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.

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.

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.

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.

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.

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.

Affinity of Cu+ for the copper-binding domain of the amyloid-ß peptide of Alzheimer's disease.

The role of metal ions in Alzheimer's disease etiology is unresolved. For the redox-active metal ions iron and copper, the formation of reactive oxygen species by metal amyloid complexes has been proposed to contribute to Alzheimer's disease neurodegeneration. For copper, reactive oxygen species are generated by copper redox cycling between its 1+ and 2+ oxidation states. Thus, the AßCu(I) complex is potentially a critical reactant associated with Alzheimer's disease etiology. Through competitive chelation, we have measured the affinity of the soluble copper-binding domain of the amyloid-ß peptide for Cu(I). The dissociation constants are in the femtomolar range for both wild-type and histidine-to-alanine mutants. These results indicate that Cu(I) binds more tightly to monomeric amyloid-ß than Cu(II) does, which leads us to propose that Cu(I) is a relevant in vivo oxidation state.

Cu K-edge X-ray absorption spectroscopy reveals differential copper coordination within amyloid-ß oligomers compared to amyloid-ß monomers.

The fatal neurological disorder Alzheimer's disease has been linked to soluble neurotoxic oligomers of amyloid-ß (Aß) peptides. Herein we demonstrate that Cu(1+) ligated within Aß(42) oligomers (Aß sequence: DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA) possesses a highly dioxygen sensitive tetrahedral coordination geometry. The biological implications of these findings are discussed.

Fluorescence of unmodified oligonucleotides: A tool to probe G-quadruplex DNA structure.

Fluorescence of unmodified oligonucleotides has not been exploited for guanine-quadruplex (G-quadruplex) characterization. We observe that G-rich sequences fluoresce more strongly than duplex or single-stranded DNA but much more weakly than fluorophores like fluorescein. This increase in the intrinsic fluorescence is not due to an increase in absorption at the excitation wavelength but rather to a change in the quantum yield. We show that unlabeled oligonucleotides that form G-quadruplexes can be differentiated on the basis of their emission spectra from similar sequences that do not contain consecutive guanines. Intermolecular quadruplexes formed by the oligonucleotides 5'-T(4)G(n)T(4)-3' (n = 4-10) display a nonlinear, but continuous, increase in emission intensity as the G content increases. The sequence 5'-GGGT-3', which has been proposed to form a monomeric quadruplex and an interlocked quadruplex (Krishnan-Ghosh et al. J Am Chem Soc 2004, 126, 11009), was compared with the similar sequence 5'-TGGG-3', the structure of which has not been characterized. Both the maximum emission intensity and the spectral shape differ for these oligonucleotides as a function of sample preparation, indicating that different types of quadruplexes form for both sequences. Our work is the first to demonstrate that the suprastructure of G-rich sequences can be probed using fluorescence signatures of unmodified oligonucleotides.

The amyloid-beta peptide of Alzheimer's disease binds Cu(I) in a linear bis-his coordination environment: insight into a possible neuroprotective mechanism for the amyloid-beta peptide.

Oxidative stress has been suggested to contribute to neuronal apoptosis associated with Alzheimer's disease (AD). Copper may participate in oxidative stress through redox-cycling between its +2 and +1 oxidation states to generate reactive oxygen species (ROS). In vitro, copper binds to the amyloid-beta peptide of AD, and in vivo, copper is associated with amyloid plaques characteristic of AD. As a result, the AbetaCu(I) complex may be a critical reactant involved in ROS associated with AD etiology. To characterize the AbetaCu(I) complex, we have pursued X-ray absorption (XAS) and electron paramagnetic resonance (EPR) spectroscopy of AbetaCu(II) and AbetaCu(I) (produced by ascorbate reduction of AbetaCu(II)). The AbetaCu(II) complex Cu K-edge XAS spectrum is indicative of a square-planar Cu(II) center with mixed N/O ligation. Multiple scattering analysis of the extended X-ray absorption fine structure (EXAFS) data for AbetaCu(II) indicates that two of the ligands are imidazole groups of histidine ligands, indicating a (N(Im))(2)(N/O)(2) Cu(II) ligation sphere for AbetaCu(II). After reduction of the AbetaCu(II) complex with ascorbate, the edge region decreases in energy by approximately 4 eV. The X-ray absorption near-edge spectrum region of AbetaCu(I) displays an intense pre-edge feature at 8984.1(2) eV. EXAFS data fitting yielded a two-coordinate geometry, with two imidazole ligands coordinated to Cu(I) at 1.877(2) A in a linear geometry. Ascorbate reduction of AbetaCu(II) under inert atmosphere and subsequent air oxidation of AbetaCu(I) to regenerate AbetaCu(II) was monitored by low-temperature EPR spectroscopy. Slow reappearance of the AbetaCu(II) EPR signal indicates that O(2) oxidation of the AbetaCu(I) complex is kinetically sluggish and Abeta damage is occurring following reoxidation of AbetaCu(I) by O(2). Together, these results lead us to hypothesize that Cu(I) is ligated by His13 and His14 in a linear coordination environment in Alphabeta, that Abeta may be playing a neuroprotective role, and that metal-mediated oxidative damage of Abeta occurs over multiple redox cycles.

Cu(II) binding to monomeric, oligomeric, and fibrillar forms of the Alzheimer's disease amyloid-beta peptide.

Copper has been proposed to play a role in Alzheimer's disease through interactions with the amyoid-beta (Abeta) peptide. The coordination environment of bound copper as a function of Cu:Abeta stoichiometry and Abeta oligomerization state are particularly contentious. Using low-temperature electron paramagnetic resonance (EPR) spectroscopy, we spectroscopically distinguish two Cu(II) binding sites on both soluble and fibrillar Abeta (for site 1, A parallel = 168 +/- 1 G and g parallel = 2.268; for site 2, A parallel = 157 +/- 2 G and g parallel = 2.303). When fibrils that have been incubated with more than 1 equiv of Cu(II) are washed, the second Cu(II) ion is removed, indicating that it is only weakly bound to the fibrils. No change in the Cu(II) coordination environment is detected by EPR spectroscopy of Cu(II) with Abeta (1:1 ratio) collected as a function of Abeta fibrillization time, which indicates that the Cu(II) environment is independent of Abeta oligomeric state. The initial Cu(II)-Abeta complexes go on to form Cu(II)-containing Abeta fibrils. Transmission electron microscopy images of Abeta fibrils before and after Cu(II) addition are the same, showing that once incorporated, Cu(II) does not affect fibrillar structure; however, the presence of Cu(II) appears to induce fibril-fibril association. On the basis of our results, we propose a model for Cu(II) binding to Abeta during fibrillization that is independent of peptide oligomeric state.

Oxidation of guanine in double-stranded DNA by [Ru(bpy)2dppz]Cl2 in cationic reverse micelles.

DNA oxidation has been investigated in the medium of cationic reverse micelles (RMs). The oxidative chemistry is photochemically initiated using the DNA intercalator bis(bipyridine)dipyridophenazine ruthenium(II) chloride ([Ru(bpy)2dppz]Cl2) bound to duplex DNA in the RMs. High-resolution polyacrylamide gel electrophoresis (PAGE) is used to reveal and quantify guanine (G) oxidation products, including 8-oxo-7,8-dihydroguanine (8OG). In buffer solution, the addition of the oxidative quenchers potassium ferricyanide or pentaamminechlorocobalt(III) dichloride leads to an increase in the amount of piperidine-labile G oxidation products generated via one-electron oxidation. In RMs, however, the yield of oxidatively generated damage is attenuated. With or without ferricyanide quencher in the RMs, the yield of oxidatively generated products is approximately the same. Inclusion of the cationic quencher [CoCl(NH3)5]2+ in the RMs increases the amount of oxidation products generated but not to the extent that it does in buffer solution. Under anaerobic conditions, all of the samples in RMs, with or without added oxidative quenchers, show decreased levels of piperidine-labile oxidation products, suggesting that the primary oxidant in RMs is singlet oxygen. G oxidation is enhanced in D2O and deuterated heptane and is diminished in the presence of sodium azide in RMs, also supporting 1O2 as the main G oxidant in RMs. Isotopic labeling experiments show that the oxygen atom in 8OG produced in RMs is not from water. The observed change in the G oxidation mechanism from a one-electron process in buffer to mostly 1O2 in RMs illustrates the importance of both DNA structure and DNA environment on the chemistry of G oxidation.

End-stacking of copper cationic porphyrins on parallel-stranded guanine quadruplexes.

Nucleic acids that contain multiple sequential guanines assemble into guanine quadruplexes (G-quadruplexes). Drugs that induce or stabilize G-quadruplexes are of interest because of their potential use as therapeutics. Previously, we reported on the interaction of the Cu(2+) derivative of 5,10,15,20-tetrakis(1-methyl-4-pyridyl)-21H,23H-porphine (CuTMpyP4), with the parallel-stranded G-quadruplexes formed by d(T(4)G( n )T(4)) (n = 4 or 8) (Keating and Szalai in Biochemistry 43:15891-15900, 2004). Here we present further characterization of this system using a series of guanine-rich oligonucleotides: d(T(4)G( n )T(4)) (n = 5-10). Absorption titrations of CuTMpyP4 with all d(T(4)G( n )G(4)) quadruplexes produce approximately the same bathochromicity (8.3 +/- 2 nm) and hypochromicity (46.2-48.6%) of the porphyrin Soret band. Induced emission spectra of CuTMpyP4 with d(T(4)G( n )T(4))(4) quadruplexes indicate that the porphyrin is protected from solvent. Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry revealed a maximum porphyrin to quadruplex stoichiometry of 2:1 for the shortest (n = 4) and longest (n = 10) quadruplexes. Electron paramagnetic resonance spectroscopy shows that bound CuTMpyP4 occupies magnetically noninteracting sites on the quadruplexes. Consistent with our previous model for d(T(4)G(4)T(4)), we propose that two CuTMpyP4 molecules are externally stacked at each end of the run of guanines in all d(T(4)G( n )T(4)) (n = 4-10) quadruplexes.