Ultrahigh-Throughput Directed Evolution of Polymer-Degrading Enzymes Using Yeast Display.

Enzymes that degrade synthetic polymers have attracted intense interest for eco-friendly plastic recycling. However, because enzymes did not evolve for the cleavage of abiotic polymers, directed evolution strategies are needed to enhance activity for plastic degradation. Previous directed evolution efforts relied on polymer degradation assays that were limited to screening ∼104 mutants. Here, we report a high-throughput yeast surface display platform to rapidly evaluate >107 enzyme mutants for increased activity in cleaving synthetic polymers. In this platform, individual yeast cells display distinct mutants, and enzyme activity is detected by a change in fluorescence upon the cleavage of a synthetic probe resembling a polymer of interest. Highly active mutants are isolated by fluorescence activated cell sorting and identified through DNA sequencing. To demonstrate this platform, we performed directed evolution of a polyethylene terephthalate (PET)-depolymerizing enzyme, leaf and branch compost cutinase (LCC). We identified activity-boosting mutations that substantially increased the kinetics of degradation of solid PET films. Biochemical assays and molecular dynamics (MD) simulations of the most active variants suggest that the H218Y mutation improves the binding of the enzyme to PET. Overall, this evolution platform increases the screening throughput of polymer-degrading enzymes by 3 orders of magnitude and identifies mutations that enhance kinetics for depolymerizing solid substrates.

Terminal Alkyne-Modified DNA Aptamers with Enhanced Protein Binding Affinities.

Nucleic acid-based receptors, known as aptamers, are relatively fast to discover and manufacture but lack the diverse functional groups of protein receptors (e.g., antibodies). The binding properties of DNA aptamers can be enhanced by attaching abiotic functional groups; for example, aromatic groups such as naphthalene slow dissociation from proteins. Although the terminal alkyne is a π-electron-rich functional group that has been used in small molecule drugs to enhance binding to proteins through noncovalent interactions, it remains unexplored for enhancing DNA aptamer binding affinity. Here, we demonstrate the utility of the terminal alkyne for improving the binding of DNA to proteins. We prepared a library of 256 terminal-alkyne-bearing variants of HD22, a DNA aptamer that binds the protein thrombin with nanomolar affinity. After a one-step thrombin-binding selection, a high-affinity aptamer containing two alkynes was discovered, exhibiting 3.2-fold tighter thrombin binding than the corresponding unmodified sequence. The tighter binding was attributable to a slower rate of dissociation from thrombin (5.2-fold slower than HD22). Molecular dynamics simulations with enhanced sampling by Replica Exchange with Solute Tempering (REST2) suggest that the π-electron-rich alkyne interacts with an asparagine side chain N-H group on thrombin, forming a noncovalent interaction that stabilizes the aptamer-protein interface. Overall, this work represents the first case of terminal alkynes enhancing the binding properties of an aptamer and underscores the utility of the terminal alkyne as an atom economical π-electron-rich functional group to enhance binding affinity with minimal steric perturbation.

DNA-Compatible Copper/TEMPO Oxidation for DNA-Encoded Libraries.

Aldehydes are important synthons for DNA-encoded library (DEL) construction, but the development of a DNA-compatible method for the oxidation of alcohols to aldehydes remains a significant challenge in the field of DEL chemistry. We report that a copper/TEMPO catalyst system enables the solution-phase DNA-compatible oxidation of DNA-linked primary activated alcohols to aldehydes. The semiaqueous, room-temperature reaction conditions afford oxidation of benzylic, heterobenzylic, and allylic alcohols in high yield, with DNA compatibility verified by mass spectrometry, qPCR, Sanger sequencing, and ligation assays. Subsequent transformations of the resulting aldehydes demonstrate the potential of this method for robust library diversification.

Switchable DNA Catalysts for Proximity Labeling at Sites of Protein-Protein Interactions.

Proximity labeling (PL) has emerged as a powerful approach to elucidate proteomes within a defined radius around a protein of interest (POI). In PL, a catalyst is attached to the POI and tags nearby endogenous proteins, which are then isolated by affinity purification and identified by mass spectrometry. Although existing PL methods have yielded numerous biological insights, proteomes with greater spatial resolution could be obtained if PL catalysts could be activated at more specific subcellular locations, such as sites where both the POI and a chemical stimulus are present or sites of protein-protein interactions (PPIs). Here, we report DNA-based switchable PL catalysts that are attached to a POI and become activated only when a secondary molecular trigger is present. The DNA catalysts consist of a photocatalyst and a spectral quencher tethered to a DNA oligomer. They are catalytically inactive by default but undergo a conformational change in response to a specific molecular trigger, thus activating PL. We designed a system in which the DNA catalyst becomes activated on living mammalian cells specifically at sites of Her2-Her3 heterodimers and c-Met homodimers, PPIs known to increase the invasion and growth of certain cancers. While this study employs a Ru(bpy)3-type complex for tagging proteins with biotin phenol, the switchable DNA catalyst design is compatible with diverse synthetic PL photocatalysts. Furthermore, the switchable DNA PL catalysts can be constructed from conformation-switching DNA aptamers that respond to small molecules, ions, and proteins, opening future opportunities for PL in highly specific subcellular locations.

Switchable DNA Photocatalysts for Radical Polymerization Controlled by Chemical Stimuli.

Polymerization catalysts that activate in response to specific chemical triggers offer spatial and temporal control over polymer synthesis, facilitating the development of responsive materials and custom polymer coatings. However, existing catalysts switch their activity through mechanisms that are not generalizable to chemically diverse stimuli. To approach the level of control exhibited in biological polymer synthesis, switchable polymerization catalysts need to be configurable for activation in response to diverse chemical stimuli. Here, we combine synthetic photocatalysts with conformation-switching DNA aptamers to create polymerization catalysts that respond to diverse chemical stimuli. We use the secondary structure of DNA to bring a photocatalyst and quencher dye into proximity, turning off photocatalysis. The DNA structure can be precisely designed to change conformation in response to a molecular trigger, moving the photocatalyst far from the quencher and activating photocatalysis. We show these photocatalysts can initiate free-radical polymerization to form bulk hydrogels in response to complementary DNA, a metal ion (Zn2+), or small molecules (glucose and hydrocortisone). We demonstrate the biocompatibility of these switchable photocatalysts by triggering their activation on the surface of yeast cells. Finally, we perform reversible-deactivation radical polymerization through photoinduced electron/energy transfer reversible addition-fragmentation chain-transfer in a dual-stimulus manner, in which catalytic activity is regulated reversibly by photoirradiation and the conformational state of the DNA catalyst. These results demonstrate that DNA conformational changes triggered by chemically diverse stimuli can regulate the activity of radical polymerization photocatalysts. This platform offers new capabilities in spatially and temporally controlled polymer synthesis, with potential applications in diagnostics, sensing, and environmentally responsive materials.

A Horseradish Peroxidase-Mediator System for Benzylic C-H Activation.

Enzyme-mediator systems generate radical intermediates that abstract hydrogen atoms under mild conditions. These systems have been employed extensively for alcohol oxidation, primarily in biomass degradation, but they are underexplored for direct activation of C(sp3)-H bonds in alkyl groups. Here, we combine horseradish peroxidase (HRP), H2O2, and redox mediator N-hydroxyphthalimide (NHPI) for C(sp3)-H functionalization of alkylbenzene-type substrates. The HRP-NHPI system is >10-fold more active than existing enzyme-mediator systems in converting alkylbenzenes to ketones and aldehydes under air, and it operates from 0-50 °C and in numerous aqueous-organic solvent mixtures. The benzylic substrate radical can be trapped through a reaction with NHPI, demonstrating the formation of benzylic products beyond ketones. Furthermore, we demonstrate a one-pot, two-step enzymatic cascade for converting alkylbenzenes to benzylic amines. Overall, the HRP-NHPI system enables the selective benzylic C-H functionalization of diverse substrates under mild conditions using a straightforward procedure.

DNA-Scaffolded Synergistic Catalysis.

We report DNA-scaffolded synergistic catalysis, a concept that combines the diverse reaction scope of synergistic catalysis with the ability of DNA to precisely preorganize abiotic groups and undergo stimuli-triggered conformational changes. As an initial demonstration of this concept, we focus on Cu-TEMPO-catalyzed aerobic alcohol oxidation, using DNA as a scaffold to hold a copper cocatalyst and an organic radical cocatalyst (TEMPO) in proximity. The DNA-scaffolded catalyst maintained a high turnover number upon dilution and exhibited 190-fold improvement in catalyst turnover number relative to the unscaffolded cocatalysts. By incorporating the cocatalysts into a DNA hairpin-containing scaffold, we demonstrate that the rate of the synergistic catalytic reaction can be controlled through a reversible DNA conformational change that alters the distance between the cocatalysts. This work demonstrates the compatibility of synergistic catalytic reactions with DNA scaffolding, opening future avenues in reaction discovery, sensing, responsive materials, and chemical biology.

Cooperative carbon capture and steam regeneration with tetraamine-appended metal-organic frameworks.

Natural gas has become the dominant source of electricity in the United States, and technologies capable of efficiently removing carbon dioxide (CO2) from the flue emissions of natural gas-fired power plants could reduce their carbon intensity. However, given the low partial pressure of CO2 in the flue stream, separation of CO2 is particularly challenging. Taking inspiration from the crystal structures of diamine-appended metal-organic frameworks exhibiting two-step cooperative CO2 adsorption, we report a family of robust tetraamine-functionalized frameworks that retain cooperativity, leading to the potential for exceptional efficiency in capturing CO2 under the extreme conditions relevant to natural gas flue emissions. The ordered, multimetal coordination of the tetraamines imparts the materials with extraordinary stability to adsorption-desorption cycling with simulated humid flue gas and enables regeneration using low-temperature steam in lieu of costly pressure or temperature swings.

Kinetics of cooperative CO2 adsorption in diamine-appended variants of the metal-organic framework Mg2(dobpdc).

Carbon capture and sequestration is a key element of global initiatives to minimize anthropogenic greenhouse gas emissions. Although many investigations of new candidate CO2 capture materials focus on equilibrium adsorption properties, it is also critical to consider adsorption/desorption kinetics when evaluating adsorbent performance. Diamine-appended variants of the metal-organic framework Mg2(dobpdc) (dobpdc4- = 4,4'-dioxidobiphenyl-3,3'-dicarboxylate) are promising materials for CO2 capture because of their cooperative chemisorption mechanism and associated step-shaped equilibrium isotherms, which enable large working capacities to be accessed with small temperature swings. However, the adsorption/desorption kinetics of these unique materials remain understudied. More generally, despite the necessity of kinetics characterization to advance adsorbents toward commercial separations, detailed kinetic studies of metal-organic framework-based gas separations remain rare. Here, we systematically investigate the CO2 adsorption kinetics of diamine-appended Mg2(dobpdc) variants using a thermogravimetric analysis (TGA) assay. In particular, we examine the effects of diamine structure, temperature, and partial pressure on CO2 adsorption and desorption kinetics. Importantly, most diamine-appended Mg2(dobpdc) variants exhibit an induction period prior to reaching the maximum rate of CO2 adsorption, which we attribute to their unique cooperative chemisorption mechanism. In addition, these materials exhibit inverse Arrhenius behavior, displaying faster adsorption kinetics and shorter induction periods at lower temperatures. Using the Avrami model for nucleation and growth kinetics, we determine rate constants for CO2 adsorption and quantitatively compare rate constants among different diamine-appended variants. Overall, these results provide guidelines for optimizing adsorbent design to facilitate CO2 capture from diverse target streams and highlight kinetic phenomena relevant for other materials in which cooperative chemisorption mechanisms are operative.

Directed Evolution of Split APEX2 Peroxidase.

APEX is an engineered peroxidase that catalyzes the oxidation of a wide range of substrates, facilitating its use in a variety of applications from subcellular staining for electron microscopy to proximity biotinylation for spatial proteomics and transcriptomics. To further advance the capabilities of APEX, we used directed evolution to engineer a split APEX tool (sAPEX). A total of 20 rounds of fluorescence activated cell sorting (FACS)-based selections from yeast-displayed fragment libraries, using 3 different surface display configurations, produced a 200-amino-acid N-terminal fragment (with 9 mutations relative to APEX2) called "AP" and a 50-amino-acid C-terminal fragment called "EX". AP and EX fragments were each inactive on their own but were reconstituted to give peroxidase activity when driven together by a molecular interaction. We demonstrate sAPEX reconstitution in the mammalian cytosol, on engineered RNA motifs within a non-coding RNA scaffold, and at mitochondria-endoplasmic reticulum contact sites.

Elucidating CO2 Chemisorption in Diamine-Appended Metal-Organic Frameworks.

The widespread deployment of carbon capture and sequestration as a climate change mitigation strategy could be facilitated by the development of more energy-efficient adsorbents. Diamine-appended metal-organic frameworks of the type diamine-M2(dobpdc) (M = Mg, Mn, Fe, Co, Ni, Zn; dobpdc4- = 4,4'-dioxidobiphenyl-3,3'-dicarboxylate) have shown promise for carbon-capture applications, although questions remain regarding the molecular mechanisms of CO2 uptake in these materials. Here we leverage the crystallinity and tunability of this class of frameworks to perform a comprehensive study of CO2 chemisorption. Using multinuclear nuclear magnetic resonance (NMR) spectroscopy experiments and van-der-Waals-corrected density functional theory (DFT) calculations for 13 diamine-M2(dobpdc) variants, we demonstrate that the canonical CO2 chemisorption products, ammonium carbamate chains and carbamic acid pairs, can be readily distinguished and that ammonium carbamate chain formation dominates for diamine-Mg2(dobpdc) materials. In addition, we elucidate a new chemisorption mechanism in the material dmpn-Mg2(dobpdc) (dmpn = 2,2-dimethyl-1,3-diaminopropane), which involves the formation of a 1:1 mixture of ammonium carbamate and carbamic acid and accounts for the unusual adsorption properties of this material. Finally, we show that the presence of water plays an important role in directing the mechanisms for CO2 uptake in diamine-M2(dobpdc) materials. Overall, our combined NMR and DFT approach enables a thorough depiction and understanding of CO2 adsorption within diamine-M2(dobpdc) compounds, which may aid similar studies in other amine-functionalized adsorbents in the future.

Overcoming double-step CO2 adsorption and minimizing water co-adsorption in bulky diamine-appended variants of Mg2(dobpdc).

Alkyldiamine-functionalized variants of the metal-organic framework Mg2(dobpdc) (dobpdc4- = 4,4'-dioxidobiphenyl-3,3'-dicarboxylate) are promising for CO2 capture applications owing to their unique step-shaped CO2 adsorption profiles resulting from the cooperative formation of ammonium carbamate chains. Primary,secondary (1°,2°) alkylethylenediamine-appended variants are of particular interest because of their low CO2 step pressures (≤1 mbar at 40 °C), minimal adsorption/desorption hysteresis, and high thermal stability. Herein, we demonstrate that further increasing the size of the alkyl group on the secondary amine affords enhanced stability against diamine volatilization, but also leads to surprising two-step CO2 adsorption/desorption profiles. This two-step behavior likely results from steric interactions between ammonium carbamate chains induced by the asymmetrical hexagonal pores of Mg2(dobpdc) and leads to decreased CO2 working capacities and increased water co-adsorption under humid conditions. To minimize these unfavorable steric interactions, we targeted diamine-appended variants of the isoreticularly expanded framework Mg2(dotpdc) (dotpdc4- = 4,4''-dioxido-[1,1':4',1''-terphenyl]-3,3''-dicarboxylate), reported here for the first time, and the previously reported isomeric framework Mg-IRMOF-74-II or Mg2(pc-dobpdc) (pc-dobpdc4- = 3,3'-dioxidobiphenyl-4,4'-dicarboxylate, pc = para-carboxylate), which, in contrast to Mg2(dobpdc), possesses uniformally hexagonal pores. By minimizing the steric interactions between ammonium carbamate chains, these frameworks enable a single CO2 adsorption/desorption step in all cases, as well as decreased water co-adsorption and increased stability to diamine loss. Functionalization of Mg2(pc-dobpdc) with large diamines such as N-(n-heptyl)ethylenediamine results in optimal adsorption behavior, highlighting the advantage of tuning both the pore shape and the diamine size for the development of new adsorbents for carbon capture applications.

Unexpected Diffusion Anisotropy of Carbon Dioxide in the Metal-Organic Framework Zn2(dobpdc).

Metal-organic frameworks are promising materials for energy-efficient gas separations, but little is known about the diffusion of adsorbates in materials featuring one-dimensional porosity at the nanoscale. An understanding of the interplay between framework structure and gas diffusion is crucial for the practical application of these materials as adsorbents or in mixed-matrix membranes, since the rate of gas diffusion within the adsorbent pores impacts the required size (and therefore cost) of the adsorbent column or membrane. Here, we investigate the diffusion of CO2 within the pores of Zn2(dobpdc) (dobpdc4- = 4,4'-dioxidobiphenyl-3,3'-dicarboxylate) using pulsed field gradient (PFG) nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulations. The residual chemical shift anisotropy for pore-confined CO2 allows PFG NMR measurements of self-diffusion in different crystallographic directions, and our analysis of the entire NMR line shape as a function of the applied field gradient provides a precise determination of the self-diffusion coefficients. In addition to observing CO2 diffusion through the channels parallel to the crystallographic c axis (self-diffusion coefficient D∥ = (5.8 ± 0.1) × 10-9 m2 s-1 at a pressure of 625 mbar CO2), we unexpectedly find that CO2 is also able to diffuse between the hexagonal channels in the crystallographic ab plane (D⊥ = (1.9 ± 0.2) × 10-10 m2 s-1), despite the walls of these channels appearing impermeable by single-crystal X-ray crystallography and flexible lattice MD simulations. Observation of such unexpected diffusion in the ab plane suggests the presence of defects that enable effective multidimensional CO2 transport in a metal-organic framework with nominally one-dimensional porosity.

Enantioselective Recognition of Ammonium Carbamates in a Chiral Metal-Organic Framework.

Chiral metal-organic frameworks have attracted interest for enantioselective separations and catalysis because of their high crystallinity and pores with tunable shapes, sizes, and chemical environments. Chiral frameworks of the type M2(dobpdc) (M = Mg, Mn, Fe, Co, Ni, Zn; dobpdc4- = 4,4'-dioxidobiphenyl-3,3'-dicarboxylate) seem particularly promising for potential applications because of their excellent stability, high internal surface areas, and strongly polarizing open metal coordination sites within the channels, but to date these materials have been isolated only in racemic form. Here, we demonstrate that when appended with the chiral diamine trans-1,2-diaminocyclohexane (dach), Mg2(dobpdc) adsorbs carbon dioxide cooperatively to form ammonium carbamate chains, and the thermodynamics of CO2 capture are strongly influenced by enantioselective interactions within the chiral pores of the framework. We further show that it is possible to access both enantiomers of Mg2(dobpdc) with high enantiopurity (≥90%) via framework synthesis in the presence of varying quantities of d-panthenol, an inexpensive chiral induction agent. Investigation of dach-M2(dobpdc) samples following CO2 adsorption-using single-crystal and powder X-ray diffraction, solid-state nuclear magnetic resonance spectroscopy, and density functional theory calculations-revealed that the ammonium carbamate chains interact extensively with each other and with the chiral M2(dobpdc) pore walls. Subtle differences in the non-covalent interactions accessible in each diastereomeric phase dramatically impact the thermodynamics of CO2 adsorption.

A Diaminopropane-Appended Metal-Organic Framework Enabling Efficient CO2 Capture from Coal Flue Gas via a Mixed Adsorption Mechanism.

A new diamine-functionalized metal-organic framework comprised of 2,2-dimethyl-1,3-diaminopropane (dmpn) appended to the Mg2+ sites lining the channels of Mg2(dobpdc) (dobpdc4- = 4,4'-dioxidobiphenyl-3,3'-dicarboxylate) is characterized for the removal of CO2 from the flue gas emissions of coal-fired power plants. Unique to members of this promising class of adsorbents, dmpn-Mg2(dobpdc) displays facile step-shaped adsorption of CO2 from coal flue gas at 40 °C and near complete CO2 desorption upon heating to 100 °C, enabling a high CO2 working capacity (2.42 mmol/g, 9.1 wt %) with a modest 60 °C temperature swing. Evaluation of the thermodynamic parameters of adsorption for dmpn-Mg2(dobpdc) suggests that the narrow temperature swing of its CO2 adsorption steps is due to the high magnitude of its differential enthalpy of adsorption (Δhads = -73 ± 1 kJ/mol), with a larger than expected entropic penalty for CO2 adsorption (Δsads = -204 ± 4 J/mol·K) positioning the step in the optimal range for carbon capture from coal flue gas. In addition, thermogravimetric analysis and breakthrough experiments indicate that, in contrast to many adsorbents, dmpn-Mg2(dobpdc) captures CO2 effectively in the presence of water and can be subjected to 1000 humid adsorption/desorption cycles with minimal degradation. Solid-state 13C NMR spectra and single-crystal X-ray diffraction structures of the Zn analogue reveal that this material adsorbs CO2 via formation of both ammonium carbamates and carbamic acid pairs, the latter of which are crystallographically verified for the first time in a porous material. Taken together, these properties render dmpn-Mg2(dobpdc) one of the most promising adsorbents for carbon capture applications.

Electron microscopy using the genetically encoded APEX2 tag in cultured mammalian cells.

Electron microscopy (EM) is the premiere technique for high-resolution imaging of cellular ultrastructure. Unambiguous identification of specific proteins or cellular compartments in electron micrographs, however, remains challenging because of difficulties in delivering electron-dense contrast agents to specific subcellular targets within intact cells. We recently reported enhanced ascorbate peroxidase 2 (APEX2) as a broadly applicable genetic tag that generates EM contrast on a specific protein or subcellular compartment of interest. This protocol provides guidelines for designing and validating APEX2 fusion constructs, along with detailed instructions for cell culture, transfection, fixation, heavy-metal staining, embedding in resin, and EM imaging. Although this protocol focuses on EM in cultured mammalian cells, APEX2 is applicable to many cell types and contexts, including intact tissues and organisms, and is useful for numerous applications beyond EM, including live-cell proteomic mapping. This protocol, which describes procedures for sample preparation from cell monolayers and cell pellets, can be completed in 10 d, including time for APEX2 fusion construct validation, cell growth, and solidification of embedding resins. Notably, the only additional steps required relative to a standard EM sample preparation are cell transfection and a 2- to 45-min staining period with 3,3-diaminobenzidine (DAB) and hydrogen peroxide (H2O2).

Controlling Cooperative CO2 Adsorption in Diamine-Appended Mg2(dobpdc) Metal-Organic Frameworks.

In the transition to a clean-energy future, CO2 separations will play a critical role in mitigating current greenhouse gas emissions and facilitating conversion to cleaner-burning and renewable fuels. New materials with high selectivities for CO2 adsorption, large CO2 removal capacities, and low regeneration energies are needed to achieve these separations efficiently at scale. Here, we present a detailed investigation of nine diamine-appended variants of the metal-organic framework Mg2(dobpdc) (dobpdc4- = 4,4'-dioxidobiphenyl-3,3'-dicarboxylate) that feature step-shaped CO2 adsorption isotherms resulting from cooperative and reversible insertion of CO2 into metal-amine bonds to form ammonium carbamate chains. Small modifications to the diamine structure are found to shift the threshold pressure for cooperative CO2 adsorption by over 4 orders of magnitude at a given temperature, and the observed trends are rationalized on the basis of crystal structures of the isostructural zinc frameworks obtained from in situ single-crystal X-ray diffraction experiments. The structure-activity relationships derived from these results can be leveraged to tailor adsorbents to the conditions of a given CO2 separation process. The unparalleled versatility of these materials, coupled with their high CO2 capacities and low projected energy costs, highlights their potential as next-generation adsorbents for a wide array of CO2 separations.

A split horseradish peroxidase for the detection of intercellular protein-protein interactions and sensitive visualization of synapses.

Intercellular protein-protein interactions (PPIs) enable communication between cells in diverse biological processes, including cell proliferation, immune responses, infection, and synaptic transmission, but they are challenging to visualize because existing techniques have insufficient sensitivity and/or specificity. Here we report a split horseradish peroxidase (sHRP) as a sensitive and specific tool for the detection of intercellular PPIs. The two sHRP fragments, engineered through screening of 17 cut sites in HRP followed by directed evolution, reconstitute into an active form when driven together by an intercellular PPI, producing bright fluorescence or contrast for electron microscopy. Fusing the sHRP fragments to the proteins neurexin (NRX) and neuroligin (NLG), which bind each other across the synaptic cleft, enabled sensitive visualization of synapses between specific sets of neurons, including two classes of synapses in the mouse visual system. sHRP should be widely applicable to studying mechanisms of communication between a variety of cell types.

Directed evolution of APEX2 for electron microscopy and proximity labeling.

APEX is an engineered peroxidase that functions as an electron microscopy tag and a promiscuous labeling enzyme for live-cell proteomics. Because limited sensitivity precludes applications requiring low APEX expression, we used yeast-display evolution to improve its catalytic efficiency. APEX2 is far more active in cells, enabling the use of electron microscopy to resolve the submitochondrial localization of calcium uptake regulatory protein MICU1. APEX2 also permits superior enrichment of endogenous mitochondrial and endoplasmic reticulum membrane proteins.