Iron-sulfur clusters are involved in post-translational arginylation.

Eukaryotic arginylation is an essential post-translational modification that modulates protein stability and regulates protein half-life. Arginylation is catalyzed by a family of enzymes known as the arginyl-tRNA transferases (ATE1s), which are conserved across the eukaryotic domain. Despite their conservation and importance, little is known regarding the structure, mechanism, and regulation of ATE1s. In this work, we show that ATE1s bind a previously undiscovered [Fe-S] cluster that is conserved across evolution. We characterize the nature of this [Fe-S] cluster and find that the presence of the [Fe-S] cluster in ATE1 is linked to its arginylation activity, both in vitro and in vivo, and the initiation of the yeast stress response. Importantly, the ATE1 [Fe-S] cluster is oxygen-sensitive, which could be a molecular mechanism of the N-degron pathway to sense oxidative stress. Taken together, our data provide the framework of a cluster-based paradigm of ATE1 regulatory control.

Recent advances in microresonators and supporting instrumentation for electron paramagnetic resonance spectroscopy.

Electron paramagnetic resonance (EPR) spectroscopy characterizes the magnetic properties of paramagnetic materials at the atomic and molecular levels. Resonators are an enabling technology of EPR spectroscopy. Microresonators, which are miniaturized versions of resonators, have advanced inductive-detection EPR spectroscopy of mass-limited samples. Here, we provide our perspective of the benefits and challenges associated with microresonator use for EPR spectroscopy. To begin, we classify the application space for microresonators and present the conceptual foundation for analysis of resonator sensitivity. We summarize previous work and provide insight into the design and fabrication of microresonators as well as detail the requirements and challenges that arise in incorporating microresonators into EPR spectrometer systems. Finally, we provide our perspective on current challenges and prospective fruitful directions.

Electron Paramagnetic Resonance Characterization of Sodium- and Carbonate-Containing Hydroxyapatite Cement.

Ionizing radiation-induced paramagnetic defects in calcified tissues like tooth enamel are indicators of irradiation dose. Hydroxyapatite (HA), the principal constituent in these materials, incorporates a variety of anions (CO32-, F-, Cl-, and SiO44-) and cations (Mn2+, Li+, Cu2+, Fe3+, Mg2+, and Na+) that directly or indirectly contribute to the formation of stable paramagnetic centers upon irradiation. Here, we used an underexploited synthesis method based on the ambient temperature setting reaction of a self-hardening calcium phosphate cement (CPC) to create carbonate-containing hydroxyapatite (CHA) and investigate its paramagnetic properties following γ-irradiation. Powder X-ray diffraction and IR spectroscopic characterization of the hardened CHA samples indicate the formation of pure B-type CHA cement. CHA samples exposed to γ-radiation doses ranging from 1 Gy to 150 kGy exhibited an electron paramagnetic resonance (EPR) signal from an orthorhombic CO2•- free radical. At γ-radiation doses from 30 to 150 kGy, a second signal emerged that is assigned to the CO3•- free radical. We observed that the formation of this second species is dose-dependent, which provided a means to extend the useful dynamic range of irradiated CHA to doses >30 kGy. These results indicate that CHA synthesized via a CPC cement is a promising substrate for EPR-based dosimetry. Further studies on the CHA cement are underway to determine the suitability of these materials for a range of biological and industrial dosimetry applications.

The YcnI protein from Bacillus subtilis contains a copper-binding domain.

Bacteria require a precise balance of copper ions to ensure that essential cuproproteins are fully metalated while also avoiding copper-induced toxicity. The Gram-positive bacterium Bacillus subtilis maintains appropriate copper homeostasis in part through the ycn operon. The ycn operon comprises genes encoding three proteins: the putative copper importer YcnJ, the copper-dependent transcriptional repressor YcnK, and the uncharacterized Domain of Unknown Function 1775 (DUF1775) containing YcnI. DUF1775 domains are found across bacterial phylogeny, and bioinformatics analyses indicate that they frequently neighbor domains implicated in copper homeostasis and transport. Here, we investigated whether YcnI can interact with copper and, using electron paramagnetic resonance and inductively coupled plasma-MS, found that this protein can bind a single Cu(II) ion. We determine the structure of both the apo and copper-bound forms of the protein by X-ray crystallography, uncovering a copper-binding site featuring a unique monohistidine brace ligand set that is highly conserved among DUF1775 domains. These data suggest a possible role for YcnI as a copper chaperone and that DUF1775 domains in other bacterial species may also function in copper homeostasis.

Challenges and Advances in the Application of Dynamic Nuclear Polarization to Liquid-State NMR Spectroscopy.

Nuclear magnetic resonance (NMR) spectroscopy is a powerful method to study the molecular structure and dynamics of materials. The inherently low sensitivity of NMR spectroscopy is a consequence of low spin polarization. Hyperpolarization of a spin ensemble is defined as a population difference between spin states that far exceeds what is expected from the Boltzmann distribution for a given temperature. Dynamic nuclear polarization (DNP) can overcome the relatively low sensitivity of NMR spectroscopy by using a paramagnetic matrix to hyperpolarize a nuclear spin ensemble. Application of DNP to NMR can result in sensitivity gains of up to four orders of magnitude compared to NMR without DNP. Although DNP NMR is now more routinely utilized for solid-state (ss) NMR spectroscopy, it has not been exploited to the same degree for liquid-state samples. This Review will consider challenges and advances in the application of DNP NMR to liquid-state samples. The Review is organized into four sections: (i) mechanisms of DNP NMR relevant to hyperpolarization of liquid samples; (ii) applications of liquid-state DNP NMR; (iii) available detection schemes for liquid-state samples; and (iv) instrumental challenges and outlook for liquid-state DNP NMR.

Scalable microresonators for room-temperature detection of electron spin resonance from dilute, sub-nanoliter volume solids.

We report a microresonator platform that allows room temperature detection of electron spins in volumes on the order of 100 pl, and demonstrate its utility to study low levels of dopants in perovskite oxides. We exploit the toroidal moment in a planar anapole, using a single unit of an anapole metamaterial architecture to produce a microwave resonance exhibiting a spatially confined magnetic field hotspot and simultaneously high quality-factor (Q-factor). To demonstrate the broad implementability of this design and its scalability to higher frequencies, we deploy the microresonators in a commercial electron paramagnetic resonance (EPR) spectrometer operating at 10 GHz and a NIST-built EPR spectrometer operating at 35 GHz. We report continuous-wave (CW) EPR spectra for various samples, including a dilute Mn2+-doped perovskite oxide, CaTiO3, and a transition metal complex, CuCl22H2O. The anapole microresonator presented here is expected to enable multifrequency EPR characterization of dopants and defects in perovskite oxide microcrystals and other volume-limited materials of technological importance.

The C-Terminus and Third Cytoplasmic Loop Cooperatively Activate Mouse Melanopsin Phototransduction.

Melanopsin, an atypical vertebrate visual pigment, mediates non-image-forming light responses including circadian photoentrainment and pupillary light reflexes and contrast detection for image formation. Melanopsin-expressing intrinsically photosensitive retinal ganglion cells are characterized by sluggish activation and deactivation of their light responses. The molecular determinants of mouse melanopsin's deactivation have been characterized (i.e., C-terminal phosphorylation and ß-arrestin binding), but a detailed analysis of melanopsin's activation is lacking. We propose that an extended third cytoplasmic loop is adjacent to the proximal C-terminal region of mouse melanopsin in the inactive conformation, which is stabilized by the ionic interaction of these two regions. This model is supported by site-directed spin labeling and electron paramagnetic resonance spectroscopy of melanopsin, the results of which suggests a high degree of steric freedom at the third cytoplasmic loop, which is increased upon C-terminus truncation, supporting the idea that these two regions are close in three-dimensional space in wild-type melanopsin. To test for a functionally critical C-terminal conformation, calcium imaging of melanopsin mutants including a proximal C-terminus truncation (at residue 365) and proline mutation of this proximal region (H377P, L380P, Y382P) delayed melanopsin's activation rate. Mutation of all potential phosphorylation sites, including a highly conserved tyrosine residue (Y382), into alanines also delayed the activation rate. A comparison of mouse melanopsin with armadillo melanopsin-which has substitutions of various potential phosphorylation sites and a substitution of the conserved tyrosine-indicates that substitution of these potential phosphorylation sites and the tyrosine residue result in dramatically slower activation kinetics, a finding that also supports the role of phosphorylation in signaling activation. We therefore propose that melanopsin's C-terminus is proximal to intracellular loop 3, and C-terminal phosphorylation permits the ionic interaction between these two regions, thus forming a stable structural conformation that is critical for initiating G-protein signaling.

The FeoC [4Fe-4S] Cluster Is Redox-Active and Rapidly Oxygen-Sensitive.

The acquisition of iron is essential to establishing virulence among most pathogens. Under acidic and/or anaerobic conditions, most bacteria utilize the widely distributed ferrous iron (Fe2+) uptake (Feo) system to import metabolically-required iron. The Feo system is inadequately understood at the atomic, molecular, and mechanistic levels, but we do know it is composed of a main membrane component (FeoB) essential for iron translocation, as well as two small, cytosolic proteins (FeoA and FeoC) hypothesized to function as accessories to this process. FeoC has many hypothetical functions, including that of an iron-responsive transcriptional regulator. Here, we demonstrate for the first time that Escherichia coli FeoC (EcFeoC) binds an [Fe-S] cluster. Using electronic absorption, X-ray absorption, and electron paramagnetic resonance spectroscopies, we extensively characterize the nature of this cluster. Under strictly anaerobic conditions after chemical reconstitution, we demonstrate that EcFeoC binds a redox-active [4Fe-4S]2+/+ cluster that is rapidly oxygen-sensitive and decays to a [2Fe-2S]2+ cluster (t1/2 ≈ 20 s), similar to the [Fe-S] cluster in the fumarate and nitrate reductase (FNR) transcriptional regulator. We further show that this behavior is nearly identical to the homologous K. pneumoniae FeoC, suggesting a redox-active, oxygen-sensitive [4Fe-4S]2+ cofactor is a general phenomenon of cluster-binding FeoCs. Finally, in contrast to FNR, we show that the [4Fe-4S]2+ cluster binding to FeoC is associated with modest conformational changes of the polypeptide, but not protein dimerization. We thus posit a working hypothesis in which the cluster-binding FeoCs may function as oxygen-sensitive iron sensors that fine-tune pathogenic ferrous iron acquisition.

Nonresonant Transmission Line Probe for Sensitive Interferometric Electron Spin Resonance Detection.

Electron spin resonance (ESR) spectroscopy measures paramagnetic free radicals, or electron spins, in a variety of biological, chemical, and physical systems. Detection of diverse paramagnetic species is important in applications ranging from quantum computation to biomedical research. Countless efforts have been made to improve the sensitivity of ESR detection. However, the improvement comes at the cost of experimental accessibility. Thus, most ESR spectrometers are limited to specific sample geometries and compositions. Here, we present a nonresonant transmission line ESR probe (microstrip geometry) that effectively couples high frequency microwave magnetic field into a wide range of sample geometries and compositions. The nonresonant transmission line probe maintains detection sensitivity while increasing availability to a wider range of applications. The high frequency magnetic field homogeneity is greatly increased by positioning the sample between the microstrip signal line and the ground plane. Sample interfacing occurs via a universal sample holder which is compatible with both solid and liquid samples. The unavoidable loss in sensitivity due to the nonresonant nature of the transmission line probe (low Q) is recuperated by using a highly sensitive microwave interferometer-based detection circuit. The combination of our sensitive interferometer and nonresonant transmission line provides similar sensitivity to a commercially available ESR spectrometer equipped with a high-Q resonator. The nonresonant probe allows for transmission, reflection, or dual-mode detection (transmission and reflection), where the dual-mode results in a √2 signal enhancement.

Mapping Electron Transfer at MoS2 Using Scanning Electrochemical Microscopy.

Understanding the role of macroscopic and atomic defects in the interfacial electron transfer properties of layered transition metal dichalcogenides is important in optimizing their performance in energy conversion and electronic devices. Means of determining the heterogeneous electron transfer rate constant, k, have relied on the deliberate exposure of specific electrode regions or additional surface characterization to correlate proposed active sites to voltammetric features. Few studies have investigated the electrochemical activity of surface features of layered dichalcogenides under the same experimental conditions. Herein, MoS2 flakes with well-defined features were mapped using scanning electrochemical microscopy (SECM). At visually flat areas of MoS2, k of hexacyanoferrate(III) ([Fe(CN)6]3-) and hexacyanoferrate(II) ([Fe(CN)6]4-) was typically smaller and spanned a larger range than that of hexaammineruthenium(III) ([Ru(NH3)6]3+), congruent with the current literature. However, in contrast to previous studies, the reduction of [Fe(CN)6]3- and the oxidation of [Fe(CN)6]4- exhibited similar rate constants, attributed to the dominance of charge transfer through surface states. The comparison of SECM with optical and atomic force microscopy images revealed that while most of the flake was electroactive, edge sites associated with freshly exposed areas that include macrosteps consisting of several monolayers as well as recessed areas exhibited the highest reactivity, consistent with the reported results.

Integration of a versatile bridge concept in a 34 GHz pulsed/CW EPR spectrometer.

We present a 34 GHz continuous wave (CW)/pulsed electron paramagnetic resonance (EPR) spectrometer capable of pulse-shaping that is based on a versatile microwave bridge design. The bridge radio frequency (RF)-in/RF-out design (500 MHz to 1 GHz input/output passband, 500 MHz instantaneous input/output bandwidth) creates a flexible platform with which to compare a variety of excitation and detection methods utilizing commercially available equipment external to the bridge. We use three sources of RF input to implement typical functions associated with CW and pulse EPR spectroscopic measurements. The bridge output is processed via high speed digitizer and an in-phase/quadrature (I/Q) demodulator for pulsed work or sent to a wideband, high dynamic range log detector for CW. Combining this bridge with additional commercial hardware and new acquisition and control electronics, we have designed and constructed an adaptable EPR spectrometer that builds upon previous work in the literature and is functionally comparable to other available systems.

Molecular Precision at Micrometer Length Scales: Hierarchical Assembly of DNA-Protein Nanostructures.

Robust self-assembly across length scales is a ubiquitous feature of biological systems but remains challenging for synthetic structures. Taking a cue from biology-where disparate molecules work together to produce large, functional assemblies-we demonstrate how to engineer microscale structures with nanoscale features: Our self-assembly approach begins by using DNA polymerase to controllably create double-stranded DNA (dsDNA) sections on a single-stranded template. The single-stranded DNA (ssDNA) sections are then folded into a mechanically flexible skeleton by the origami method. This process simultaneously shapes the structure at the nanoscale and directs the large-scale geometry. The DNA skeleton guides the assembly of RecA protein filaments, which provides rigidity at the micrometer scale. We use our modular design strategy to assemble tetrahedral, rectangular, and linear shapes of defined dimensions. This method enables the robust construction of complex assemblies, greatly extending the range of DNA-based self-assembly methods.

Fabrication of Scanning Electrochemical Microscopy-Atomic Force Microscopy Probes to Image Surface Topography and Reactivity at the Nanoscale.

Concurrent mapping of chemical reactivity and morphology of heterogeneous electrocatalysts at the nanoscale allows identification of active areas (protrusions, flat film surface, or cracks) responsible for productive chemistry in these materials. Scanning electrochemical microscopy (SECM) can map surface characteristics, record catalyst activity, and identify chemical products at solid-liquid electrochemical interfaces. It lacks, however, the ability to distinguish topographic features where surface reactivity occurs. Here, we report the design and fabrication of scanning probe tips that combine SECM with atomic force microscopy (AFM) to perform measurements at the nanoscale. Our probes are fabricated by integrating nanoelectrodes with quartz tuning forks (QTFs). Using a calibration standard fabricated in our lab to test our probes, we obtain simultaneous topographic and electrochemical reactivity maps with a lateral resolution of 150 nm.

Two-Step Cycle for Producing Multiple Anodic Aluminum Oxide (AAO) Films with Increasing Long-Range Order.

Nanoporous anodic aluminum oxide (AAO) membranes are being used for an increasing number of applications. However, the original two-step anodization method in which the first anodization is sacrificial to pre-pattern the second is still widely used to produce them. This method provides relatively low throughput and material utilization as half of the films are discarded. An alternative scheme that relies on alternating anodization and cathodic delamination is demonstrated that allows for the fabrication of several AAO films with only one sacrificial layer thus greatly improving total aluminum to alumina yield. The thickness for which the cathodic delamination performs best to yield full, unbroken AAO sheets is around 85 µm. Additionally, an image analysis method is used to quantify the degree of long-range ordering of the unit cells in the AAO films which was found to increase with each successive iteration of the fabrication cycle.

Optical Fluorescence Microscopy for Spatially Characterizing Electron Transfer across a Solid-Liquid Interface on Heterogeneous Electrodes.

Heterogeneous catalytic materials and electrodes are used for (electro)chemical transformations, including those important for energy storage and utilization.1, 2 Due to the heterogeneous nature of these materials, activity measurements with sufficient spatial resolution are needed to obtain structure/activity correlations across the different surface features (exposed facets, step edges, lattice defects, grain boundaries, etc.). These measurements will help lead to an understanding of the underlying reaction mechanisms and enable engineering of more active materials. Because (electro)catalytic surfaces restructure with changing environments,1 it is important to perform measurements in operando. Sub-diffraction fluorescence microscopy is well suited for these requirements because it can operate in solution with resolution down to a few nm. We have applied sub-diffraction fluorescence microscopy to a thin cell containing an electrocatalyst and a solution containing the redox sensitive dye p-aminophenyl fluorescein to characterize reaction at the solid-liquid interface. Our chosen dye switches between a nonfluorescent reduced state and a one-electron oxidized bright state, a process that occurs at the electrode surface. This scheme is used to investigate the activity differences on the surface of polycrystalline Pt, in particular to differentiate reactivity at grain faces and grain boundaries. Ultimately, this method will be extended to study other dye systems and electrode materials.

Design considerations for enhancing absorption in semiconductors on metals through surface plasmon polaritons.

Surface plasmon polaritons have attracted attention for energy applications such as photovoltaic and photoelectrochemical cells because of their ability to improve optical absorption in thin films. We show that surface plasmon polaritons enhance absorption most significantly in materials with small positive real permittivity and large positive imaginary permittivity, e.g. organics or CdTe. Additional losses, accounting for dissipation in the metal and the existence of a cutoff frequency above which polaritons are no longer bound, are incorporated into efficiency calculations. Owing to these losses, devices with optical absorption based solely on SPPs will necessarily always have a lower efficiency than that predicted by the Shockley-Queisser limit. Calculations are presented for specific materials, including crystalline and amorphous Si, GaAs, CdTe, a P3HT:PCBM blend, α-Fe2O3 and rutile TiO2, as well as for general materials of arbitrary permittivity. Guidelines for selecting absorber materials and determining whether specific materials are good candidates for improving optical absorption with SPPs are presented.

N-methylmesoporphyrin IX fluorescence as a reporter of strand orientation in guanine quadruplexes.

Guanine quadruplexes (GQ) are four-stranded DNA structures formed by guanine-rich DNA sequences. The formation of GQs inhibits cancer cell growth, although the detection of GQs in vivo has proven difficult, in part because of their structural diversity. The development of GQ-selective fluorescent reporters would enhance our ability to quantify the number and location of GQs, ultimately advancing biological studies of quadruplex relevance and function. N-methylmesoporphyrin IX (NMM) interacts selectively with parallel-stranded GQs; in addition, its fluorescence is sensitive to the presence of DNA, making this ligand a possible candidate for a quadruplex probe. In the present study, we investigated the effect of DNA secondary structure on NMM fluorescence. We found that NMM fluorescence increases by about 60-fold in the presence of parallel-stranded GQs and by about 40-fold in the presence of hybrid GQs. Antiparallel GQs lead to lower than 10-fold increases in NMM fluorescence. Single-stranded DNA, duplex, or i-motif, induce no change in NMM fluorescence. We conclude that NMM shows promise as a 'turn-on' fluorescent probe for detecting quadruplex structures, as well as for differentiating them on the basis of strand orientation.

Tunable electrical conductivity in metal-organic framework thin-film devices.

We report a strategy for realizing tunable electrical conductivity in metal-organic frameworks (MOFs) in which the nanopores are infiltrated with redox-active, conjugated guest molecules. This approach is demonstrated using thin-film devices of the MOF Cu3(BTC)2 (also known as HKUST-1; BTC, benzene-1,3,5-tricarboxylic acid) infiltrated with the molecule 7,7,8,8-tetracyanoquinododimethane (TCNQ). Tunable, air-stable electrical conductivity over six orders of magnitude is achieved, with values as high as 7 siemens per meter. Spectroscopic data and first-principles modeling suggest that the conductivity arises from TCNQ guest molecules bridging the binuclear copper paddlewheels in the framework, leading to strong electronic coupling between the dimeric Cu subunits. These ohmically conducting porous MOFs could have applications in conformal electronic devices, reconfigurable electronics, and sensors.

Synapsable quadruplex-mediated fibers.

We have fabricated a DNA-based nanofiber created by self-assembly of guanine quadruplex (Hoogsteen base pairing) and double-stranded DNA (Watson-Crick base pairing). When duplexes containing a long stretch of contiguous guanines and single-stranded overhangs are incubated in potassium-containing buffer, the preformed duplexes create high molecular weight species that contain quadruplexes. In addition to observation of these larger species by gel electrophoresis, solutions were analyzed by atomic force microscopy to reveal nanofibers. Analysis of the atomic force microscopy images indicates that fibers form with lengths ranging from 250 to 2,000 nm and heights from 0.45 to 4.0 nm. This work is a first step toward the creation of new structurally heterogeneous (quadruplex/duplex), yet controllable, DNA-based materials exhibiting novel properties suitable for a diverse array of nanotechnology applications.

Local structure and global patterning of Cu2+ binding in fibrillar amyloid-ß [Aß(1-40)] protein.

The amyloid-ß (Aß) protein forms fibrils and higher-order plaque aggegrates in Alzheimer's disease (AD) brain. The copper ion, Cu(2+), is found at high concentrations in plaques, but its role in AD etiology is unclear. We use high-resolution pulsed electron paramagnetic resonance spectroscopy to characterize the coordination structure of Cu(2+) in the fibrillar form of full-length Aß(1-40). The results reveal a bis-cis-histidine (His) equatorial Cu(2+) coordination geometry and participation of all three N-terminal His residues in Cu(2+) binding. A model is proposed in which Cu(2+)-His6/His13 and Cu(2+)-His6/His14 sites alternate along the fibril axis on opposite sides of the ß-sheet fibril structure. The local intra-ß-strand coordination structure is not conducive to Cu(2+)/Cu(+) redox-linked coordination changes, and the global arrangement of Cu sites precludes facile multielectron and bridged-metal site reactivity. This indicates that the fibrillar form of Aß suppresses Cu redox cycling and reactive oxygen species production. The configuration suggests application of Cu(2+)-Aß fibrils as an amyloid architecture for switchable electron charge/spin coupling and redox reactivity.