Single Zinc Atom Research Articles

Boosted hydrogen evolution kinetics of heteroatom-doped carbons with isolated Zn as an accelerant

Carbon-based single-atom catalysts, a promising candidate in electrocatalysis, offer insights into electron-donating effects of metal center on adjacent atoms. Herein, we present a practical strategy to rationally design a model catalyst with a single zinc (Zn) atom coordinated with nitrogen and sulfur atoms in a multilevel carbon matrix. The Zn site exhibits an atomic interface configuration of ZnN4S1, where Zn's electron injection effect enables thermal-neutral hydrogen adsorption on neighboring atoms, pushing the activity boundaries of carbon electrocatalysts toward electrochemical hydrogen evolution to an unprecedented level. Experimental and theoretical analyses confirm the low-barrier Volmer-Tafel mechanism of proton reduction, while the multishell hollow structures facilitate the hydrogen evolution even at high current intensities. This work provides insights for understanding the actual active species during hydrogen evolution reaction and paves the way for designing high-performance electrocatalysts.

Rational design of MXene-based single atom catalysts for Na-Se batteries from sabatier principle.

Na-Se batteries have attracted great attention because of their high-energy density and low cost, though the shuttle effect of polyselenides and sluggish reaction dynamics still limit their practical applications. Herein, MXenes were decorated with single zinc atom as selenium hosts, and the effect of interfacial electrochemical reaction was studied via first-principles simulation. The embedding of single zinc atom into MXenes was found to enhance the anchoring ability to inhibit the shuttle effect. However, Zn-MXenes as single atom catalysts had different effects on interfacial electrochemical reactions, which can be attributed to the increased interaction strengths between Zn-MXenes and polyselenides. For Ti-based MXenes, the enhanced interaction was found to be beneficial for the electrochemical reaction, whereas the overly strong anchoring strength of Zn-Cr2CO2 would inhibit charging-discharging kinetics. Therefore, the matching of MXenes and metal atoms should be considered to adjust the anchoring ability based on the Sabatier principle. This work provides new insights into the design of SACs and high-performance Na-Se batteries.

Single Zinc Atom Aggregates: Synergetic Interaction to Boost Fast Polysulfide Conversion in Lithium-Sulfur Batteries.

Single-atom catalysts (SACs) pave new possibilities to improve the utilization efficiency of sulfur electrodes arising from polysulfide shuttle effects and sluggish kinetics due to their excellent applicability in atomic-scale reaction mechanisms and structure-activity relationships. Herein, nitrogen (N)-anchored SACs on the highly ordered N-doped carbon nanotube arrays are reported as the sulfur host for fast redox conversion in lithium-sulfur (Li-S) batteries. The cube structure of the aligned carbon nanotubes can promote the rapid mass transfer under high sulfur loadings, and abundant single-atom active sites further accelerate the conversion of lithium polysulfides (LiPSs). The synergistic enhancement effect induced by adjacent single atoms with interatomic distances <1nm further accelerates the rapid multi-step reaction of sulfur at high sulfur loadings. As a result, the obtained Li-S batteries exhibit outstanding cycle stability with a high areal capacity of 5.6mAhcm-2 after 100 cycles under a high sulfur loading of 7.2mgcm-2 (electrolyte to sulfur ratio is ≈3.7mLg-1 ). Even assembled into a pouch cell, it still delivers a high capacity of 953.4mAhg-1 after 100 cycles at 0.1C, contributing to the development of the practically viable Li-S batteries.

Chemical transformation and dissociation of amino acids on metal sulfide surface: Insights from DFT into the effect of surface vacancies on alanine-sphalerite system

The affinity of amino acid species toward mineral surfaces is a favorable phenomenon in green material science applications. To this end, we performed a DFT calculation to study adsorption of alanine on the sphalerite (ZnS) surface, focusing on the role of surface vacancy defects by removing single sulfur, single zinc and double neighboring congruent atoms from the outermost layer. Removal of single sulfur or zinc atom led to surface reconstruction by forming favorable local Zn-Zn and S-S bonds around the defect sites, respectively. The presence of double neighboring congruent vacancies and their relative positions altered the adsorption configuration to more complicated modes. The lowest-energy configuration was predicted when both sulfur and zinc atoms were removed from the upper hill of the surface. In this case, alanine was dissociatively adsorbed on the surface, and a tridentate bond was formed between alanine and two zinc atoms on the surface. The alanine affinity was also significantly more stable than the defect-free surface with an energy difference of about 55 kcal/mol. In another special case with two Zn and S vacancies, respectively, on the downward and upper hills, alanine underwent a chemical transformation from neutral to zwitterionic form according to an intramolecular dissociation reaction.

Zinc 1s Valence-to-Core X-ray Emission Spectroscopy of Halozincate Complexes.

The Zn 1s valence-to-core (VtC) X-ray emission spectra of seven ionic liquids have been measured experimentally and simulated on the basis of time-dependent density-functional theory (TDDFT) calculations. Six of the ionic liquids were made by mixing [C8C1Im]X and Zn(II)X2 at three different ZnX2 mole fractions (0.33, 0.50, or 0.67) for X = Cl or Br, and a further ionic liquid was made by mixing [P6,6,6,14]Cl and a mole fraction of ZnCl2 of 0.33. Calculations were performed for the [ZnX4]2-, [Zn2X6]2-, and [Zn4X10]2- ions to capture the expected metal complex speciation. The VtC emission spectra showed three bands arising from single-electron processes that can be assigned to emission from ligand p-type orbitals, zinc d-orbitals, and ligand s-type orbitals. For all seven ionic liquids, the highest occupied molecular orbital arises from the ligand p orbitals, and the spectra for the different size metal complexes for the same X were found to be very similar, in terms of both relative peak intensities and peak energies. For both experiments and TDDFT calculations, there was an energy difference of 0.5 eV between the Cl-based and Br-based metal complexes for the ligand s and p orbitals, while the Zn 3d orbital energies were relatively unaffected by the identity of the ligand. The TDDFT calculations find that for the ions with symmetrically equivalent zinc atoms ([Zn2X6]2- and [Zn4X10]2-), the most appropriate core-ionized reference state has a core-hole that is localized on a single zinc atom. In this framework, the spectra for the larger ions can be viewed as a sum of spectra for the tetrahedral complex with a single zinc atom with small variations in the structure of the coordinating ligands. Because the spectra are relatively insensitive to small changes in the geometry of the ligands, this is consistent with the small variation in the spectra measured in the experiment.

Mega High Utilization of Sodium Metal Anodes Enabled by Single Zinc Atom Sites.

Low utilization of active metallic sodium (Na) and uncontrollable growth of Na dendrites remain significant challenges for high-performance Na metal batteries, which are limited to inefficient Na utilization (<1%) and shallow cycling conditions (0.25-1.0 mAh cm-2). In this work, a kind of Na metal anode with record-high utilization and long-term cycling stability is reported, using carbon-substrate-supported nitrogen-anchored zinc (Zn) single atoms as a current collector. Single Zn atom sites whichserve as a strong "magnet" for Na ions,can guide the metallic Na uniform nucleation and free from dendrite-induced short circuit. The nucleation overpotential of our strategy is essentially zero, where most of the reported modified substrates weregreatly exceed 20 mV. Specifically, the Na anodes exhibit a high Na stripping/plating Coulombic efficiency with 99.8% over 350 cycles and a stable voltage response with small voltage hysteresis after cycling 1000 h. The full cell exhibits high Na utilization up to 100% and superior long-term cycling stability for more than 1000 cycles with excellent capacity retention. In terms of lifetime and Na utilization, the Na metal anodes based onour strategysignificantly outperforms the reported state-of-the-art Na metal anodes. Moreover, this affords new insights into the controllable Na nucleation behavior and high Na utilization and sheds fresh light on atomic level design of an electrode for Na metal anodes.

Characterisation of the membrane topology and molecular structure of LITAF to provide insights into the molecular pathogenesis of Charcot-Marie-Tooth disease type 1C

BackgroundThe Charcot-Marie-Tooth (CMT) diseases are the commonest inherited neuromuscular disorders. These disorders are characterised by progressive degenerative peripheral neuropathies and are associated with mutations in over 80 genes. Mutations in LITAF cause CMT type 1C (CMT1C), an autosomal dominant demyelinating subtype of the disease. Why mutations in LITAF cause disease remains unclear, so we aimed to gather data about its molecular structure, which are lacking despite being highly conserved throughout evolution. MethodsWe applied biochemical and cell biological techniques (differential membrane preparation, semi-permeabilisation and immunofluorescence microscopy, and cell-free protein expression) to determine the membrane topology of LITAF. Micro particle-induced X-ray emission (microPIXE) was used to establish whether LITAF associates with metal ions. We then analysed recombinantly expressed LITAF contructs by solution nuclear magnetic resonance (NMR) spectroscopy to gain structural insights to the LITAF protein, thereby allowing us to use the Rosetta software to generate a structural molecular model. FindingsWe found that LITAF behaves as an integral membrane protein and that membrane association is dependent on six leucine residues found within the C-terminal LITAF domain. The membrane topology of the LITAF domain was such that both N-terminals and C-terminals were found on the cytosolic surface of endosomes and that these regions coordinated a single zinc atom. NMR spectroscopy was then used to characterise the secondary structural motifs of the LITAF domain, resulting in a correlation spectrum which was over 90% assigned. These data enabled us to create a structural model using NMR restraints and Rosetta. A LITAF construct harbouring a CMT1C-associated pathogenic mutation was then analysed by NMR, revealing marked structural shifts across the LITAF domain. InterpretationWe present the first structural model, to our knowledge, of the highly-conserved LITAF domain, which is a membrane-anchoring motif forming a zinc-binding structure on the surface of endosomes. We also show that a CMT1C-associated mutation causes large structural shifts across the LITAF domain, suggesting that structural instability on the surface of Scwann cell endosomes underpins the neuropathy seen in CMT1C. These insights will provide a platform for the development of future treatments. FundingWellcome Trust.

Structure of N-formimino-L-glutamate iminohydrolase from Pseudomonas aeruginosa.

N-Formimino-l-glutamate iminohydrolase (HutF), from Pseudomonas aeruginosa with a locus tag of Pa5106 (gi|15600299), is a member of the amidohydrolase superfamily. This enzyme catalyzes the deamination of N-formimino-l-glutamate to N-formyl-l-glutamate and ammonia in the histidine degradation pathway. The crystal structure of Pa5106 was determined in the presence of the inhibitors N-formimino-l-aspartate and N-guanidino-l-glutaric acid at resolutions of 1.9 and 1.4 Å, respectively. The structure of an individual subunit is composed of two domains with the larger domain folding as a distorted (β/α)8-barrel. The (β/α)8-barrel domain is composed of eight β-strands flanked by 11 α-helices, whereas the smaller domain is made up of eight β-strands. The active site of Pa5106 contains a single zinc atom that is coordinated by His-56, His-58, His-232, and Asp-320. The nucleophilic solvent water molecule coordinates with the zinc atom at a distance of 2.0 Å and is hydrogen bonded to Asp-320 and His-269. The α-carboxylate groups of both inhibitors are hydrogen bonded to the imidazole moiety of His-206, the hydroxyl group of Tyr-121, and the side chain amide group of Gln-61. The side chain carboxylate groups of the two inhibitors are ion-paired with the guanidino groups of Arg-209 and Arg-82. Computational docking of high-energy tetrahedral intermediate forms of the substrate, N-formimino-l-glutamate, to the three-dimensional structure of Pa5106 suggests that this compound likely undergoes a re-faced nucleophilic attack at the formimino group by the metal-bound hydroxide. A catalytic mechanism of the reaction catalyzed by Pa5106 is proposed.

Toxic and nontoxic components of botulinum neurotoxin complex are evolved from a common ancestral zinc protein

Zinc atoms play an essential role in a number of enzymes. Botulinum neurotoxin (BoNT), the most potent toxin known in nature, is a zinc-dependent endopeptidase. Here we identify the nontoxic nonhemagglutinin (NTNHA), one of the BoNT-complex constituents, as a zinc-binding protein, along with BoNT. A protein structure classification database search indicated that BoNT and NTNHA share a similar domain architecture, comprising a zinc-dependent metalloproteinase-like, BoNT coiled-coil motif and concanavalin A-like domains. Inductively coupled plasma-mass spectrometry analysis demonstrated that every single NTNHA molecule contains a single zinc atom. This is the first demonstration of a zinc atom in this protein, as far as we know. However, the NTNHA molecule does not possess any known zinc-coordinating motif, whereas all BoNT serotypes possess the classical HEXXH motif. Homology modeling of the NTNHA structure implied that a consensus K-C-L-I-K-X35-D sequence common among all NTNHA serotype molecules appears to coordinate a single zinc atom. These findings lead us to propose that NTNHA and BoNT may have evolved distinct functional specializations following their branching out from a common ancestral zinc protein.

A Two-Site Mechanism for the Inhibition of IAPP Amyloidogenesis by Zinc

Human islet amyloid polypeptide (hIAPP) is a highly amyloidogenic protein co-secreted with insulin in response to glucose levels. The formation of hIAPP amyloid plaques near islet cells has been linked to the death of insulin-secreting β-cells in humans and the progression of type II diabetes. Since both healthy individuals and those with type II diabetes produce and secrete hIAPP, it is reasonable to look for factors involved in storing hIAPP and preventing amyloidosis. We have previously shown that zinc inhibits the formation of insoluble amyloid plaques of hIAPP; however, there remains significant ambiguity in the underlying mechanisms. In this study, we show that zinc binds unaggregated hIAPP at micromolar concentrations similar to those found in the extracellular environment. By contrast, the fibrillar amyloid form of hIAPP has low affinity for zinc. The binding stoichiometry obtained from isothermal titration calorimetry experiments indicates that zinc favors the formation of hIAPP hexamers. High-resolution NMR structures of hIAPP bound to zinc reveal changes in the electron environment along residues that would be located along one face of the amphipathic hIAPP α-helix proposed as an intermediate for amyloid formation. Results from electrospray ionization mass spectroscopy investigations showed that a single zinc atom is predominantly bound to hIAPP and revealed that zinc inhibits the formation of the dimer. At higher concentrations of zinc, a second zinc atom binds to hIAPP, suggesting the presence of a low-affinity secondary binding site. Combined, these results suggest that zinc promotes the formation of oligomers while creating an energetic barrier for the formation of amyloid fibers.

Zinc Binding in Pestivirus N pro Is Required for Interferon Regulatory Factor 3 Interaction and Degradation

Pestiviruses, such as bovine viral diarrhea virus and classical swine fever virus (CSFV), use the viral protein N pro to subvert host cell antiviral responses. N pro is the first protein encoded by the single large open reading frame of the pestivirus positive-sense RNA genome and has an autoproteolytic activity, cleaving itself off from the polyprotein. N pro also targets interferon regulatory factor 3 (IRF3), a transcription factor for alpha/beta interferon genes, and promotes its proteasomal degradation, a process that is independent of the proteolytic activity of N pro. We determined that N pro contains a novel metal-binding TRASH motif consisting of Cys-X 21-Cys-X 3-Cys (where X is any amino acid) at its C-terminus. We also found that N pro coordinates a single zinc atom as determined by graphite furnace–atomic absorption spectrophotometry and inductively coupled plasma–mass spectrometry. Mutational and biochemical analyses show that the cysteine residues in the TRASH motif are required for zinc binding and protein stability. Individual substitutions of the cysteines in the TRASH motif of CSFV N pro abolished the interaction of N pro with IRF3 and resulted in the loss of virus-mediated IRF3 degradation in CSFV-infected cells. Thus, the zinc-binding ability of N pro in pestiviruses appears to be essential for the virus-mediated degradation of IRF3.

Dihydroorotase from the Hyperthermophile Aquifiex aeolicus Is Activated by Stoichiometric Association with Aspartate Transcarbamoylase and Forms a One-Pot Reactor for Pyrimidine Biosynthesis

In prokaryotes, the first three enzymes in pyrimidine biosynthesis, carbamoyl phosphate synthetase (CPS), aspartate transcarbamoylase (ATC), and dihydroorotase (DHO), are commonly expressed separately and either function independently (Escherichia coli) or associate into multifunctional complexes (Aquifex aeolicus). In mammals the enzymes are expressed as a single polypeptide chain (CAD) in the order CPS-DHO-ATC and associate into a hexamer. This study presents the three-dimensional structure of the noncovalent hexamer of DHO and ATC from the hyperthermophile A. aeolicus at 2.3 A resolution. It is the first structure of any multienzyme complex in pyrimidine biosynthesis and is a possible model for the core of mammalian CAD. The structure has citrate, a near isosteric analogue of carbamoyl aspartate, bound to the active sites of both enzymes. Three active site loops that are intrinsically disordered in the free, inactive DHO are ordered in the complex. The reorganization also changes the peptide bond between Asp153, a ligand of the single zinc atom in DHO, and Gly154, to the rare cis conformation. In the crystal structure, six DHO and six ATC chains form a hollow dodecamer, in which the 12 active sites face an internal reaction chamber that is approximately 60 A in diameter and connected to the cytosol by narrow tunnels. The entrances and the interior of the chamber are both electropositive, which suggests that the architecture of this nanoreactor modifies the kinetics of the bisynthase, not only by steric channeling but also by preferential escape of the product, dihydroorotase, which is less negatively charged than its precursors, carbamoyl phosphate, aspartate, or carbamoyl aspartate.

Characterisation of zinc-binding domains of peroxisomal RING finger proteins using size exclusion chromatography/inductively coupled plasma-mass spectrometry

We determined the zinc binding stoichiometry of peroxisomal RING finger proteins by measuring sulfur/metal ratios using inductively coupled plasma-mass spectrometry coupled to size exclusion chromatography, a strategy that provides a fast and quantitative overview on the binding of metals in proteins. As a quality control, liquid chromatography-electrospray ionisation-time of flight-mass spectrometry was used to measure the molar masses of the intact proteins. The RING fingers of Pex2p, Pex10p, and Pex12p showed a stoichiometry of 2.0, 2.1, and 1.2 mol zinc/mol protein, respectively. Thus, Pex2p and Pex10p possess a typical RING domain with two coordinated zinc atoms, whereas that of Pex12p coordinates only a single zinc atom.

Solution Structure of the THAP Domain from Caenorhabditis elegans C-terminal Binding Protein (CtBP)

The THAP (Thanatos-associated protein) domain is a recently discovered zinc-binding domain found in proteins involved in transcriptional regulation, cell-cycle control, apoptosis and chromatin modification. It contains a single zinc atom ligated by cysteine and histidine residues within a Cys-X 2-4-Cys-X 35-53-Cys-X 2-His consensus. We have determined the NMR solution structure of the THAP domain from Caenorhabditis elegans C-terminal binding protein (CtBP) and show that it adopts a fold containing a treble clef motif, bearing similarity to the zinc finger-associated domain (ZAD) from Drosophila Grauzone. The CtBP THAP domain contains a large, positively charged surface patch and we demonstrate that this domain can bind to double-stranded DNA in an electrophoretic mobility-shift assay. These data, together with existing reports, indicate that THAP domains might exhibit a functional diversity similar to that observed for classical and GATA-type zinc fingers.

The Unorthodox SNAP50 Zinc Finger Domain Contributes to Cooperative Promoter Recognition by Human SNAPC

Human small nuclear RNA gene transcription by RNA polymerases II and III depends upon promoter recognition by the SNAPC general transcription factor. DNA binding by SNAPC involves direct DNA contacts by the SNAP190 subunit in cooperation with SNAP50 and SNAP43. The data presented herein shows that SNAP50 plays an important role in DNA binding by SNAPC through its zinc finger domain. The SNAP50 zinc finger domain contains 15 cysteine and histidine residues configured in two potential zinc coordination arrangements. Individual alanine substitution of each cysteine and histidine residue demonstrated that eight sites are important for DNA binding by SNAPC. However, metal binding studies revealed that SNAPC contains a single zinc atom indicating that only one coordination site functions as a zinc finger. Of the eight residues critical for DNA binding, four cysteine residues were also essential for both U1 and U6 transcription by RNA polymerase II and III, respectively. Surprisingly, the remaining four residues, although critical for U1 transcription could support partial U6 transcription. DNA binding studies showed that defects in DNA binding by SNAPC alone could be suppressed through cooperative DNA binding with another member of the RNA polymerase III general transcription machinery, TFIIIB. These results suggest that these eight cysteine and histidine residues perform different functions during DNA binding with those residues involved in zinc coordination likely performing a dominant role in domain stabilization and the others involved in DNA binding. These data further define the unorthodox SNAP50 zinc finger region as an evolutionarily conserved DNA binding domain.

Characterization of a Byproduct in the Alkylation of DMIT: Alkylation on the Least Nucleophilic Sulfur Atom

AbstractIt is generally accepted that the alkylation of (Et4N)2[Zn(DMIT)2] (1) is a very clean reaction that produces 4,5‐bis(alkylthio)‐1,3‐dithiole‐2‐thiones 4 in very high yields. Although this procedure has been widely used, we have found that, in addition to the 4,5‐bis(alkylthio)‐1,3‐dithiole‐2‐thione products 4, highly unexpected byproducts are formed during the alkylation reaction in which the thione functionalities of DMIT in (Et4N)2[Zn(DMIT)2] (1) are alkylated instead of the more reactive thiolate groups. These byproducts were identified as a novel type of compounds, namely bis(2‐alkylthio‐1,3‐dithiol‐1‐ium‐4,5‐dithiolato)zinc compounds 3, being composed of two identical 2‐alkylthio‐1,3‐dithiol‐1‐ium‐4,5‐dithiolate moieties coordinated to a single zinc(II) atom and were characterized by elemental analyses and different spectroscopic techniques as well as single‐crystal X‐ray structure analyses. A comparison of the physical properties of compounds 3 and (Et4N)2[Zn(DMIT)2] (1) has been carried out. The outcome of these investigations revealed significant differences in the physical properties of these two systems. (© Wiley‐VCH Verlag GmbH &amp; Co. KGaA, 69451 Weinheim, Germany, 2006)

The NS5A Protein of Bovine Viral Diarrhea Virus Contains an Essential Zinc-Binding Site Similar to That of the Hepatitis C Virus NS5A Protein

The recent demonstration that the NS5A protein of hepatitis C virus (HCV) contains an unconventional zinc-binding site with the format Cx(17)CxCx(20)C and the presence of a similar sequence element in the NS5A proteins of members of the Pestivirus genus has led to the hypothesis that the NS5A protein of the pestivirus bovine viral diarrhea virus (BVDV) is a zinc-binding protein. A method for the expression and partial purification of BVDV NS5A was developed, and the partially purified protein was analyzed for zinc content by atomic absorption spectroscopy. BVDV NS5A was found to coordinate a single zinc atom per protein molecule. Mutation of any of the four cysteines of the predicted zinc-binding motif eliminated zinc coordination. Furthermore, analysis of mutations at these cysteine residues in the context of a BVDV replicon system indicated that these residues were absolutely essential for RNA replication. The recently determined crystal structure of the N-terminal zinc-binding domain of the HCV NS5A protein, combined with secondary structure predictions of the region surrounding the mapped BVDV zinc-binding region, indicates that the BVDV zinc-binding motif fits the general template Cx(22)CxCx(24)C and likely comprises a three-stranded antiparallel beta-sheet fold. These data highlight the similarities between the Hepacivirus and Pestivirus NS5A proteins and suggest that both proteins perform a not-yet-defined function in RNA replication that requires coordination of a single zinc atom.

Solution Structure of the RBCC/TRIM B-box1 Domain of Human MID1: B-box with a RING

B-box domains are a defining feature of the tripartite RBCC (RING, B-box, coiled-coil) or TRIM proteins, many of which are E3 ubiquitin ligases. However, little is known about the biological function of B-boxes. In some RBCC/TRIM proteins there is only a single B-box (type 2) domain, while others have both type 1 and type 2 B-box domains in tandem adjacent to their RING domain. These two types of B-boxes share little sequence similarity, except the presence of cysteine and histidine residues: eight in most B-box1 domains and seven in B-box2 domains. We report here the high-resolution solution structure of the first B-box1 domain (from the human RBCC protein, MID1) based on 670 nuclear Overhauser effect (NOE)-derived distance restraints, 12 hydrogen bonds, and 44 dihedral angles. The domain consists of a three-turn alpha-helix, two short beta-strands, and three beta-turns, encompassing Val117 to Pro164, which binds two zinc atoms. One zinc atom is coordinated by cysteine residues 119, 122, 142, 145, while cysteine 134, 137 and histidine 150, 159 coordinate the other. This topology is markedly different from the only other B-box structure reported; that of a type 2 B-box from Xenopus XNF7, which binds a single zinc atom. Of note, the B-box1 structure closely resembles the folds of the RING, ZZ and U-box domains of E3 and E4 ubiquitin enzymes, raising the possibility that the B-box1 domain either has E3 activity itself or enhances the activity of RING type E3 ligases (i.e. confers E4 enzyme activity). The structure of the MID1 B-box1 also reveals two potential protein interaction surfaces. One of these is likely to provide the binding interface for Alpha 4 that is required for the localized turnover of the catalytic subunit of PP2A, the major Ser/Thr phosphatase.

Gas-phase experiments on the chemistry and coordination of Zn(II) by aprotic solvent molecules

Experiments have been performed in the gas phase on a series of doubly charged zinc–ligand complexes to elucidate their solvation structure and available fragmentation pathways. Production of such complexes was achieved by the formation of neutral argon–ligand clusters followed by the subsequent addition of a single zinc atom using a pickup technique. Multiply charged ions were then produced by electron impact within a high resolution, double-focusing mass spectrometer. Studies have been undertaken on a number of zinc(II) – aprotic solvent complexes including those consisting of argon and carbon dioxide in association with the zinc cation. Investigation of these novel metal–solvent clusters took the form of recorded parent ion intensity distributions and the measurement of fragmentation patterns promoted via collision-induced dissociation (CID). Discussion of the intensity distributions is presented in terms of the solvation of Zn(II) by each solvent, drawing on existing theoretical and experimental data from the gaseous and condensed phases. Investigation of collision-induced dissociation processes includes identifying charge transfer reactions in each solvated system, and analysis of the results in terms of kinetic energy release as well as possible mechanisms for fragmentation pathways. Key words: zinc, clusters, dications, gas phase, solvation.

Structure of the zinc-binding domain of an essential component of the hepatitis C virus replicase

Hepatitis C virus (HCV) is a human pathogen affecting nearly 3% of the world's population. Chronic infections can lead to cirrhosis and liver cancer. The RNA replication machine of HCV is a multi-subunit membrane-associated complex. The non-structural protein NS5A is an active component of HCV replicase, as well as a pivotal regulator of replication and a modulator of cellular processes ranging from innate immunity to dysregulated cell growth. NS5A is a large phosphoprotein (56-58 kDa) with an amphipathic alpha-helix at its amino terminus that promotes membrane association. After this helix region, NS5A is organized into three domains. The N-terminal domain (domain I) coordinates a single zinc atom per protein molecule. Mutations disrupting either the membrane anchor or zinc binding of NS5A are lethal for RNA replication. However, probing the role of NS5A in replication has been hampered by a lack of structural information about this multifunctional protein. Here we report the structure of NS5A domain I at 2.5-A resolution, which contains a novel fold, a new zinc-coordination motif and a disulphide bond. We use molecular surface analysis to suggest the location of protein-, RNA- and membrane-interaction sites.