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NADH:ubiquinone oxidoreductase, respiratory complex I, plays a central role in cellular energy metabolism. As a major source of reactive oxygen species (ROS) it affects ageing and mitochondrial dysfunction. The novel inhibitor NADH-OH specifically blocks NADH oxidation and ROS production by complex I in nanomolar concentrations. Attempts to elucidate its structure by NMR spectroscopy have failed. Here, by using X-ray crystallographic analysis, we report the structure of NADH-OH bound in the active site of a soluble fragment of complex I at 2.0 Å resolution. We have identified key amino acid residues that are specific and essential for binding NADH-OH. Furthermore, the structure sheds light on the specificity of NADH-OH towards the unique Rossmann-fold of complex I and indicates a regulatory role in mitochondrial ROS generation. In addition, NADH-OH acts as a lead-structure for the synthesis of a novel class of ROS suppressors.

Nitrogenases catalyze the biological fixation of inert N2 into bioavailable ammonium. They are bipartite systems consisting of the catalytic dinitrogenase and a complementary reductase, the Fe protein that is also the site where ATP is hydrolyzed to drive the reaction forward. Three different subclasses of dinitrogenases are known, employing either molybdenum, vanadium or only iron at their active site cofactor. Although in all these classes the mode and mechanism of interaction with Fe protein is conserved, each one encodes its own orthologue of the reductase in the corresponding gene cluster. Here we present the 2.2 A resolution structure of VnfH from Azotobacter vinelandii, the Fe protein of the alternative, vanadium-dependent nitrogenase system, in its ADP-bound state. VnfH adopts the same conformation that was observed for NifH, the Fe protein of molybdenum nitrogenase, in complex with ADP, representing a state of the functional cycle that is ready for reduction and subsequent nucleotide exchange. The overall similarity of NifH and VnfH confirms the experimentally determined cross-reactivity of both ATP-hydrolyzing reductases.

Inorganic polyphosphate is a ubiquitous, linear biopolymer built of up to thousands of phosphate residues that are linked by energyrich phosphoanhydride bonds. Polyphosphate kinases of the family 2 (PPK2) use polyphosphate to catalyze the reversible phosphorylation of nucleotide phosphates and are highly relevant as targets for new pharmaceutical compounds and as biocatalysts for cofactor regeneration. PPK2s can be classified based on their preference for nucleoside mono- or diphosphates or both. The detailedmechanism of PPK2s and the molecular basis for their substrate preference is unclear, which is mainly due to the lack of high-resolution structures with substrates or substrate analogs. Here, we report the structural analysis and comparison of a class I PPK2 (ADP-phosphorylating) and a class III PPK2 (AMP- and ADP-phosphorylating), both complexed with polyphosphate and/or nucleotide substrates. Together with complementarybiochemical analyses, these define the molecular basis of nucleotidespecificity and are consistent with a Mg2+ catalyzed in-line phosphoryl transfer mechanism. This mechanistic insight will guide the development of PPK2 inhibitors as potential antibacterials or genetically modified PPK2s that phosphorylate alternative substrates.

The S-adenosyl-L-methionine-dependent methyltransferase Rv0560c of Mycobacterium tuberculosis belongs to an orthologous group of heterocyclic toxin methyltransferases (Htm) which likely contribute to resistance of mycobacteria towards antimicrobial natural compounds as well as drugs. HtmM.t. catalyzes the methylation of the Pseudomonas aeruginosa toxin 2-heptyl-1-hydroxyquinolin-4(1H)-one (also known as 2-heptyl-4-hydroxyquinoline N-oxide), a potent inhibitor of respiratory electron transfer, its 1-hydroxyquinolin-4(1H)-one core (QNO), structurally related (iso)quinolones, and some mycobactericidal compounds. In this study, crystal structures of HtmM.t. in complex with S-adenosyl-L-homocysteine (SAH) and the methyl-accepting substrates QNO or 4-hydroxyisoquinoline-1(2H)-one, or the methylated product 1-methoxyquinolin-4(1H)-one, were determined at

Bromodomain and extra-terminal domain (BET) inhibitors are widely used both as chemical tools to study the biological role of their targets in living organisms and as candidates for drug development against several cancer variants and human disorders. However, non-BET bromodomains such as those in p300 and CBP are less studied. XDM-CBP is a highly potent and selective inhibitor for the bromodomains of CBP and p300 derived from a pan-selective BET BRD-binding fragment. Along with X-ray crystal-structure analysis and thermodynamic profiling, XDM-CBP was used in screenings of several cancer cell lines in vitro to study its inhibitory potential on cancer cell proliferation. XDM-CBP is demonstrated to be a potent and selective CBP/p300 inhibitor that acts on specific cancer cell lines, in particular malignant melanoma, breast cancer, and leukemia.

Epitaxially grown self-assembled quantum dots (QDs) are promising candidates for an efficient single-photon generation. In order to maximize the number of photons extracted from the device, QDs are frequently embedded into photonic structures such as micro-cavities. Once the QD is positioned inside an optical cavity, the Purcell effect ensures that light is emitted predominantly into the cavity mode. In this contribution, we demonstrate results of emission tuning of QDs inserted in micro-cavities. A sample containing an InAs/GaAs QDs embedded in a planar cavity based on Bragg reflectors has been integrated onto the PMN-PT piezo crystal. Subsequently, micro-cavities have been fabricated by electron-beam lithography and reactive ion etching. The application of external stress produces linear shifts of QDs emission which could be tuned into the resonance with fundamental cavity mode and allow enhancement of QD emission due to the Purcell effect.

Strain tuning emerged as an appealing tool for tuning of fundamental optical properties of solid-state quantum emitters. In particular, the wavelength and fine structure of quantum dot states can be tuned using hybrid semiconductor-piezoelectric devices. Here, we show how an applied external stress can directly impact the polarization properties of coupled InAs quantum dot-micropillar cavity systems. In our experiment, we find that we can reversibly tune the anisotropic polarization splitting of the fundamental microcavity mode by approximately 60 μeV. We discuss the origin of this tuning mechanism, which arises from an interplay between elastic deformation and the photoelastic effect in our micropillar. Finally, we exploit this effect to tune the quantum dot polarization optomechanically via the polarization-anisotropic Purcell effect. Our work paves the way for optomechanical and reversible tuning of the polarization and spin properties of light-matter-coupled solid-state systems., We acknowledge funding by the DFG within the Projects No. SCHN1376-5.1 and No. PR1749/1-1. Further, we acknowledge financial support by the State of Bavaria and the German Ministry of Education and Research (BMBF) within the project Q.Link.X (FKZ 16KIS0871). Project HYPER-U-P-S has received funding from the QuantERA ERA-NET Cofund in Quantum Technologies implemented within the European Union’s Horizon 2020 Programme. A.P. would like to thank the Swedish Research Council and Carl Tryggers Stiftelse. J.M.-S. acknowledges financial support from the Ramón y Cajal Program from the Government of Spain (RYC2018-026196-I) and the ClarínProgramme from the Government of the Principality of Asturias and a Marie Curie-COFUND Grant (PA-18-ACB17-29).

Scalable quantum photonic architectures demand highly efficient, high-purity single-photon sources, which can be frequency matched via external tuning. We demonstrate a single-photon source based on an InAs quantum dot embedded in a micropillar resonator, which is frequency tunable via externally-applied stress. Our platform combines the advantages of a Bragg micropillar cavity and the piezo-strain-tuning technique enabling single photon spontaneous emission enhancement via the Purcell effect and quantum dot (QD) with tunable wavelength. Our optomechanical platform has been implemented by integration of semiconductor-based QD-micropillars on a piezoelectric substrate. The fabricated device exhibits spontaneous emission enhancement with a Purcell factor of 4.4$\pm$0.7 and allows for a pure triggered single-photon generation with $g^{(2)}(0)$ < 0.07 under resonant excitation. A quantum dot emission energy tuning range of 0.75 meV for 27 kV/cm applied to the piezo substrate has been achieved. Our results pave the way towards the scalable implementation of single-photon quantum photonic technologies using optoelectronic devices., 16 pages, 4 figures

An optimal single-photon source should deterministically deliver one and only one photon at a time, with no trade-off between the source's efficiency and the photon indistinguishability. However, all reported solid-state sources of indistinguishable single photons had to rely on polarization filtering which reduced the efficiency by 50%, which fundamentally limited the scaling of photonic quantum technologies. Here, we overcome this final long-standing challenge by coherently driving quantum dots deterministically coupled to polarization-selective Purcell microcavities--two examples are narrowband, elliptical micropillars and broadband, elliptical Bragg gratings. A polarization-orthogonal excitation-collection scheme is designed to minimize the polarization-filtering loss under resonant excitation. We demonstrate a polarized single-photon efficiency of 0.60+/-0.02 (0.56+/-0.02), a single-photon purity of 0.975+/-0.005 (0.991+/-0.003), and an indistinguishability of 0.975+/-0.006 (0.951+/-0.005) for the micropillar (Bragg grating) device. Our work provides promising solutions for truly optimal single-photon sources combining near-unity indistinguishability and near-unity system efficiency simultaneously., 28 pages, 16 figures. partial overlap with arXiv:1809.10992 which was unpublished

Mg2+ -dependent catechol-O-methyltransferases occur in animals as well as in bacteria, fungi and plants, often with a pronounced selectivity towards one of the substrate's hydroxyl groups. Here, we show that the bacterial MxSafC exhibits excellent regioselectivity for para as well as for meta methylation, depending on the substrate's characteristics. The crystal structure of MxSafC was solved in apo and in holo form. The structure complexed with a full set of substrates clearly illustrates the plasticity of the active site region. The awareness that a wide range of factors influences the regioselectivity will aid the further development of catechol-O-methyltransferases as well as other methyltransferases as selective and efficient biocatalysts for chemical synthesis.

Phloroglucinol reductases (PGRs) are involved in anaerobic degradation in bacteria, in which they catalyze the dearomatization of phloroglucinol into dihydrophloroglucinol. We identified three PGRs, from different bacterial species, that are members of the family of NAD(P)H-dependent short-chain dehydrogenases/reductases (SDRs). In addition to catalyzing the reduction of the physiological substrate, the three enzymes exhibit activity towards 2,4,6-trihydroxybenzaldehyde, 2,4,6-trihydroxyacetophenone, and methyl 2,4,6-trihydroxybenzoate. Structural elucidation of PGRcl and comparison to known SDRs revealed a high degree of conservation. Several amino acid positions were identified as being conserved within the PGR subfamily and might be involved in substrate differentiation. The results enable the enzymatic dearomatization of monoaromatic phenol derivatives and provide insight into the functional diversity that may be found in families of enzymes displaying a high degree of structural homology.

Scalable quantum photonic applications require wavelength reproducibility and high quality of the emitted photons. To address these issues, we investigate strain-tuning ofself-assembled semiconductor quantum dots embedded into micropillar cavities

Several human diseases, including cancer, show altered signaling pathways resulting from changes in the activity levels of epigenetic modulators. In the past few years, small-molecule inhibitors against specific modulators, including the bromodomain and extra-terminal (BET) bromodomain family of acetylation readers, have shown early promise in the treatment of the genetically defined midline carcinoma and hematopoietic malignancies. We have recently developed a novel potent inhibitor of BET proteins, 1 (XD14[ Angew. Chem., Int. Ed. 2013, 52, 14055]), which exerts a strong inhibitory potential on the proliferation of specific leukemia cell lines. In the study presented here, we designed analogues of 1 to study the potential of substitutions on the 4-acyl pyrrole backbone to occupy additional sites within the substrate recognition site of BRD4(1). The compounds were profiled using ITC, DSF, and X-ray crystallography. We could introduce several substitutions that address previously untargeted areas of the substrate recognition site. This work may substantially contribute to the development of therapeutics with increased target specificity against BRD4-related malignancies.

Prostate cancer evolution is driven by a combination of epigenetic and genetic alterations such as coordinated chromosomal rearrangements, termed chromoplexy. TMPRSS2-ERG gene fusions found in human prostate tumors are a hallmark of chromoplexy. TMPRSS2-ERG fusions have been linked to androgen signaling and depend on androgen receptor (AR)-coupled gene transcription. Here, we show that dimethylation of KDM1A at K114 (to form K114me2) by the histone methyltransferase EHMT2 is a key event controlling androgen-dependent gene transcription and TMPRSS2-ERG fusion. We identified CHD1 as a KDM1A K114me2 reader and characterized the KDM1A K114me2-CHD1 recognition mode by solving the cocrystal structure. Genome-wide analyses revealed chromatin colocalization of KDM1A K114me2, CHD1 and AR in prostate tumor cells. Together, our data link the assembly of methylated KDM1A and CHD1 with AR-dependent transcription and genomic translocations, thereby providing mechanistic insight into the formation of TMPRSS2-ERG gene fusions during prostate-tumor evolution.

Sirtuins are NAD(+)-dependent protein deacylases that cleave off acetyl but also other acyl groups from the ε-amino group of lysines in histones and other substrate proteins. Dysregulation of human Sirt2 (hSirt2) activity has been associated with the pathogenesis of cancer, inflammation, and neurodegeneration, which makes the modulation of hSirt2 activity a promising strategy for pharmaceutical intervention. The sirtuin rearranging ligands (SirReals) have recently been discovered by us as highly potent and isotype-selective hSirt2 inhibitors. Here, we present a well-defined structure-activity relationship study, which rationalizes the unique features of the SirReals and probes the limits of modifications on this scaffold regarding inhibitor potency. Moreover, we present a crystal structure of hSirt2 in complex with an optimized SirReal derivative that exhibits an improved in vitro activity. Lastly, we show cellular hyperacetylation of the hSirt2 targeted tubulin caused by our improved lead structure.

In the present study, microseed matrix seeding was successfully applied to obtain a large number of crystals of the human sirtuin isotypes Sirt2 and Sirt3. These crystals appeared predictably in diverse crystallization conditions, diffracted to a higher resolution than reported in the literature and were subsequently used to study the protein–ligand interactions of two indole inhibitors., Sirtuins constitute a family of NAD+-dependent enzymes that catalyse the cleavage of various acyl groups from the ∊-amino group of lysines. They regulate a series of cellular processes and their misregulation has been implicated in various diseases, making sirtuins attractive drug targets. To date, only a few sirtuin modulators have been reported that are suitable for cellular research and their development has been hampered by a lack of structural information. In this work, microseed matrix seeding (MMS) was used to obtain crystals of human Sirt3 in its apo form and of human Sirt2 in complex with ADP ribose (ADPR). Crystal formation using MMS was predictable, less error-prone and yielded a higher number of crystals per drop than using conventional crystallization screening methods. The crystals were used to solve the crystal structures of apo Sirt3 and of Sirt2 in complex with ADPR at an improved resolution, as well as the crystal structures of Sirt2 in complex with ADPR and the indoles EX527 and CHIC35. These Sirt2–ADPR–indole complexes unexpectedly contain two indole molecules and provide novel insights into selective Sirt2 inhibition. The MMS approach for Sirt2 and Sirt3 may be used as the basis for structure-based optimization of Sirt2/3 inhibitors in the future.

We study the optical properties of coupled quantum dot-microcavity systems with elliptical cross section. First, we develop an analytic model that describes the spectrum of the cavity modes that are split due to the reduced symmetry of the resonator. By coupling the QD emission to the polarized fundamental cavity modes, we observe the vectorial nature of the Purcell enhancement, which depends on the intrinsic polarization of the quantum dot and its relative alignment with respect to the cavity axis. The variable interaction strength of the QD with the polarized cavity modes leads to the observation of strong and weak coupling. Finally, we demonstrate the capability of elliptical micropillars to emit single and highly indistinguishable photons (visibility over 93 \%)., Comment: 12 pages, 10 figures

The structures of the O-glycosyltransferase LanGT2 and the engineered, C-C bond-forming variant LanGT2S8Ac show how the replacement of a single loop can change the functionality of the enzyme. Crystal structures of the enzymes in complex with a nonhydrolyzable nucleotide-sugar analogue revealed that there is a conformational transition to create the binding sites for the aglycon substrate. This induced-fit transition was explored by molecular docking experiments with various aglycon substrates.

Strukturen der O-Glycosyltransferase LanGT2 und der generierten C-Glycosyltransferase LanGT2S8Ac zeigen, dass der Austausch eines kurzen Peptidfragments die Funktionalitat eines Enzyms deutlich verandern kann. Eine synthetische TDP-Carbaolivose wurde mit den Enzymen kokristallisiert. Es zeigte sich, dass die Bindung des Carbazuckers zu einer Konformationsanderung des Enzyms fuhrt, die dann eine Bindestelle fur ein Aglykonsubstrat erzeugt. Obwohl eine Bindung des Aglykons nicht experimentell nachgewiesen wurde, deuten Docking-Studien auf unterschiedliche Bindungsmodi im Fall von O- bzw. C-Glycosylierung hin.

We report a joint experimental and theoretical study of the interference properties of a single photon source based on a In(Ga)As quantum dot embedded in a quasi-planar GaAs-microcavity. Using resonant laser excitation with a pulse separation of 2 ns, we find near-perfect interference of the emitted photons, and a corresponding indistinguishability of $\mathcal{I} = (99.6\,^{+\,0.4}_{-\,1.4})\%$. For larger pulse separations, quasi-resonant excitation conditions, increasing pump power or with increasing temperature, the interference contrast is progressively and notably reduced. We present a systematic study of the relevant dephasing mechanisms, and explain our results in the framework of a microscopic model of our system. For strictly resonant excitation, we show that photon indistinguishability is independent of pump power, but strongly influenced by virtual phonon assisted processes which are not evident in excitonic Rabi oscillations., 12 pages, 6 figures

Deterministic techniques enabling the implementation and engineering of bright and coherent solid-state quantum light sources are key for the reliable realization of a next generation of quantum devices. Such a technology, at best, should allow one to significantly scale up the number of implemented devices within a given processing time. In this work, we discuss a possible technology platform for such a scaling procedure, relying on the application of nanoscale quantum dot imaging to the pillar microcavity architecture, which promises to combine very high photon extraction efficiency and indistinguishability. We discuss the alignment technology in detail, and present the optical characterization of a selected device which features a strongly Purcell-enhanced emission output. This device, which yields an extraction efficiency of $\eta=(49\pm4)~\%$, facilitates the emission of photons with $(94\pm2.7)~\%$ indistinguishability., Comment: 8 pages, 5 figures. arXiv admin note: text overlap with arXiv:1512.07453

The importance and structural diversity of chiral amines is well-demonstrated by the myriad nonenzymatic methods for their chemical production. In nature, the production of amines is performed by transamination rather than by reduction of an imine precursor derived from the corresponding ketone. Imine reductases, however, show great potential in the reduction of cyclic imines that are stable towards hydrolysis in aqueous reaction media. Here, we report the catalytic activity of two S-selective imine reductases towards 3,4-dihydroisoquinolines and 3,4-dihydro-β-carbolines and their activity in the direct reductive amination of ketone substrates. The crystal structures of the enzyme from Streptomyces sp. GF3546 in complex with the cofactor NADPH and from Streptomyces aurantiacus in native form have been solved and refined to a resolution of 1.9 A.

4-Hydroxyphenylpyruvate dioxygenase (Hpd, EC 1.13.11.27) catalyzes the conversion of 4-hydroxyphenylpyruvate into homogentisate in the second step of oxidative tyrosine catabolism. This pathway is known from bacteria and eukaryotes, but so far no archaeal Hpd has been described. Here, we report the biochemical characterization of an Hpd from the extremophilic archaeon Picrophilus torridus (Pt_Hpd), together with its three-dimensional structure at a resolution of 2.6 Å. Two pH optima were observed at 50 °C: pH 4.0 (close to native conditions) and pH 7.0. The enzyme showed only moderate thermostability and was inactivated with a half-life of ~1.5 h even under optimal reaction conditions. At the ideal physiological growth conditions of P. torridus, Pt_Hpd was inactive after 1 h, showing that the enzyme is protected in vivo from denaturation and/or is only partially adapted to the harsh environmental conditions in the cytosol of P. torridus. The influence of different additives on the activity was investigated. Pt_Hpd exhibited a turnover number k(cat) of 9.9 ± 0.6 s(-1) and a substrate binding affinity K(m) of 142 ± 23 µM. In addition, substrate inhibition with a binding affinity K(i) of 1.9 ± 0.3 mM was observed. Pt_Hpd is compared with isoenzymes from other species and the putative bacterial origin of the gene is discussed.

3,3-Di-methyl-azetidin-2-ones were identified as potent and selective 11β-HSD1 inhibitors against the human and mouse forms of the enzyme. Structure guided optimisation of LLE was conducted, utilising a key polar interaction and identifying stereochemical preference for the 4S isomer. Metabolic stability was improved to afford oral exposure, providing tool compounds suitable for pre-clinical evaluation.

We present the efficient generation of coherent single photons based on quantum dots in micropillars. We utilize a scalable lithography scheme leading to quantum dot-micropillar devices with 74% extraction efficiency. Via pulsed strict resonant pumping, we show an indistinguishability of consecutively emitted photons up to 98.5%.

A novel, potent and selective quinazolinone series of inhibitors of p38α MAP kinase has been identified. Modifications designed to address the issues of poor aqueous solubility and high plasma protein binding as well as embedded aniline functionalities resulted in the identification of a clinical candidate N-cyclopropyl-4-methyl-3-[6-(4-methylpiperazin-1-yl)-4-oxoquinazolin-3(4H)-yl]benzamide (AZD6703). Optimisation was guided by understanding of the binding modes from X-ray crystallographic studies which showed a switch from DFG 'out' to DFG 'in' as the inhibitor size was reduced to improve overall properties.

The metal-reducing δ-proteobacterium Geobacter sulfurreducens produces a large number of c-type cytochromes, many of which have been implicated in the transfer of electrons to insoluble metal oxides. Among these, the dihemic MacA was assigned a central role. Here we have produced G. sulfurreducens MacA by recombinant expression in Escherichia coli and have solved its three-dimensional structure in three different oxidation states. Sequence comparisons group MacA into the family of diheme cytochrome c peroxidases, and the protein indeed showed hydrogen peroxide reductase activity with ABTS(-2) as an electron donor. The observed K(M) was 38.5 ± 3.7 μM H(2)O(2) and v(max) was 0.78 ± 0.03 μmol of H(2)O(2)·min(-1)·mg(-1), resulting in a turnover number k(cat) = 0.46 · s(-1). In contrast, no Fe(III) reductase activity was observed. MacA was found to display electrochemical properties similar to other bacterial diheme peroxidases, in addition to the ability to electrochemically mediate electron transfer to the soluble cytochrome PpcA. Differences in activity between CcpA and MacA can be rationalized with structural variations in one of the three loop regions, loop 2, that undergoes conformational changes during reductive activation of the enzyme. This loop is adjacent to the active site heme and forms an open loop structure rather than a more rigid helix as in CcpA. For the activation of the protein, the loop has to displace the distal ligand to the active site heme, H93, in loop 1. A H93G variant showed an unexpected formation of a helix in loop 2 and disorder in loop 1, while a M297H variant that altered the properties of the electron transfer heme abolished reductive activation.

Directed screening has identified a novel series of non-zinc binding MMP13 inhibitors that possess good levels of activity whilst demonstrating excellent selectivity over related MMPs. A lead optimisation campaign has delivered compounds with enhanced MMP13 potency, good selectivity and acceptable bioavailability profiles leading to a predicted twice-a-day dosing regimen in man.

Directed screening has identified a novel series of MMP13 inhibitors that possess good levels of activity whilst possessing excellent selectivity over related MMPs. The binding mode of the series has been solved by co-crystallisation and demonstrates an interesting mode of inhibition without interaction with the catalytic zinc atom.

Two series of N-hydroxyformamide inhibitors of ADAM-TS4 were identified from screening compounds previously synthesised as inhibitors of matrix metalloproteinase-13 (collagenase-3). Understanding of the binding mode of this class of compound using ADAM-TS1 as a structural surrogate has led to the discovery of potent and very selective inhibitors with favourable DMPK properties. Synthesis, structure-activity relationships, and strategies to improve selectivity and lower in vivo metabolic clearance are described.

GlnB and GlnK are ancient signalling proteins that play a crucial role in the regulation of nitrogen assimilation. Both protein types can be present in the same genome as either single or multiple copies. However, the gene product of glnK is always found in an operon together with an amt gene encoding an ammonium-transport (Amt) protein. Complex formation between GlnK and Amt blocks ammonium uptake and depends on the nitrogen level in the cell, which is regulated through the binding of specific effector molecules to GlnK. In particular, an ammonium shock to a cell culture previously starved in this nitrogen source or the binding of ATP to purified GlnK can stimulate effective complex formation. While the binding of ATP/ADP and 2-oxoglutarate (as a signal for low intracellular nitrogen) to GlnK have been reported and several GlnB/K protein structures are available, essential functional questions remain un­answered. Here, the crystal structure of A. fulgidus GlnK1 at 2.28 A resolution and a comparison with the crystal structures of other GlnK proteins, in particular with that of its paralogue GlnK2 from the same organism, is reported.

The displacement of probes that bind selectively to subdomains IIA or IIIA on human serum albumin (HSA) by competing compounds has been followed using fluorescence spectroscopy, and has therefore been used to assign a primary binding site for these compounds in the presence and absence of fatty acids. The crystal structures have also been solved for three compounds: a matched pair of carboxylic acids whose binding strength to HSA unexpectedly decreased as the lipophilicity increased; and a highly bound sulphonamide that appeared not to displace the probes in the displacement assay. The crystallography results support the findings from the fluorescence displacement assay. The results indicate that drug binding to subdomain IB might also be important location for certain compounds.

A number of molecular recognition features have been exploited in structure-based design of selective Cathepsin inhibitors.

A series of potent Cathepsin L inhibitors with good selectivity with respect to other cysteine Cathepsins is described and SAR is discussed with reference to the crystal structure of a protein-ligand complex.

We report on the observation of bright emission of single photons under pulsed resonance fluorescence conditions from a single quantum dot (QD) in a micropillar cavity. The brightness of the QD fluorescence is greatly enhanced via the coupling to the fundamental mode of a micropillar, allowing us to determine a single photon extraction efficiency of $(20.7\pm0.8)~\%$ per linear polarization basis. This yields an overall extraction efficiency of $(41.4\pm1.5)~\%$ in our device. We observe the first Rabi-oscillation in a weakly coupled quantum dot-micropillar system under coherent pulsed optical excitation, which enables us to deterministically populate the excited QD state. In this configuration, we probe the single photon statistics of the device yielding $g^{(2)}(0)=0.072\pm0.011$ at a QD-cavity detuning of $75~\mu$eV., Comment: accepted for publication in Optics Express

The two-component metalloprotein nitrogenase catalyzes the reductive fixation of atmospheric dinitrogen into bioavailable ammonium in diazotrophic prokaryotes. The process requires an efficient energy metabolism, so that although the metal clusters of nitrogenase rapidly decompose in the presence of dioxygen, many free-living diazotrophs are obligate aerobes. In order to retain the functionality of the nitrogen-fixing enzyme, some of these are able to rapidly "switch-off" nitrogenase, by shifting the enzyme into an inactive but oxygen-tolerant state. Under these conditions the two components of nitrogenase form a stable, ternary complex with a small [2Fe:2S] ferredoxin termed FeSII or the "Shethna protein II". Here we have produced and isolated Azotobacter vinelandii FeS II and have determined its three-dimensional structure to 2.1 Å resolution by X-ray diffraction. In the crystals, the dimeric protein was present in two distinct states that differ in the conformation of an extended loop in close proximity to the iron-sulfur cluster. We show that this rearrangement is redox-dependent and forms the molecular basis for oxygen-dependent conformational protection of nitrogenase. Protection assays highlight that FeSII binds to a preformed complex of MoFe and Fe protein upon activation, primarily through electrostatic interactions. The surface properties and known complexes of nitrogenase component proteins allow us to propose a model of the conformationally protected ternary complex of nitrogenase.

Sirtuins are NAD(+)-dependent protein deacylases that cleave off acetyl groups, as well as other acyl groups, from the ɛ-amino group of lysines in histones and other substrate proteins. Dysregulation of human Sirt2 activity has been associated with the pathogenesis of cancer, inflammation, and neurodegeneration, thus making Sirt2 a promising target for pharmaceutical intervention. Here, based on a crystal structure of Sirt2 in complex with an optimized sirtuin rearranging ligand (SirReal) that shows improved potency, water solubility, and cellular efficacy, we present the development of the first Sirt2-selective affinity probe. A slow dissociation of the probe/enzyme complex offers new applications for SirReals, such as biophysical characterization, fragment-based screening, and affinity pull-down assays. This possibility makes the SirReal probe an important tool for studying sirtuin biology.

Septins are a conserved family of GTP-binding proteins that assemble into a highly ordered array of filaments at the mother bud neck in Saccharomyces cerevisiae cells. Many molecular functions and mechanisms of the septins in S. cerevisiae were already uncovered. However, structural information is only available from modeling the crystallized subunits of the human septins into the EM cryomicroscopy data of the yeast hetero-octameric septin rod. Octameric rods are the building block of septin filaments in yeast. We present here the first crystal structure of Cdc11, the terminal subunit of the octameric rod and discuss its structure in relation to its human homologues. Size exclusion chromatography analysis revealed that Cdc11 forms homodimers through its C-terminal coiled coil tail.

Whereas eubacterial and eukaryotic riboflavin synthases form homotrimers, archaeal riboflavin synthases from Methanocaldococcus jannaschii and Methanothermobacter thermoautrophicus are homopentamers with sequence similarity to the 6,7-dimethyl-8-ribityllumazine synthase catalyzing the penultimate step in riboflavin biosynthesis. Recently it could be shown that the complex dismutation reaction catalyzed by the pentameric M. jannaschii riboflavin synthase generates riboflavin with the same regiochemistry as observed for trimeric riboflavin synthases. Here we present crystal structures of the pentameric riboflavin synthase from M. jannaschii and its complex with the substrate analog inhibitor, 6,7-dioxo-8-ribityllumazine. The complex structure shows five active sites located between adjacent monomers of the pentamer. Each active site can accommodate two substrate analog molecules in anti-parallel orientation. The topology of the two bound ligands at the active site is well in line with the known stereochemistry of a pentacyclic adduct of 6,7-dimethyl-8-ribityllumazine that has been shown to serve as a kinetically competent intermediate. The pentacyclic intermediates of trimeric and pentameric riboflavin synthases are diastereomers.

Inhibition of p38alpha MAP kinase is a potential approach for the treatment of inflammatory disorders. MKK6-dependent phosphorylation on the activation loop of p38alpha increases its catalytic activity and affinity for ATP. An inhibitor, BIRB796, binds at a site used by the purine moiety of ATP and extends into a "selectivity pocket", which is not used by ATP. It displaces the Asp168-Phe169-Gly170 motif at the start of the activation loop, promoting a "DFG-out" conformation. Some other inhibitors bind only in the purine site, with p38alpha remaining in a "DFG-in" conformation. We now demonstrate that selectivity pocket compounds prevent MKK6-dependent activation of p38alpha in addition to inhibiting catalysis by activated p38alpha. Inhibitors using only the purine site do not prevent MKK6-dependent activation. We present kinetic analyses of seven inhibitors, whose crystal structures as complexes with p38alpha have been determined. This work includes four new crystal structures and a novel assay to measure K(d) for nonactivated p38alpha. Selectivity pocket compounds associate with p38alpha over 30-fold more slowly than purine site compounds, apparently due to low abundance of the DFG-out conformation. At concentrations that inhibit cellular production of an inflammatory cytokine, TNFalpha, selectivity pocket compounds decrease levels of phosphorylated p38alpha and beta. Stabilization of a DFG-out conformation appears to interfere with recognition of p38alpha as a substrate by MKK6. ATP competes less effectively for prevention of activation than for inhibition of catalysis. By binding to a different conformation of the enzyme, compounds that prevent activation offer an alternative approach to modulation of p38alpha.

A system of electrodes and Langmuir probes has been developed for the measurement of plasma flow damping in the Helically Symmetric Experiment (HSX) stellarator. A biased electrode is used to apply a J×B torque to the plasma. The fast switching electrode power supply allows the electrode voltage to be applied in ∼1 μs, which is much faster than any of the plasma time scales; the electrode current is terminated on a similar time scale at the end of the electrode pulse. A pair of multitipped Mach probes have been designed to measure the plasma flows in a magnetic surface with good spatial and time resolution (Δt>20 μs). The unmagnetized model by Hutchinson is used to analyze the Mach probe data, and radial force balance is found to be well satisfied during electrode bias. These probes allow for measurements of the transient response of the plasma flows and floating potential when the electrode is energized. An example of using the system for the estimation of viscous damping times in HSX is provided.

Thomson scattering and diamagnetic loop measurements in a hot electron plasma in the Helically Symmetric Experiment (HSX) indicate that the central electron temperature and stored energy increase linearly with power. Experimentally it is found that the central electron temperature is roughly independent of plasma density. The ASTRA code is used to model electron cyclotron heating for a magnetic configuration that is quasi-symmetric as well as for a configuration in which the symmetry is broken. The experimental results are consistent with an anomalous thermal conductivity that scales inversely with the density. However, the experimental scaling of the stored energy against density is not usually in agreement with the model. From the measured X-ray flux and the high absorbed power, as well as from the calculated low single-pass absorption efficiency, it is concluded that at low densities, a nonthermal electron population accounts for a significant fraction of the stored energy. With the ASTRA code, it is also possible to model under what conditions the central electron temperature in the quasi-symmetric configuration will be measurably greater than the temperature in the nonsymmetric configuration. These calculations depend greatly on the radial electric field of the nonsymmetric plasma but suggest that at somewhat higher densitymore » and higher power than achieved to date, differences in the central electron temperature may be observed.« less

A system of absolutely calibrated Hα detectors for neutral hydrogen monitoring has been designed and implemented for the Helically Symmetric eXperiment. An array of detectors at fixed poloidal angle but many toroidal angles yields a measurement of the toroidal asymmetry in Hα emission and is used for monitoring hydrogen recycling. A multichord array at the toroidal angle of the gas puff allows an accurate determination of neutral gas penetration. Preliminary neutral gas modeling allows for the estimation of the density of atomic and molecular hydrogen from the Hα emission, which are used for particle transport and flow damping studies.

The amino acid residue tryptophan 27 of 6,7-dimethyl-8-ribityllumazine synthase of the yeast Schizosaccharomyces pombe was replaced by tyrosine. The structures of the W27Y mutant protein in complex with riboflavin, the substrate analogue 5-nitroso-6-ribitylamino-2,4(1H,3H)-pyrimidinedione, and the product analogue 6-carboxyethyl-7-oxo-8-ribityllumazine, were determined by X-ray crystallography at resolutions of 2.7–2.8 A. Whereas the indole system of W27 forms a coplanar π-complex with riboflavin, the corresponding phenyl ring in the W27Y mutant establishes only peripheral contact with the heterocyclic ring system of the bound riboflavin. These findings provide an explanation for the absence of the long wavelength shift in optical absorption spectra of riboflavin bound to the mutant enzyme. The structures of the mutants are important tools for the interpretation of the unusual physical properties of riboflavin in complex with lumazine synthase.

The gene specifying plastid transketolase (TK) of maize (Zea mays) was cloned from a cDNA library by southern blotting using a heterologous probe from sorghum (Sorghum bicolor). A recombinant fusion protein comprising thioredoxin of Escherichia coli and mature TK of maize was expressed at a high level in E. coli and cleaved with thrombin, affording plastid TK. The protein in complex with thiamine pyrophoshate was crystallized, and its structure was solved by molecular replacement. The enzyme is a C2 symmetric homodimer closely similar to the enzyme from yeast (Saccharomyces cerevisiae). Each subunit is folded into three domains. The two topologically equivalent active sites are located in the subunit interface region and resemble those of the yeast enzyme.