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Nitrene transfer reactions catalyzed by heme proteins have broad potential for the stereoselective formation of carbon-nitrogen bonds. However, competition between productive nitrene transfer and the undesirable reduction of nitrene precursors limits the broad implementation of such biocatalytic methods. Here, we investigated the reduction of azides by the model heme protein myoglobin to gain mechanistic insights into the factors that control the fate of key reaction intermediates. In this system, the reaction proceeds via a proposed nitrene intermediate that is rapidly reduced and protonated to give a reactive ferrous amide species, which we characterized by UV/vis and Mössbauer spectroscopies, quantum mechanical calculations, and X-ray crystallography. Rate-limiting protonation of the ferrous amide to produce the corresponding amine is the final step in the catalytic cycle. These findings contribute to our understanding of the heme protein-catalyzed reduction of azides and provide a guide for future enzyme engineering campaigns to create more efficient nitrene transferases. Moreover, harnessing the reduction reaction in a chemoenzymatic cascade provided a potentially practical route to substituted pyrroles.

The third variable (V3) loop on the human immunodeficiency virus 1 (HIV-1) envelope glycoprotein trimer is indispensable for virus cell entry. Conformational masking of V3 within the trimer allows efficient neutralization via V3 only by rare, broadly neutralizing glycan-dependent antibodies targeting the closed prefusion trimer but not by abundant antibodies that access the V3 crown on open trimers after CD4 attachment. Here, we report on a distinct category of V3-specific inhibitors based on designed ankyrin repeat protein (DARPin) technology that reinstitute the CD4-bound state as a key neutralization target with up to >90% breadth. Broadly neutralizing DARPins (bnDs) bound V3 solely on open envelope and recognized a four-turn amphipathic α-helix in the carboxy-terminal half of V3 (amino acids 314-324), which we termed 'αV3C'. The bnD contact surface on αV3C was as conserved as the CD4 binding site. Molecular dynamics and escape mutation analyses underscored the functional relevance of αV3C, highlighting the potential of αV3C-based inhibitors and, more generally, of postattachment inhibition of HIV-1., (© 2023. The Author(s).)

High protein stability is an important feature for proteins used as therapeutics, as diagnostics, and in basic research. We have previously employed consensus design to engineer optimized Armadillo repeat proteins (ArmRPs) for sequence-specific recognition of linear epitopes with a modular binding mode. These designed ArmRPs (dArmRPs) feature high stability and are composed of M-type internal repeats that are flanked by N- and C-terminal capping repeats that protect the hydrophobic core from solvent exposure. While the overall stability of the designed ArmRPs is remarkably high, subsequent biochemical and biophysical experiments revealed that the N-capping repeat assumes a partially unfolded, solvent-accessible conformation for a small fraction of time that renders it vulnerable to proteolysis and aggregation. To overcome this problem, we have designed new N-caps starting from an M-type internal repeat using the Rosetta software. The superior stability of the computationally refined models was experimentally verified by circular dichroism and nuclear magnetic resonance spectroscopy. A crystal structure of a dArmRP containing the novel N-cap revealed that the enhanced stability correlates with an improved packing of this N-cap onto the hydrophobic core of the dArmRP. Hydrogen exchange experiments further show that the level of local unfolding of the N-cap is reduced by several orders of magnitude, resulting in increased resistance to proteolysis and weakened aggregation. As a first application of the novel N-cap, we determined the solution structure of a dArmRP with four internal repeats, which was previously impeded by the instability of the original N-cap.

Anthocyanins are ubiquitous plant pigments used in a variety of technological applications. Yet, after over a century of research, the penultimate biosynthetic step to anthocyanidins attributed to the action of leucoanthocyanidin dioxygenase has never been efficiently reconstituted outside plants, preventing the construction of heterologous cell factories. Through biochemical and structural analysis, here we show that anthocyanin-related glutathione transferases, currently implicated only in anthocyanin transport, catalyse an essential dehydration of the leucoanthocyanidin dioxygenase product, flavan-3,3,4-triol, to generate cyanidin. Building on this knowledge, introduction of anthocyanin-related glutathione transferases into a heterologous biosynthetic pathway in baker's yeast results in >35-fold increased anthocyanin production. In addition to unravelling the long-elusive anthocyanin biosynthesis, our findings pave the way for the colourants' heterologous microbial production and could impact the breeding of industrial and ornamental plants., Competing Interests: Competing interestsM.N. is employed by Lantana Bio. The other authors declare no competing interests., (© The Author(s) 2023.)

Peptides comprising many hydrophobic amino acids are almost insoluble under physiological buffer conditions, which complicates their structural analysis. To investigate the three-dimensional structure of the hydrophobic leucinostatin derivative ZHAWOC6027, the previously developed host lattice display technology was applied. Two designed ankyrin-repeat proteins (DARPins) recognizing a biotinylated ZHAWOC6027 derivative were selected from a diverse library by ribosome display under aqueous buffer conditions. ZHAWOC6027 was immobilized by means of the DARPin in the host lattice and the structure of the complex was determined by X-ray diffraction. ZHAWOC6027 adopts a distorted α-helical conformation. Comparison with the structures of related compounds that have been determined in organic solvents reveals elevated flexibility of the termini, which might be functionally important.

While native scaffolds offer a large diversity of shapes and topologies for enzyme engineering, their often unpredictable behavior in response to sequence modification makes de novo generated scaffolds an exciting alternative. Here we explore the customization of the backbone and sequence of a de novo designed eight stranded β-barrel protein to create catalysts for a retro-aldolase model reaction. We show that active and specific catalysts can be designed in this fold and use directed evolution to further optimize activity and stereoselectivity. Our results support previous suggestions that different folds have different inherent amenability to evolution and this property could account, in part, for the distribution of natural enzymes among different folds., (© 2022 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

High-throughput engineering has the potential to revolutionize the customization of biosynthetic assembly lines for the sustainable production of pharmaceutically relevant natural product analogs. Here, we show that the substrate specificity of gatekeeper adenylation domains of nonribosomal peptide synthetases can be switched from an α-amino acid to an α-hydroxy acid in a single round of combinatorial mutagenesis and selection using yeast cell surface display. In addition to shedding light on how such proteins discriminate between amino and hydroxy groups, the remodeled domains function in a pathway context to produce α-hydroxy acid-containing linear peptides and cyclic depsipeptides with high efficiency. Site-specific replacement of backbone amines with oxygens by an engineered synthetase provides the means to probe and tune the activities of diverse peptide metabolites in a simple and predictable fashion.

α-adrenergic receptors (αARs) are G protein-coupled receptors that regulate vital functions of the cardiovascular and nervous systems. The therapeutic potential of αARs, however, is largely unexploited and hampered by the scarcity of subtype-selective ligands. Moreover, several aminergic drugs either show off-target binding to αARs or fail to interact with the desired subtype. Here, we report the crystal structure of human α 1B AR bound to the inverse agonist (+)-cyclazosin, enabled by the fusion to a DARPin crystallization chaperone. The α 1B AR structure allows the identification of two unique secondary binding pockets. By structural comparison of α 1B AR with α 2 ARs, and by constructing α 1B AR-α 2C AR chimeras, we identify residues 3.29 and 6.55 as key determinants of ligand selectivity. Our findings provide a basis for discovery of α 1B AR-selective ligands and may guide the optimization of aminergic drugs to prevent off-target binding to αARs, or to elicit a selective interaction with the desired subtype., (© 2022. The Author(s).)

Mammalian adenoviruses (AdVs) comprise more than ~350 types including over 100 human (HAdVs) and just three mouse AdVs (MAdVs). While most HAdVs initiate infection by high affinity/avidity binding of their fiber knob (FK) protein to either coxsackievirus AdV receptor (CAR), CD46 or desmoglein (DSG)-2, MAdV-1 (M1) infection requires arginine-glycine-aspartate (RGD) binding integrins. To identify the receptors mediating MAdV infection we generated five novel reporter viruses for MAdV-1/-2/-3 (M1, M2, M3) transducing permissive murine (m) CMT-93 cells, but not B16 mouse melanoma cells expressing mCAR, human (h) CD46 or hDSG-2. Recombinant M1 or M3 FKs cross-blocked M1 and M3 but not M2 infections. Profiling of murine and human cells expressing RGD-binding integrins suggested that αvβ6 and αvβ8 heterodimers are associated with M1 and M3 infections. Ectopic expression of mβ6 in B16 cells strongly enhanced M1 and M3 binding, infection, and progeny production comparable with mαvβ6-positive CMT-93 cells, whereas mβ8 expressing cells were more permissive to M1 than M3. Anti-integrin antibodies potently blocked M1 and M3 binding and infection of CMT-93 cells and hαvβ8-positive M000216 cells. Soluble integrin αvβ6, and synthetic peptides containing the RGDLXXL sequence derived from FK-M1, FK-M3 and foot and mouth disease virus coat protein strongly interfered with M1/M3 infections, in agreement with high affinity interactions of FK-M1/FK-M3 with αvβ6/αvβ8, determined by surface plasmon resonance measurements. Molecular docking simulations of ternary complexes revealed a bent conformation of RGDLXXL-containing FK-M3 peptides on the subunit interface of αvβ6/β8, where the distal leucine residue dips into a hydrophobic pocket of β6/8, the arginine residue ionically engages αv aspartate215, and the aspartate residue coordinates a divalent cation in αvβ6/β8. Together, the RGDLXXL-bearing FKs are part of an essential mechanism for M1/M3 infection engaging murine and human αvβ6/8 integrins. These integrins are highly conserved in other mammals, and may favour cross-species virus transmission., Competing Interests: The authors have declared that no competing interests exist

The members of the human epidermal growth factor receptor (HER) family are among the most intensely studied oncological targets. HER3 (ErbB3), which had long been neglected, has emerged as a key oncogene, regulating the activity of other receptors and being involved in progression and tumor escape in multiple types of cancer. Designed ankyrin-repeat proteins (DARPins) serve as antibody mimetics that have proven to be useful in the clinic, in diagnostics and in research. DARPins have previously been selected against EGFR (HER1), HER2 and HER4. In particular, their combination into bivalent binders that separate or lock receptors in their inactive conformation has proved to be a promising strategy for the design of potent anticancer therapeutics. Here, the selection of DARPins targeting extracellular domain 4 of HER3 (HER3d4) is described. One of the selected DARPins, D5, in complex with HER3d4 crystallized in two closely related crystal forms that diffracted to 2.3 and 2.0 Å resolution, respectively. The DARPin D5 epitope comprises HER3d4 residues 568-577. These residues also contribute to interactions within the tethered (inactive) and extended (active) conformations of the extracellular domain of HER3., (open access.)

Changing the primary metal coordination sphere is a powerful strategy for tuning metalloprotein properties. Here we used amber stop codon suppression with engineered pyrrolysyl-tRNA synthetases, including two newly evolved enzymes, to replace the proximal histidine in myoglobin with N δ -methylhistidine, 5-thiazoylalanine, 4-thiazoylalanine and 3-(3-thienyl)alanine. In addition to tuning the heme redox potential over a >200 mV range, these noncanonical ligands modulate the protein's carbene transfer activity with ethyl diazoacetate. Variants with increased reduction potential proved superior for cyclopropanation and N-H insertion, whereas variants with reduced E o values gave higher S-H insertion activity. Given the functional importance of histidine in many enzymes, these genetically encoded analogues could be valuable tools for probing mechanism and enabling new chemistries., (© 2021 Wiley-VCH GmbH.)

Neurotensin receptor 1 (NTSR1) and related G protein-coupled receptors of the ghrelin family are clinically unexploited, and several mechanistic aspects of their activation and inactivation have remained unclear. Enabled by a new crystallization design, we present five new structures: apo-state NTSR1 as well as complexes with nonpeptide inverse agonists SR48692 and SR142948A, partial agonist RTI-3a, and the novel full agonist SRI-9829, providing structural rationales on how ligands modulate NTSR1. The inverse agonists favor a large extracellular opening of helices VI and VII, undescribed so far for NTSR1, causing a constriction of the intracellular portion. In contrast, the full and partial agonists induce a binding site contraction, and their efficacy correlates with the ability to mimic the binding mode of the endogenous agonist neurotensin. Providing evidence of helical and side-chain rearrangements modulating receptor activation, our structural and functional data expand the mechanistic understanding of NTSR1 and potentially other peptidergic receptors., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Stimulation of regulatory T (T reg ) cells holds great promise for the treatment of autoimmune, chronic inflammatory, and certain metabolic diseases. Recent clinical trials with low-dose interleukin-2 (IL-2) to expand T reg cells led to beneficial results in autoimmunity, but IL-2 immunotherapy can activate both T reg cells and pathogenic T cells. Use of IL-2 receptor α (IL-2Rα, CD25)-biased IL-2/anti-IL-2 antibody complexes improves IL-2 selectivity for T reg cells; however, the mechanism of action of such IL-2 complexes is incompletely understood, thus hampering their translation into clinical trials. Using a cell-based and dynamic IL-2R platform, we identified a particular anti-human IL-2 antibody, termed UFKA-20. When bound to UFKA-20, IL-2 failed to stimulate cells expressing IL-2Rβ (CD122) and IL-2Rγ (CD132), unless these cells also expressed high amounts of CD25. CD25 allowed IL-2/UFKA-20 complexes to bind, and binding to CD25 in the presence of CD122 and CD132 was followed by rapid dissociation of UFKA-20 from IL-2, delivery of IL-2 to CD122 and CD132, and intracellular signaling. IL-2/UFKA-20 complexes efficiently and preferentially stimulated CD4 + T reg cells in freshly isolated human T cells ex vivo and in mice and rhesus macaques in vivo. The crystal structure of the IL-2/UFKA-20 complex demonstrated that UFKA-20 interfered with IL-2 binding to CD122 and, to a lesser extent, also CD25. Together, we translated CD25-biased IL-2 complexes from mice to nonhuman primates and extended our mechanistic understanding of how CD25-biasing anti-human IL-2 antibodies work, which paves the way to clinical trials of CD25-biased IL-2 complexes., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Designed armadillo repeat proteins (dArmRPs) bind extended peptides in a modular way. The consensus version recognises alternating arginines and lysines, with one dipeptide per repeat. For generating new binding specificities, the rapid and robust analysis by crystallography is key. Yet, we have previously found that crystal contacts can strongly influence this analysis, by displacing the peptide and potentially distorting the overall geometry of the scaffold. Therefore, we now used protein design to minimise these effects and expand the previously described concept of shared helices to rigidly connect dArmRPs and designed ankyrin repeat proteins (DARPins), which serve as a crystallisation chaperone. To shield the peptide-binding surface from crystal contacts, we rigidly fused two DARPins to the N- and C-terminal repeat of the dArmRP and linked the two DARPins by a disulfide bond. In this ring-like structure, peptide binding, on the inside of the ring, is very regular and undistorted, highlighting the truly modular binding mode. Thus, protein design was utilised to construct a well crystallising scaffold that prevents interference from crystal contacts with peptide binding and maintains the equilibrium structure of the dArmRP. Rigid DARPin-dArmRPs fusions will also be useful when chimeric binding proteins with predefined geometries are required.

To overcome the laborious identification of crystallisation conditions for protein X-ray crystallography, we developed a method where the examined protein is immobilised as a guest molecule in a universal host lattice. We applied crystal engineering to create a generic crystalline host lattice under reproducible, predefined conditions and analysed the structures of target guest molecules of different size, namely two 15-mer peptides and green fluorescent protein (sfGFP). A fusion protein with an N-terminal endo-α-N-acetylgalactosaminidase (EngBF) domain and a C-terminal designed ankyrin repeat protein (DARPin) domain establishes the crystal lattice. The target is recruited into the host lattice, always in the same crystal form, through binding to the DARPin. The target structures can be determined rapidly from difference Fourier maps, whose quality depends on the size of the target and the orientation of the DARPin.

Temperature influences the reaction kinetics and evolvability of all enzymes. To understand how evolution shapes the thermodynamic drivers of catalysis, we optimized the modest activity of a computationally designed enzyme for an elementary proton-transfer reaction by nearly 4 orders of magnitude over 9 rounds of mutagenesis and screening. As theorized for primordial enzymes, the catalytic effects of the original design were almost entirely enthalpic in origin, as were the rate enhancements achieved by laboratory evolution. However, the large reductions in Δ H ⧧ were partially offset by a decrease in T Δ S ⧧ and unexpectedly accompanied by a negative activation heat capacity, signaling strong adaptation to the operating temperature. These findings echo reports of temperature-dependent activation parameters for highly evolved natural enzymes and are relevant to explanations of enzymatic catalysis and adaptation to changing thermal environments.

De novo drug discovery is still a challenge in the search for potent and selective modulators of therapeutically relevant target proteins. Here, we disclose the unexpected discovery of a peptidic ligand 1 by X-ray crystallography, which was auto-tailored by the therapeutic target MMP-13 through partial self-degradation and subsequent structure-based optimization to a highly potent and selective β-sheet peptidomimetic inhibitor derived from the endogenous tissue inhibitors of metalloproteinases (TIMPs). The incorporation of non-proteinogenic amino acids in combination with a cyclization strategy proved to be key for the de novo design of TIMP peptidomimetics. The optimized cyclic peptide 4 (ZHAWOC7726) is membrane permeable with an IC 50 of 21 nm for MMP-13 and an attractive selectivity profile with respect to a polypharmacology approach including the anticancer targets MMP-2 (IC 50 : 170 nm) and MMP-9 (IC 50 : 140 nm)., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Primordial sequence signatures in modern proteins imply ancestral origins tracing back to simple peptides. Although short peptides seldom adopt unique folds, metal ions might have templated their assembly into higher-order structures in early evolution and imparted useful chemical reactivity. Recapitulating such a biogenetic scenario, we have combined design and laboratory evolution to transform a zinc-binding peptide into a globular enzyme capable of accelerating ester cleavage with exacting enantiospecificity and high catalytic efficiency ( k cat / K M ~ 10 6 M -1 s -1 ). The simultaneous optimization of structure and function in a naïve peptide scaffold not only illustrates a plausible enzyme evolutionary pathway from the distant past to the present but also proffers exciting future opportunities for enzyme design and engineering., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

To untangle the complex signaling of the c-Jun N-terminal kinase (JNK) isoforms, we need tools that can selectively detect and inhibit individual isoforms. Because of the high similarity between JNK1, JNK2 and JNK3, it is very difficult to generate small-molecule inhibitors with this discriminatory power. Thus, we have recently selected protein binders from the designed ankyrin repeat protein (DARPin) library which were indeed isoform-specific inhibitors of JNK1 with low nanomolar potency. Here we provide the structural basis for their isotype discrimination and their inhibitory action. All our previous attempts to generate crystal structures of complexes had failed. We have now made use of a technology we recently developed which consists of rigid fusion of an additional special DARPin, which acts as a crystallization enhancer. This can be rigidly fused with different geometries, thereby generating a range of alternative crystal packings. The structures reveal the molecular basis for isoform specificity of the DARPins and their ability to prevent JNK activation and may thus form the basis of further investigation of the JNK family as well as novel approaches to drug design., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Designed armadillo repeat proteins (dArmRPs) were developed to create a modular peptide binding technology where each of the structural repeats binds two residues of the target peptide. An essential prerequisite for such a technology is a dArmRP geometry that matches the peptide bond length. To this end, we determined a large set (n=27) of dArmRP X-ray structures, of which 12 were previously unpublished, to calculate curvature parameters that define their geometry. Our analysis shows that consensus dArmRPs exhibit curvatures close to the optimal range for modular peptide recognition. Binding of peptide ligands can induce a curvature within the desired range, as confirmed by single-molecule FRET experiments in solution. On the other hand, computationally designed ArmRPs, where side chains have been chosen with the intention to optimally fit into a geometrically optimized backbone, turned out to be more divergent in reality, and thus not suitable for continuous peptide binding. Furthermore, we show that the formation of a crystal lattice can induce small but significant deviations from the curvature adopted in solution, which can interfere with the evaluation of repeat protein scaffolds when high accuracy is required. This study corroborates the suitability of consensus dArmRPs as a scaffold for the development of modular peptide binders., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Expanding the range of genetically encoded metal coordination environments accessible within tunable protein scaffolds presents excellent opportunities for the creation of metalloenzymes with augmented properties and novel activities. Here, we demonstrate that installation of a noncanonical N δ -methyl histidine (NMH) as the proximal heme ligand in the oxygen binding protein myoglobin (Mb) leads to substantial increases in heme redox potential and promiscuous peroxidase activity. Structural characterization of this catalytically modified myoglobin variant (Mb NMH) revealed significant changes in the proximal pocket, including alterations to hydrogen-bonding interactions involving the prosthetic porphyrin cofactor. Further optimization of Mb NMH via a combination of rational modification and several rounds of laboratory evolution afforded efficient peroxidase biocatalysts within a globin fold, with activities comparable to those displayed by nature's peroxidases.

Designed armadillo repeat proteins (dArmRP) are α-helical solenoid repeat proteins with an extended peptide binding groove that were engineered to develop a generic modular technology for peptide recognition. In this context, the term "peptide" not only denotes a short unstructured chain of amino acids, but also an unstructured region of a protein, as they occur in termini, loops, or linkers between folded domains. Here we report two crystal structures of dArmRPs, in complex with peptides fused either to the N-terminus of Green Fluorescent Protein or to the C-terminus of a phage lambda protein D. These structures demonstrate that dArmRPs bind unfolded peptides in the intended conformation also when they constitute unstructured parts of folded proteins, which greatly expands possible applications of the dArmRP technology. Nonetheless, the structures do not fully reflect the binding behavior in solution, that is, some binding sites remain unoccupied in the crystal and even unexpected peptide residues appear to be bound. We show how these differences can be explained by restrictions of the crystal lattice or the composition of the crystallization solution. This illustrates that crystal structures have to be interpreted with caution when protein-peptide interactions are characterized, and should always be correlated with measurements in solution., (© 2017 The Protein Society.)

Multivalent binding proteins can gain biological activities beyond what is inherent in the individual binders, by bringing together different target molecules, restricting their conformational flexibility or changing their subcellular localization. In this study, we demonstrate a method to build up rigid multivalent and multispecific scaffolds by exploiting the modular nature of a repeat protein scaffold and avoiding flexible linkers. We use DARPins (Designed Ankyrin Repeat Proteins), synthetic binding proteins based on the Ankyrin-repeat protein scaffold, as binding units. Their ease of in vitro selection, high production yield and stability make them ideal specificity-conferring building blocks for the design of more complex constructs. C- and N-terminal DARPin capping repeats were re-designed to be joined by a shared helix in such a way that rigid connector modules are formed. This allows us to join two or more DARPins in predefined geometries without compromising their binding affinities and specificities. Nine connector modules with distinct geometries were designed; for eight of these we were able to confirm the structure by X-ray crystallography, while only one did not crystallize. The bispecific constructs were all able to bind both target proteins simultaneously.

Armadillo repeat proteins (ArmRPs) recognize their target peptide in extended conformation and bind, in a first approximation, two residues per repeat. Thus, they may form the basis for building a modular system, in which each repeat is complementary to a piece of the target peptide. Accordingly, preselected repeats could be assembled into specific binding proteins on demand and thereby avoid the traditional generation of every new binding molecule by an independent selection from a library. Stacked armadillo repeats, each consisting of 42 aa arranged in three α-helices, build an elongated superhelical structure. Here, we analyzed the curvature variations in natural ArmRPs and identified a repeat pair from yeast importin-α as having the optimal curvature geometry that is complementary to a peptide over its whole length. We employed a symmetric in silico design to obtain a uniform sequence for a stackable repeat while maintaining the desired curvature geometry. Computationally designed ArmRPs (dArmRPs) had to be stabilized by mutations to remove regions of higher flexibility, which were identified by molecular dynamics simulations in explicit solvent. Using an N-capping repeat from the consensus-design approach, two different crystal structures of dArmRP were determined. Although the experimental structures of dArmRP deviated from the designed curvature, the insertion of the most conserved binding pockets of natural ArmRPs onto the surface of dArmRPs resulted in binders against the expected peptide with low nanomolar affinities, similar to the binders from the consensus-design series., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Fluorescent probes constitute a valuable toolbox to address a variety of biological questions and they have become irreplaceable for imaging methods. Commonly, such probes consist of fluorescent proteins or small organic fluorophores coupled to biological molecules of interest. Recently, a novel class of fluorescence-based probes, fluorogen-activating proteins (FAPs), has been reported. These binding proteins are based on antibody single-chain variable fragments and activate fluorogenic dyes, which only become fluorescent upon activation and do not fluoresce when free in solution. Here we present a novel class of fluorogen activators, termed FADAs, based on the very robust designed ankyrin repeat protein scaffold, which also readily folds in the reducing environment of the cytoplasm. The FADA generated in this study was obtained by combined selections with ribosome display and yeast surface display. It enhances the fluorescence of malachite green (MG) dyes by a factor of more than 11,000 and thus activates MG to a similar extent as FAPs based on single-chain variable fragments. As shown by structure determination and in vitro measurements, this FADA was evolved to form a homodimer for the activation of MG dyes. Exploiting the favorable properties of the designed ankyrin repeat protein scaffold, we created a FADA biosensor suitable for imaging of proteins on the cell surface, as well as in the cytosol. Moreover, based on the requirement of dimerization for strong fluorogen activation, a prototype FADA biosensor for in situ detection of a target protein and protein-protein interactions was developed. Therefore, FADAs are versatile fluorescent probes that are easily produced and suitable for diverse applications and thus extend the FAP technology., (Copyright © 2016 Elsevier Ltd. All rights reserved.)