Journal of the American Chemical Society

Radical S-adenosyl-L-methionine (SAM) enzymes catalyze various free radical-mediated reactions. In these enzymes, the rate-determining SAM cleavage kinetically masks all the subsequent steps. Due to this kinetic masking, detailed mechanistic characterization of radical transformations catalyzed by these enzymes is very difficult. Here, we report a successful kinetic characterization of the radical C-C bond formation catalyzed by a MoaA radical SAM enzyme. MoaA catalyzes an unprecedented 3',8-cyclization of GTP into 3',8-cyclo-7,8-dihydro-GTP (3',8-cH2GTP) during the molybdenum cofactor (Moco) biosynthesis. Through a series of EPR and biochemical characterization, we found that MoaA accumulates a 5'-deoxyadenos-4'-yl radical (5'-dA-C4'*) under the turnover conditions, and forms (4'S)-5'-deoxyadenosine ((4'S)-5'-dA), which is a C-4' epimer of the naturally occurring (4'R)-5'-dA. Together with kinetic characterizations, these observations revealed the presence of a shunt pathway in which an on-pathway intermediate, GTP C-3' radical, abstracts H-4' atom from 5'-dA to transiently generate 5'-dA-C4'* that is subsequently reduced stereospecifically to yield (4'S)-5'-dA. Detailed kinetic characterization of the shunt and the main pathways provided the comprehensive view of MoaA kinetics, and determined the rate of the on-pathway 3',8-cyclization step as 2.7 {+/-} 0.7 s-1. Together with DFT calculations, this observation suggested that the 3',8-cyclization is accelerated by 6 [~] 9 orders of magnitude by MoaA. Potential contributions of the active-site amino acid residues, and their potential relationships with human Moco deficiency disease are discussed. This is the first determination of the magnitude of catalytic rate acceleration by a radical SAM enzyme, and provides the foundation for understanding how radical SAM enzymes achieve highly specific radical catalysis.

Reversibly photoswitchable fluorophores have enabled a broad range of applications in advanced fluorescence bioimaging. Here, we provide an entirely new class of representatives based on a pair: a biomolecular host and a dark photoisomerizable guest, which becomes bright upon complexation. Hence, we introduce RSpFAST, which delivers the first non-covalent chemogenetic reversibly photoswitchable fluorescent proteins from combining photoisomerizable fluorogens with the FAST protein tag. Our experimental strategy involving thermokinetic, photochemical, and structural investigations provides a comprehensive mechanistic and kinetic understanding of RSpFAST. Building on this theoretical framework, we demonstrate in both live and fixed cells that RSpFAST exhibits an unprecedented dual behavior: a stable and wash-free fluorescent labeling tag turns into a negative reversible photoswitcher by simply lowering the fluorogen concentration and increasing light intensity. In this photoejection-driven kinetic regime, RSpFAST is shown to be an efficient marker for dynamic contrast and super-resolution microscopy.

High-valent metal-nitrido species are powerful nitrogen-atom transfer intermediates but remain difficult to access and control due to intrinsic instability and bimolecular N-N coupling pathways. Herein, we report the first formation of a high-valent Mn(V)-nitrido complex within a de novo designed protein scaffold and demonstrate that a reactive precursor to this species can be catalytically intercepted for enantioselective aziridination. A Mn(V){equiv}N unit derived from an abiological diphenyl porphyrin is confined within a designed helical bundle protein, where the protein environment suppresses bimolecular decay and enables detailed spectroscopic characterization. Electron paramagnetic resonance, resonance Raman, and circular dichroism spectroscopies confirm formation of a low-spin Mn(V)-nitrido species that is stable for weeks at room temperature and exhibits minimal perturbation of the Mn{equiv}N unit upon modulation of the axial histidine ligand, while catalytic activity and stereochemical outcome are sensitive to its presence. Mechanistic studies identify monochloramine (NH2Cl) as the operative nitrogen-atom donor and support the involvement of a transient Mn-bound N-transfer intermediate en route to nitrido formation. Under catalytic conditions, this intermediate is inter-cepted to perform aziridination with TON {approx} 180 and an enantiomeric ratio of 65:35. Together, these results establish de novo protein design as a platform for stabilizing high-valent metal-nitrido species and harnessing their reactivity for nitrogen-atom transfer chemistry beyond the limits of natural metalloenzymes and small-molecule catalysts.

De novo protein design has advanced such that many peptide assemblies and protein structures can be generated predictably and quickly. The drive now is to bring functions to these structures, for example, small-molecule binding and catalysis. The formidable challenge of binding and orienting multiple small molecules to direct chemistry is particularly important for paving the way to new functionalities. To address this, here we describe the design, characterization, and application of small-molecule:peptide ternary complexes in aqueous solution. This uses -helical barrel (HB) peptide assemblies, which comprise 5 or more -helices arranged around central channels. These channels are solvent accessible, and their internal dimensions and chemistries can be altered predictably. Thus, HBs are analogous to molecular flasks made in supramolecular, polymer, and materials chemistry. Using Forster resonance energy transfer as a readout, we demonstrate that specific HBs can accept two different organic dyes, 1,6-diphenyl-1,3,5-hexatriene and Nile Red in close proximity. In addition, two anthracene molecules can be accommodated within an HB to promote photocatalytic anthracene-dimer formation. However, not all ternary complexes are productive, either in energy transfer or photocatalysis, illustrating the control that can be exerted by judicious choice and design of the HB.

Molecular hydrogen (H2) is considered an eco-friendly future energy-carrier and an alternative to fossil fuel1 and thus, major efforts are directed towards identifying efficient and economical hydrogen catalysts.2,3 Efficient hydrogen catalysis is used by many microorganisms, some of them producing H2 from organic materials and others consuming it.4-6 To metabolize H2, these microorganisms use enzymes called hydrogenases.7,8 For the future development of efficient catalysts a detailed analysis of the catalytic mechanisms of such hydrogenases is required and existing analytical techniques could not provide a full understanding.9 Consequently, new analytical technologies are of utmost importance to unravel natures blueprints for highly efficient hydrogen catalysts. Here, we introduce signal-enhanced or hyperpolarized, nuclear magnetic resonance (NMR) to study hydrogenases under turnover conditions. So far undiscovered hydrogen species of the catalytic cycle of [Fe]-hydrogenases, are revealed and thus, extend the knowledge regarding this class of enzymes. These findings pave new pathways for the exploration of novel hydrogen metabolisms in vivo. We furthermore envision that the results contribute to the rational design of future catalysts to solve energy challenges of our society.

Photo-induced protein labelling strategies have become essential tools in chemical biology, but most strategies require high energy wavelengths of light as input to drive reactivity. Recently, we reported a biocompatible method for engaging photo-induced electron transfer to drive protein labelling using biaryl pyridinium salts and, here, we report the design of a series of aromatic cation salts that trigger this process using longer wavelengths of light while maintaining a sterically minimal profile. We achieved this through the systematic study of structure-reactivity relationships of various donor-acceptor pyridinium salts possessing extended conjugation, and these studies revealed the need of a constrained trans-stilbene relationship between the probes donor and acceptor substituents in order to achieve protein labelling. Probes with chromene-based donor groups in particular showed either robust protein labelling, significant fluorescence quantum yields, or state-dependent photophysical properties; in turn enabling the same probes to be used for both photo-induced protein labelling and wash-free live-cell imaging. We also demonstrate that these enhanced probes possess robust reactivity in complex biological environments through green light-triggered intracellular labelling in live HeLa cells, resulting in the identification of 659 enriched proteins. This series of experiments not only demonstrates the ability of this latest generation of probes to engage in photo-induced labelling using lower energy light in complex proteomes, but also reveals new capabilities for photophysical state-dependent reactivity and measurements. Table of Contents O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/681063v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1be9a0eorg.highwire.dtl.DTLVardef@741221org.highwire.dtl.DTLVardef@525596org.highwire.dtl.DTLVardef@170ed11_HPS_FORMAT_FIGEXP M_FIG C_FIG

Activity-based probes (ABPs) are widely used to profile serine protease activity - enzymes central to diverse physiological and pathological processes - but most rely on covalent modification of the conserved catalytic serine residue, often resulting in poor selectivity across related proteases. Here, we introduce covalent macrocyclic activity-based probes (cmABPs) that selectively target non-catalytic residues within serine protease active sites. By combining phage display with systematic electrophile scanning, we identify macrocyclic scaffolds that position sulfur(VI) fluoride (SuFEx) electrophiles to covalently engage alternative nucleophiles such as lysine and tyrosine. Applied to plasma kallikrein, this approach yielded a macrocyclic scaffold that was converted into covalent probes via fluorosulfate scanning. Remarkably, small changes in electrophile structure produced large, tuneable differences in covalent kinetics, with benzenesulfonyl fluoride derivative 23 achieving rapid and complete protein modification. Biochemical and mass spectrometry analyses confirmed selective modification of an active-site lysine by 23, along with robust performance in complex biological samples. Extension to urokinase plasminogen activator further demonstrates the generality of this strategy. More broadly, this work establishes electrophile scanning within macrocyclic scaffolds as a general approach for tuning covalent reactivity and provides a blueprint for designing selective probes that move beyond catalytic-residue targeting.

Caspases are a highly conserved family of cysteine-aspartyl proteases known for their essential roles in regulating apoptosis, inflammation, cell differentiation, and proliferation. Complementary to genetic approaches, small-molecule probes have emerged as useful tools for modulating caspase activity. However, due to the high sequence and structure homology of all twelve human caspases, achieving selectivity remains a central challenge for caspase-directed small-molecule inhibitor development efforts. Here, using mass spectrometry-based chemoproteomics, we first identify a highly reactive non-catalytic cysteine that is unique to caspase-2. By combining both gel-based activity-based protein profiling (ABPP) and a tobacco etch virus (TEV) protease activation assay, we then identify covalent lead compounds that react preferentially with this cysteine and afford a complete blockade of caspase-2 activity. Inhibitory activity is restricted to the zymogen or precursor form of monomeric caspase-2. Focused analogue synthesis combined with chemoproteomic target engagement analysis in cellular lysates and in cells yielded both pan-caspase reactive molecules and caspase-2 selective lead compounds together with a structurally matched inactive control. Application of this focused set of tool compounds to stratify caspase contributions to initiation of intrinsic apoptosis, supports compensatory caspase-9 activity in the context of caspase-2 inactivation. More broadly, our study highlights future opportunities for the development of proteoform-selective caspase inhibitors that target non-conserved and non-catalytic cysteine residues.

The arrangement of histidine sidechains in influenza A M2 tetramer determines their pKa values, which define pH controlled proton conduction critical to the virus lifecycle. Both water associated and hydrogen bonded Imidazole-Imidazolium histidine quaternary structures have been proposed, based on crystal structures, and NMR chemical shifts, respectively. Here we show, using the conduction domain construct of M2 in lipid bilayers, that the imidazole rings are hydrogen bonded even at a pH of 7.8 in the neutral charge state.\n\nAn intermolecular 8.9 {+/-} 0.3 Hz 2hJNN hydrogen bond is observed between H37 N{varepsilon} and N{delta} recorded in a fully protonated sample with 100 kHz magic-angle spinning. This interaction could not be detected in the drug-bound sample.

Photosensory protein function spans multiple time and length scales, demanding integrative approaches. We introduce 4-diacetylenyl-phenylalanine (DAF), a dual-purpose non-canonical amino acid (ncAA) that enables both chemical control and spectroscopic readout of photoreceptor dynamics. Genetically encoded in E. coli, DAF combines a reactive diyne for bioorthogonal ligations (thiols, azides, tetrazines) with a strong, solvatochromic Raman signal in the cell-silent region. Applied to the light-oxygen-voltage (LOV) transcription factor EL222, DAF enables multifaceted interrogation of its photocycle. We engineer a covalently cross-linked variant that suppresses light-driven conformational changes and DNA binding, and generate a donor-acceptor construct for Forster resonance energy transfer (FRET) tracking of photoinduced structural dynamics. Time-resolved stimulated Raman spectroscopy following flavin mononucleotide (FMN) excitation reveals additional processes -from vibrational energy transfer to local unfolding-spanning femtoseconds to milliseconds. DAF thus constitutes a versatile tool to resolve protein dynamics with high spatiotemporal resolution.

The nonenzymatic replication of ribonucleic acid (RNA) oligonucleotides may have enabled the propagation of genetic information during the origin of life. RNA copying can be initiated in the laboratory with chemically activated nucleotides, but continued copying requires a source of chemical energy for in situ nucleotide activation. Recent work has illuminated a potentially prebiotic cyanosulfidic chemistry that activates nucleotides, but its application to nonenzymatic RNA copying remains a challenge. Here we report a novel pathway that enables the activation of RNA nucleotides in a manner that is compatible with template-directed nonenzymatic polymerization. We show that this pathway selectively yields the reactive imidazolium-bridged dinucleotide intermediate required for nonenzymatic template-directed RNA copying. Our results will enable more realistic prebiotic chemical simulations of RNA copying based on continuous in situ nucleotide activation.

Protein labelling by covalent attachment of a specific substrate to a self-labelling protein tag has become a regular in the life sciences. Herein, we report the design of a two-component labelling system, comprised of a non-fluorescent difluorinated xanthene, called F2X, and a HaloTag mutant engineered for targeted reactivity towards F2X. Upon primary covalent locking of the ligand at the canonical aspartate residue, two proximal lysine residues located at the protein surface can undergo nucleophilic aromatic substitution with the F2X core, building a fluorescent rhodamine via triple-covalent fusion. We used a generalizable in silico pipeline for heuristic conformational sampling of covalent protein-ligand complexes to find suitable mutation sites, culminating in the curation of 7 double-lysine HaloTag mutants for targeted in vitro testing. Reaction with the best-performing mutant, HTPL161K_Q165K, is characterized by full protein mass spectrometry, fluorescence polarization fluorescence lifetime, and fluorescence anisotropy and rationalized by computational modelling. We showcase the system in single molecule microscopy, where obviation of post-labelling purification is a prime advantage when targeting recombinant proteins that may not be expressed in larger quantities, and employ F2X in living cells with reduced photobleaching. Lastly, a cell-impermeable version was obtained by means of sulfonation, exclusively targeting extracellularly exposed HTPKK fused to the neuromodulatory G protein-coupled receptor metabotropic glutamate receptor 2.

Developing enzymatic mechanisms for C-F bond formation remains a long-standing challenge. Here, we repurposed the biosynthetic nonheme Fe enzyme EgtB, which features a three-histidine facial triad, to catalyze C(sp3)-H fluorination reactions. Directed evolution of EgtB afforded two new-to-nature fluorine atom transferases with opposite enantiopreference, EgtBCHF1 and EgtBCHF2, with up to 28-fold improved total activity. In contrast to our previously evolved nonheme Fe fluorine atom transfer biocatalyst ACCOCHF, which contains a two-histidine-one-carboxylate facial triad, the evolved EgtBCHF variants displayed unexpected hydroxylation activity. 18O-labeling experiments showed that the hydroxy group originated from water rather than residual O2. Computational studies suggested that the three-histidine-supported Fe(III) center exhibits enhanced Lewis acidity compared to the two-histidine-one-carboxylate system, allowing deprotonation of Fe(III)-bound water to form a Fe(III)-OH species to catalyze radical hydroxylation. Primary coordination-sphere mutagenesis in EgtB and ACCO further supported the critical role of Fe coordination chemistry in controlling radical rebound reactivity and selectivity. Computational studies revealed that Fe coordination chemistry strongly influences both fluorine atom abstraction and radical rebound, with the intrinsic C-X (X = F, OH, and N3) bond forming radical rebound preference following the order N3 > OH > F. Furthermore, multivariate linear regression analysis revealed that fluorine atom abstraction is primarily governed by the intrinsic Fe-F bond strength, whereas fluorine rebound is predominantly controlled by the electronic structure of the Fe(III) intermediate. Together, these findings provide mechanistic insights into nonheme Fe enzymology and reprogramming toward selective radical rebound reactions, including challenging C-H fluorination. Table of Contents (TOC) O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/737789v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1ad85b2org.highwire.dtl.DTLVardef@1248bd4org.highwire.dtl.DTLVardef@58268dorg.highwire.dtl.DTLVardef@14b2da0_HPS_FORMAT_FIGEXP M_FIG C_FIG

High Mannose Glycans (HMGs) play key roles in eukaryotic biology, regulating processes ranging from protein folding to host pathogen defense. Lectins have evolved to interact with these glycans through multivalent interactions facilitated by the multiple sugars displayed on glycans and via multiple binding sites on each lectin. Using Fe(II) iminopyridine complexes, we generated chemically defined multivalent glycan displays where the valency, arm length, and spatial display of mannose residues can be controlled via subcomponent synthesis. Due to its sensitivity towards the geometric display of mannose residues, monomeric Griffithsin (mGRFT) was utilized as a model lectin. Interactions between the Fe(II) glycan assemblies and mGRFT were characterized using biolayer interferometry (BLI), isothermal titration calorimetry (ITC), and NMR spectroscopy. Our results display a >1000-fold range in KD for Fe(II) iminopyridine complexes that can be tuned by factors such as saccharide tether length and number of sugars displayed. Through leveraging systematic molecular-level modifications, we demonstrate that tunable Fe(II) glycan assemblies can be used both as mimetics for high mannose glycans as well as competitive inhibitors for native glycan binding.

Catalytic molecular templating, wherein a copolymer molecule serves as a sequence-specific template to propagate genetic information to a daughter copolymer, is fundamental to cells. Templating underlies DNA replication, RNA transcription and protein translation, underpinning the molecular basis of heredity, evolution, and biological function, and allowing staggering complexity to arise from simple building blocks. It has hitherto been challenging to emulate templating without highly evolved enzymes, largely due to product inhibition of catalytic turnover, which is a major challenge for templated dimerization and prohibitive for longer products. We present an enzyme-free DNA-based templated dimerization reaction enabled and controlled by a fuel strand that actively displaces the product from the template only once dimerization is complete, overcoming product inhibition. We systematically investigate design variants to optimise catalytic turnover, and demonstrate information propagation through the action of distinct templates that assemble specific products from the same pool of building blocks. We also show that the fuel represents an input by which the templating can be controlled, allowing the coupling of catalytic turnover to the output of upstream DNA circuitry. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/706517v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@e82eeeorg.highwire.dtl.DTLVardef@1612b85org.highwire.dtl.DTLVardef@706911org.highwire.dtl.DTLVardef@217d6_HPS_FORMAT_FIGEXP M_FIG C_FIG

Reversibly photoswitchable fluorophores are widely used in advanced bioimaging but their design remains demanding. Here, we introduce a new series spanning the whole visible range, which results from combining a large set of fluorogens with the FAST protein scaffold. We first demonstrate that these well-established labeling fluorescent protein tags turn into negative reversible photoswitchers upon decreasing the fluorogen concentration and increasing light intensity. We then show that using not anymore one but two fluorogens adds new responses to illumination. Thus, we obtain positive reversible photoswitchers, that increase their brightness under illumination. We also generate a palette of non-covalent reversibly photoconvertible fluorescent proteins changing their fluorescence color upon illumination, a reversible behavior that still remains absent in regular fluorescent proteins. This light-induced color change opens the possibility to discriminate six spectrally similar FAST variants in live cells upon demonstrating the superiority of using multiple spectral channels for exploiting the time dependence of the fluorescence response to illumination.

Glycation crosslinks account for more than 40% of all known advanced glycation end products (AGEs) and are correlated with many age-related diseases. Despite much interest, crosslinking AGEs (xl-AGEs) remain poorly understood, as they have been challenging to discover, prepare, and quantify. Here we describe a peptide platform that is ideally suited for the study of xl-AGEs, which not only facilitates direct comparisons between the prevalence of known xl-AGEs and other AGEs, but also enables the discovery of previously unknown xl-AGEs. In this study, we use this platform to discover the first known Arg-Arg xl-AGEs, a pair of methylglyoxal-derived dihydroxyimidazolidine hemiacetal crosslink, or MIDAL, isomers. We show that MIDAL can become the major AGE, exceeding levels of all other AGEs, for substrates in which two Arg glycation sites are optimally positioned. We further demonstrate that MIDAL is readily and reversibly generated in biocompatible conditions, persisting with a half-life of more than three days. We also demonstrate that MIDAL can form in living mammalian cells, suggesting that it has the potential to be a dynamic, physiologically relevant and functional xl-AGE. This work therefore offers important insights about MIDAL formation and describes a versatile platform to enable the study of xl-AGEs under a variety of conditions. We expect that it will be highly useful for further discovery of biologically relevant glycation crosslinks that are yet to be identified.

The water-soluble inositol phosphates (InsPs) represent a functionally diverse group of small-molecule messengers central to a myriad of cellular processes. However, we have an incomplete understanding of InsP metabolism because the available analytical toolset for inositol phosphates is rather limited. Here, we have synthesized and utilized fully and unsymmetrically 13C-labeled myo-inositol and inositol phosphates. These probes were applied in combination with nuclear magnetic resonance spectroscopy (NMR) and capillary electrophoresis mass spectrometry (CE-MS) to further annotate central aspects of InsP metabolism in human cells. The labeling strategy provided detailed structural information via NMR - down to individual enantiomers - which overcomes a crucial blind spot in the analysis of InsPs. We uncovered a novel branch of InsP dephosphorylation in human cells which is dependent on MINPP1, a phytase-like enzyme, that contributes to cellular homeostasis. Full characterization of MINPP1 activity in vitro and in cells, provided a clear picture of this multifunctional phosphatase. Metabolic labeling with stable isotopomers thus constitutes a powerful tool for investigating InsP networks in a variety of different biological contexts.

Activity-based detection of hydrogen sulfide in live cells can expand our understanding of its reactivity and complex physiological effects. We have discovered a highly efficient method for fluorescent probe activation, which is driven by H2S-triggered 1,6-elimination of an -CF3-benzyl to release resorufin. In detecting intracellular H2S, 4-azido-(-CF3)-benzyl resorufin offers significantly faster signal generation and improved sensitivity compared to 4-azidobenzyl resorufin. Computed free energy profiles for the 1,6-elimination process support the hypothesis that a benzylic CF3 group can reduce the activation energy barrier toward probe activation. This novel probe design allows for near-real-time detection of H2S in HeLa cells under stimulation conditions.

Intermediates along the fibrillation pathway are generally considered to be the toxic species responsible for the pathologies of amyloid diseases. However, structural studies of these species have been hampered by heterogeneity and poor stability in standard aqueous conditions. Here, we report a novel methodology for producing stable, on-pathway oligomers of the human Type-2 Diabetes-associated islet amyloid polypeptide (hIAPP, or amylin) using the mechanical forces associated with magic angle spinning (MAS). The species were a heterogeneous mixture of globular and short rod-like species with significant {beta}-sheet content and the capability of seeding hIAPP fibrillation. We used MAS NMR to demonstrate that the nature of the species was sensitive to sample conditions including peptide concentration, ionic strength, and buffer. The methodology should be suitable for studies of other aggregating systems.