is demonstrated toward high performing deep‐red biohybrid LEDs (Bio‐HLEDs). At first, the best protein pocket (aromaticity, polarity, charge, etc.) to stabilize S in water is determined using four LmrR variants (position 96 with tryptophan, histidine, phenylalanine, and alanine). Computational and time‐resolved spectroscopic findings suggest that the tryptophan is instrumental toward achieving artificial red‐emitting FPs with ϕ > 50% stable over weeks. These features are further enhanced in the polymer coating (ϕ > 65% stable over months) without affecting emission color. Finally, deep‐red Bio‐HLEDs are fabricated featuring external quantum efficiencies of 7% and stabilities of ≈800 h. This represents threefold enhancement compared to reference devices with S‐polymer color filters. Overall, this work highlights a new design for highly emissive deep‐red biophosphors, achieving record performance in deep‐red protein‐LEDs. [ABSTRACT FROM AUTHOR]

Gold nanoclusters (AuNCs) are nanomaterials with interesting photoluminescent properties that can be endowed with biomolecular recognition and biocompatibility when stabilized with proteins. The interplay between the optical features of AuNCs and the function added by the protein makes them perfect candidates for generating hybrid protein‐inorganic nanomaterials. Focusing on protein stabilized‐AuNCs, hitherto most of the work has covered the use of natural proteins for in situ growth of AuNCs. However, the exploitation of design proteins for such endeavors enables fine‐tuning of the photoluminescent assets of AuNCs. In this work, rational protein engineering of modular protein scaffolds is applied for capping of non‐emissive, non‐passivated naked AuNCs, resulting in a fast and easy method for the synthesis of customizable and emissive protein‐AuNC nanomaterials. Tuning of the photoluminescent properties of the final hybrid module is obtained by appropriate choice of the coordination residues grafted on the same protein scaffold. The effects of ligands and coordination bonds are studied using time‐resolved photoluminescence and X‐ray absorbance spectroscopies, shedding light on the mechanisms behind the emerging properties of these hybrid materials. Moreover, the described versatile strategy opens new avenues for the synthesis of on‐demand photoluminescent hybrids for a wide spectrum of optical applications. [ABSTRACT FROM AUTHOR]

Metal nanoclusters (NCs) are considered ideal nanomaterials for biological applications owing to their strong photoluminescence (PL), excellent photostability, and good biocompatibility. This study presents a simple and versatile strategy to design proteins, via incorporation of a di‐histidine cluster coordination site, for the sustainable synthesis and stabilization of metal NCs with different metal composition. The resulting protein‐stabilized metal NCs (Prot‐NCs) of gold, silver, and copper are highly photoluminescent and photostable, have a long shelf life, and are stable under physiological conditions. The biocompatibility of the clusters was demonstrated in cell cultures in which Prot‐NCs showed efficient cell internalization without affecting cell viability or losing luminescence. Moreover, the approach is translatable to other proteins to obtain Prot‐NCs for various biomedical applications such as cell imaging or labeling. [ABSTRACT FROM AUTHOR]

Abstract: The development of sophisticated ordered functional materials is one of the important challenges in current science. One of the keys to enhance the properties of these materials is the control of the organization and morphology at different scales. This work presents a novel bioinspired methodology to achieve highly ordered donor/acceptor bio‐nanohybrids using a designed repeat protein as scaffold, endowed with photoactive and electron donating porphyrin (P) units, to efficiently wrap around electron accepting single wall carbon nanotubes (SWCNT). A systematic experimental and theoretical study to evaluate the effect of the length of the protein reveals that longer proteins wrap around the SWCNT in a more efficient manner due to the stronger supramolecular interaction existing between the inner concave surface of the protein (namely Trp and His residues) and the convex surface of the (7,6)‐SWCNT. Interestingly, spectroscopy and X‐ray diffraction data further confirm that the so‐called protein‐P–SWCNT donor–acceptor bio‐nanohybrids retain the original protein structure. Finally, the new bio‐nanohybrids show a remarkable enhancement on the photoconductivity values by flash‐photolysis microwave conductivity (FP‐TRMC technique) demonstrating that the major charge carriers of electrons are injected into the SWCNTs and move along the 1D‐structures. [ABSTRACT FROM AUTHOR]

Abstract: Recent advances in bioorthogonal catalysis promise to deliver new chemical tools for performing chemoselective transformations in complex biological environments. Herein, we report how FAD (flavin adenine dinucleotide), FMN (flavin mononucleotide), and four flavoproteins act as unconventional photocatalysts capable of converting PtIV and RuII complexes into potentially toxic PtII or RuII−OH2 species. In the presence of electron donors and low doses of visible light, the flavoproteins mini singlet oxygen generator (miniSOG) and NADH oxidase (NOX) catalytically activate PtIV prodrugs with bioorthogonal selectivity. In the presence of NADH, NOX catalyzes PtIV activation in the dark as well, indicating for the first time that flavoenzymes may contribute to initiating the activity of PtIV chemotherapeutic agents. [ABSTRACT FROM AUTHOR]

Abstract: Nanotechnology has brought countless novel applications regarding therapeutic, diagnostic, and sensing innovations. In particular, nanoparticle engineering enables the synthesis of nanocrystals with customized physical, chemical, and biological activities, which are generally well validated in vitro. However, when nanoparticles are tested in vivo they often show a disappointing loss of efficacy or increase of toxicity. These phenomena are related to the interaction of the nanoparticle surface with proteins, lipids, and ions present in the blood stream. Herein, we assess the physicochemical properties of an array of iron oxide nanoparticles coated with 15 different biocompatible materials dispersed in physiological aqueous media and human blood plasma. Our findings establish valid criteria for the prediction of the colloidal stability of nanoparticles dispersed in physiological aqueous media and human blood plasma. The criteria are based on the hydrophilicity and the net surface charge of the pristine nanoparticle coating in water dispersion. Hence, the methodology to select the most adequate nanoparticle coating for in vivo studies will be improved. [ABSTRACT FROM AUTHOR]

Repeat proteins are tandem arrays of a small structural motif, in which tertiary structure is stabilized by interactions within a repeat and between neighboring repeats. Several studies have shown that this modular structure is manifest in modular thermodynamic properties. Specifically, the global stability of a repeat protein can be described by simple linear models, considering only two parameters: the stability of the individual repeated units ( H) and the coupling interaction between the units ( J). If the repeat units are identical, single values of H and J, together with the number of repeated units, is sufficient to completely describe the thermodynamic behavior of any protein within a series. In this work, we demonstrate how the global stability of a repeat protein can be changed, in a predictable fashion, by modifying only the H parameter. Taking a previously characterized series of consensus tetratricopeptide repeats (TPR) (CTPRa) proteins, we introduced mutations into the basic repeating unit, such that the stability of the individual repeat unit was increased, but its interaction with neighboring units was unchanged. In other words, we increased H but kept J constant. We demonstrated that the denaturation curves for a series of such repeat proteins can be fit and additional curves can be predicted by the one-dimensional Ising model in which only H has changed from the original fit for the CTPRa series. Our results show that we can significantly increase the stability of a repeat protein by rationally increasing the stability of the units ( H), whereas the interaction between repeats ( J) remains unchanged. [ABSTRACT FROM AUTHOR]

Repeat proteins comprise tandem arrays of a small structural motif. Their structure is defined and stabilized by interactions between residues that are close in the primary sequence. Several studies have investigated whether their structural modularity translates into modular thermodynamic properties. Tetratricopeptide repeat proteins (TPRs) are a class in which the repeated unit is a 34 amino acid helix-turn-helix motif. In this work, we use differential scanning calorimetry (DSC) to study the equilibrium stability of a series of TPR proteins with different numbers of an identical consensus repeat, from 2 to 20, CTPRa2 to CTPRa20. The DSC data provides direct evidence that the folding/unfolding transition of CTPR proteins does not fit a two-state folding model. Our results confirm and expand earlier studies on TPR proteins, which showed that apparent two-state unfolding curves are better fit by linear statistical mechanics models: 1D Ising models in which each repeat is treated as an independent folding unit. [ABSTRACT FROM AUTHOR]

Tetratricopeptide repeats (TPRs) are protein domains that mediate key protein–protein interactions in cells. Several TPR domains bind the C-termini of the chaperones heat shock protein (Hsp)90 and/or Hsp70, and exchange of such binding partners is key for the heat shock response. We have previously described the design of a TPR protein that binds tightly and specifically to the C-terminus of Hsp90, and in doing so, is able to inhibit chaperone function in vivo. Here we present the X-ray crystal structure of the designed TPR domain (CTPR390) in complex with its peptide ligand – the C-terminal residues of Hsp90 (peptide MEEVD). This structure reveals two interesting aspects of the TPR modules. First, a new packing arrangement of 3-TPR modules is observed. The TPR units stack against each other in an unusual fashion to form infinite superhelices in the crystal. Second, the structure provides insights into the molecular basis of TPR–ligand recognition. [ABSTRACT FROM AUTHOR]

The structure and stability of repeat proteins has been little studied in comparison to the properties of the more familiar globular proteins. Here, the structure and stability of designed tetratricopeptide-repeat (TPR) proteins is described. The TPR is a 34-amino-acid motif which adopts a helix–turn–helix structure and occurs as tandem repeats. The design of a consensus TPR motif (CTPR) has previously been described. Here, the crystal structures and stabilities of proteins that contain eight or 20 identical tandem repeats of the CTPR motif (CTPR8 and CTPR20) are presented. Both CTPR8 and CTPR20 adopt a superhelical overall structure. The structures of the different-length CTPR proteins are compared with each other and with the structures of natural TPR domains. Also, the unusual and perhaps unique crystal-packing interactions resulting in pseudo-infinite crystalline superhelices observed in the different crystal forms of CTPR8 and CTPR20 are discussed. Finally, it is shown that the thermodynamic behavior of CTPR8 and CTPR20 can be predicted from the behavior of other TPRs in this series using an Ising model-based analysis. The designed protein series CTPR2–CTPR20 covers the natural size repertoire of TPR domains and as such is an excellent model system for natural TPR proteins. [ABSTRACT FROM AUTHOR]

Tetratricopeptide repeat (TPR) domains bind specific peptide ligands and are thought to mediate protein-protein interactions in a variety of biological systems. Here we compare peptide ligand-binding by several different TPR domains. We present specific examples that demonstrate that TPR domains typically undergo little or no structural rearrangement upon ligand binding. Our data suggest that, contrary to a recent proposal, coupled folding and binding is not the common mechanism of ligand recognition by TPR domains. [ABSTRACT FROM AUTHOR]

α-Haemolysin is a protein toxin secreted by pathogenic strains of Escherichia coli and requires sub-millimolar Ca2+ for optimum lytic activity. As a member of the so-called RTX toxin family it contains a Gly-rich, Asp-rich Ca2+-binding domain, consisting of a series of nonapeptides repeated in tandem. Asp-863 is located immediately after the last-but-one nonapeptide. A mutant in which Asp-863 has been substituted by Gly displays a requirement for Ca2+ that is 100-fold higher than the wild-type. Membrane lytic activity, as well as a conformational change revealed through an increase in intrinsic fluorescence, and the appearance of Ca2+-bound protein monomers resolvable by fast protein liquid chromatography, are all three dependent on Ca2+ concentrations in the 2–20 mM range. Most RTX toxins have an Asp or Glu residue located at a position homologous to Asp-863, thus the key role of this residue for Ca2+ requirements of α-haemolysin may be a general feature of this family of toxins. [Copyright &y& Elsevier]

Color down‐converting filters with fluorescent proteins (FPs) embedded in a polymer matrix have led to new bio‐hybrid light‐emitting diodes (Bio‐HLEDs), featuring stabilities of 100 h and <1 min at low and high applied currents, respectively. Herein, the FP deactivation mechanism in Bio‐HLEDs at high driving currents is deciphered. Primarily, the nonradiative energy relaxation of FPs upon excitation promotes the release of excess energy to the polymer matrix, reaching 60 °C and, in turn, a significant thermal emission quenching. This is circumvented by changing the device architecture, achieving stabilities of >300 h at high driving currents. Here, the photoinduced deactivation mechanism takes place, consisting of a slow and reversible partial dehydration followed by a quick and irreversible deactivation of the highly emissive ionic form. This is supported by steady‐state/time‐resolved emission, circular dichroism, and electrochemical impedance spectroscopic techniques. Overall, the limitations of Bio‐HLEDs concerning matrix, buffers, device design, and FP stability are highlighted as key aspects to achieve efficient and stable devices. [ABSTRACT FROM AUTHOR]

The deposition of protein thin films on (in)organic surfaces is a key approach to incorporate new functionalities into these materials for a broad number of applications. However, most of the current methods used for the controlled assembly of such biomolecules and eventual film formation are limiting since entail either the chemical modification of the proteins, which leads sometimes to impaired materials, or the sequential layer‐by‐layer deposition of charged macromolecules. In this work, a facile bioinspired method for the versatile fabrication of robust catalytic films is developed. The herein shown approach involves the one‐pot synthesis of porous enzyme nanogels decorated with imidazole motifs. These nanogels act as small nanoreactors that are further self‐assembled into continuous catalytic nanocoatings through metal‐imidazole coordination. An extensive structural, chemical, and functional characterization of the biocatalytic coatings evidences the integrity, stability in organic solvents and high temperatures, and the reusability of the deposits. Moreover, the thickness and metal composition of the nanocoatings can be tuned on demand. Finally, interesting applications for the bioinspired films are demonstrated, i.e., the fabrication of a glucose electrochemical biosensor and the completion of tandem enzymatic reactions, which suggest a broad applicability potential of this methodology. [ABSTRACT FROM AUTHOR]

A detailed investigation of the effect of electron injection on the chiral plasmon modes of helical nanorod assemblies is presented. An increased surface plasmon frequency of the electron gas due to the addition of electrons leads to a blue‐shift in the corresponding chiral surface plasmon modes. The mechanism behind the shift in plasmonic chirality due to nanorod charging is investigated using theoretical simulations. Charging of the nanorods alters the surface electron density, thereby modifying the plasma frequency and causing a change in the dielectric function. The nature of the plasmon shift and the intensity of chiral surface plasmons are found to be largely dependent on the extent of electron addition. At extended periods of time, the blue shifted band slowly shifts back toward the red, due to transfer of electrons back to the medium, leading to discharging of the nanorods. [ABSTRACT FROM AUTHOR]