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At least 811 records · Page 45

Neutron Imaging of Advanced Transportation Technologies

Unlike X-rays, neutrons are very sensitive to light elements such as hydrogen (H) atoms and can penetrate through thick layers of metals (Figure 1a). These two properties suggest neutrons are well suited to probe engine parts such as diesel particulate filters, exhaust gas recirculation coolers, fuel injectors, oil in engines, oil residues in filters, batteries, advanced materials, etc. Neutron imaging is based on the interactions of a sample with a neutron beam. The interactions are dependent on sample thickness/density and elemental make-up and result in absorption and scattering of neutrons within the sample. A two-dimensional position-sensitive detector placed behind the sample can measure the transmitted neutron flux (Figure 1b). When combined with a high-precision (~1/100 of a degree) rotational stage, it is possible to perform computed tomography (CT) scans and thus generate 3-dimensional images of working fluids inside real devices. Samples can be analyzed at one position or a complete reconstruction can provide a cross-section of the entire sample at a resolution of the detector; the detector resolution is currently at ~50–80 microns (at the detector).

47 OTHER INSTRUMENTATION↗

Ionospheric range-rate effects in satellite-to-satellite tracking

Investigation of ionospheric range and range-rate corrections in satellite-to-satellite tracking were investigated. Major problems were cited and the magnitude of errors that have to be considered for communications between satellites and related experiments was defined. The results point to the need of using a sophisticated modeling approach incorporating daily solar data, and where possible actual ionospheric measurements as update information, as a simple median model cannot possibly account for the complex interaction of the many variables. The findings provide a basis from which the residual errors can be estimated after ionospheric modeling is incorporated in the reduction. Simulations were performed for satellites at various heights: Apollo, Geos, and Nimbus tracked by ATS-6; and in two different geometric configurations: coplanar and perpendicular orbits.

Lipofsky, J. R.↗

Observation of Beam ION Instability in Spear3

Weak vertical coupled bunch instability with oscillation amplitude at {mu}m level has been observed in SPEAR3. The instability becomes stronger when there is a vacuum pressure rise by partially turning off vacuum pumps and it becomes weaker when the vertical beam emittance is increased by turning off the skew quadrupole magnets. These confirmed that the instability was driven by ions in the vacuum. The threshold of the beam ion instability when running with a single bunch train is just under 200 mA. This paper presents the comprehensive observations of the beam ion instability in SPEAR3. The effects of vacuum pressure, beam current, beam filling pattern, chromaticity, beam emittance and bunch-by-bunch feedback are investigated in great detail. In an electron accelerator, ions generated from the residual gas molecules can be trapped by the beam. Then these trapped ions interact resonantly with the beam and cause beam instability and emittance blow-up. Most existing light sources use a long single bunch train filling pattern, followed by a long gap to avoid multi-turn ion trapping. However, such a gap does not preclude ions from accumulating during one passage of the single bunch train beam, and those ions can still cause a Fast Ion Instability (FII) as predicted by Raubenheimer and Zimmermann. FII has been observed in ALS, and PLS by artificially increasing the vacuum pressure by injecting helium gas into the vacuum chamber or by turning off the ion pumps in order to observe the beam ion instability. In some existing rings, for instance B factory, the beam ion instability was observed at the beginning of the machine operation after a long period of shutdown and then it automatically disappeared when the vacuum was better. However, when the beam emittance becomes smaller, the FII can occur at nominal conditions as observed in PLS, SOLEIL and SSRF. This paper reports the observations of beam ion instabilities in SPEAR3 under different condition during a period of one year, which includes single bunch train instability (FII) and multi-bunch train instability. Note that the instability may be not the same even with the same beam due to the change of the vacuum with time. SPEAR3 has a circumference of 234 m with a harmonic number of 372. SPEAR3 runs with six bunch train filling pattern in order to suppress the possible beam ion instability. Table 1 lists the main parameters of SPEAR3. The vacuum of SPEAR3 ranges from 0.1 to 0.5 nTorr, which varies from section to section.

43 PARTICLE ACCELERATORS↗

Characterization of a Cross-Linked Protein-Nucleic Acid Substrate Radical in the Reaction Catalyzed by RlmN

RlmN and Cfr are methyltransferases/methylsynthases that belong to the radical S-adenosylmethionine superfamily of enzymes. RlmN catalyzes C2 methylation of adenosine 2503 (A2503) of 23S rRNA, while Cfr catalyzes C8 methylation of the exact same nucleotide, and will subsequently catalyze C2 methylation if the site is unmethylated. A key feature of the unusual mechanisms of catalysis proposed for these enzymes is the attack of a methylene radical, derived from a methylcysteine residue, onto the carbon center undergoing methylation to generate a paramagnetic protein–nucleic acid cross-linked species. This species has been thoroughly characterized during Cfr-dependent C8 methylation, but does not accumulate to detectible levels in RlmN-dependent C2 methylation. Herein, we show that inactive C118S/A variants of RlmN accumulate a substrate-derived paramagnetic species. Characterization of this species by electron paramagnetic resonance spectroscopy in concert with strategic isotopic labeling shows that the radical is delocalized throughout the adenine ring of A2503, although predominant spin density is on N1 and N3. Moreover, 13 C hyperfine interactions between the radical and the methylene carbon of the formerly [methyl- 13 C]Cys355 residue show that the radical species exists in a covalent cross-link between the protein and the nucleic acid substrate. X-ray structures of RlmN C118A show that, in the presence of SAM, the substitution does not alter the active site structure compared to that of the wild-type enzyme. Together, these findings have new mechanistic implications for the role(s) of C118 and its counterpart in Cfr (C105) in catalysis, and suggest involvement of the residue in resolution of the cross-linked species via a radical mediated process

Silakov, Alexey↗

Structural basis for the specificity of PPM1H phosphatase for Rab GTPases

LRRK2 serine/threonine kinase is associated with inherited Parkinson’s disease. LRRK2 phosphorylates a subset of Rab GTPases within their switch 2 motif to control their interactions with effectors. Recent work has shown that the metal-dependent protein phosphatase PPM1H counteracts LRRK2 by dephosphorylating Rabs. PPM1H is highly selective for LRRK2 phosphorylated Rabs, and closely related PPM1J exhibits no activity towards substrates such as Rab8a phosphorylated at Thr72 (pThr72). Here, we have identified the molecular determinant of PPM1H specificity for Rabs. The crystal structure of PPM1H reveals a structurally conserved phosphatase fold that strikingly has evolved a 110-residue flap domain adjacent to the active site. The flap domain distantly resembles tudor domains that interact with histones in the context of epigenetics. Cellular assays, crosslinking and 3-D modelling suggest that the flap domain encodes the docking motif for phosphorylated Rabs. Consistent with this hypothesis, a PPM1J chimaera with the PPM1H flap domain dephosphorylates pThr72 of Rab8a both in vitro and in cellular assays. Therefore, PPM1H has acquired a Rab-specific interaction domain within a conserved phosphatase fold.

60 APPLIED LIFE SCIENCES↗

Energetics of the lattice: packing elements in crystals of four-stranded intercalated cytosine-rich DNA molecules

Condensation of single molecules from solution into crystals represents a transition between distinct energetic states. In solution, the atomic interactions within the molecule dominate. In the crystalline state, however, a set of additional interactions are formed between molecules in close contact in the lattice--these are the packing interactions. The crystal structures of d(CCCT), d(TAACCC), d(CCCAAT), and d(AACCCC) have in common a four-stranded intercalated cytosine segment, built by stacked layers of cytosine.cytosine+ (C.C+) base pairs coming from two parallel duplexes that intercalate into each other with opposite polarity. The intercalated cytosine segments in these structures are similar in their geometry, even though the sequences crystallized in different space groups. In the crystals, adenine and thymine residues of the sequences are used to build the three-dimensional crystal lattice by elaborately interacting with symmetry-related molecules. The packing elements observed provide novel insight about the copious ways in which nucleic acid molecules can interact with each other--for example, when folded in more complicated higher order structures, such as mRNA and chromatin.

Non-NASA Center↗

Structure of FcγRI in complex with Fc reveals the importance of glycan recognition for high-affinity IgG binding

Fc gamma receptor I (FcγRI) contributes to protective immunity against bacterial infections, but exacerbates certain autoimmune diseases. The sole high-affinity IgG receptor, FcγRI plays a significant role in immunotherapy. To elucidate the molecular mechanism of its high-affinity IgG binding, we determined the crystal structure of the extracellular domains of human FcγRI in complex with the Fc domain of human IgG 1 . FcγRI binds to the Fc in a similar mode as the low-affinity FcγRII and FcγRIII receptors. In addition to many conserved contacts, FcγRI forms additional hydrogen bonds and salt bridges with the lower hinge region of Fc. Unique to the high-affinity receptor-Fc complex, however, is the conformation of the receptor D2 domain FG loop, which enables a charged KHR motif to interact with proximal carbohydrate units of the Fc glycans. Both the length and the charge of the FcγRI FG loop are well conserved among mammalian species. Ala and Glu mutations of the FG loop KHR residues showed significant contributions of His-174 and Arg-175 to antibody binding, and the loss of the FG loop–glycan interaction resulted in an ~20- to 30-fold decrease in FcγRI affinity to all three subclasses of IgGs. Furthermore, deglycosylation of IgG 1 resulted in a 40-fold loss in FcγRI binding, demonstrating involvement of the receptor FG loop in glycan recognition. Here, these results highlight a unique glycan recognition in FcγRI function and open potential therapeutic avenues based on antibody glycan engineering or small molecular glycan mimics to target FcγRI for certain autoimmune diseases.

59 BASIC BIOLOGICAL SCIENCES↗

Enhanced Guest@MOF Interaction via Stepwise Thermal Annealing: TCNQ@Cu 3 (BTC) 2

Confinement of guest molecules in porous materials such as metal organic frameworks (MOFs) promises to deliver emergent properties separate from those of the individual components. Understanding the confinement mechanism is therefore important for the development of new synthesis routes that adjust MOF properties for specific applications. In this work, we developed a new synthetic method to confine guest molecules into MOF pores through a stepwise thermal annealing process, wherein the confinement of 7,7,8,8-tetracyanoquinodimethane (TCNQ) guest molecules into Cu 3 (BTC) 2 (BTC = benzene-1,3,5-tricarboxylic acid) MOF host is used as an example of how novel materials can be created with new physical properties. The stepwise thermal annealing process includes 1) an activation process of pristine Cu 3 (BTC) 2 MOF to maximizes the TCNQ guest loading in the MOF host by effectively removing the residual solvents and 2) post-annealing of the TCNQ infiltrated MOF to enhances the interaction of the confined guest molecules with the MOF host. Obtained experimental results based on thermogravimetric analysis, N 2 gas adsorption, electron microscopy, X-ray diffraction and infrared absorption, combined with density functional theory calculations provide evidence that the use of a stepwise thermal annealing process yields enhancements in the guest loading, packing and interaction between the TCNQ guest and the MOF host. The new hybrid TCNQ@Cu 3 (BTC) 2 system is stable and shows no significant signs of structural degradation even after submersion in water. This is due to the presence of significantly stronger interactions of TCNQ with the frame-work metal ions compared to those of the water molecules competing for the same framework binding sites. It was also found that TCNQ@Cu 3 (BTC) 2 system maintains a significant CO 2 and CH 4 adsorption potential compared to the pristine MOF. The synthetic route developed in this work yields novel guest@MOF hybrid systems that will be useful for many MOF-based applications such as gas separations and chemical sensors performed under humid conditions.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Optical model methods of predicting nuclide production cross sections from heavy ion fragmentation

Quantum mechanical optical potential methods for calculating inclusive isotope and element production cross sections from the fragmenting of heavy nuclei by intermediate- and high-energy protons and heavy ions are presented based upon a modified abrasion-ablation-FSI (frictional spectator interaction) collision model. The abrasion stage is treated as a quantum mechanical knockout process that leaves the residual prefragment in an excited state. Prefragment excitation energies are estimated using a combined liquid drop and FSI method. In ablation the prefragment deexcites by particle and photon emission to produce the final fragment. Contributions from electromagnetic dissociation to single nucleon removal cross sections are incorporated using a Weiszacker-Williams theory that includes electric dipole and electric quadrupole interactions. Estimates of elemental and isotopic production cross sections are in good agreement with published cross section measurements for a variety of projectile-target-beam energy combinations.

NASA Discipline Radiation Health↗

Intracellular segment between transmembrane helices S0 and S1 of BK channel α subunit contains two amphipathic helices connected by a flexible loop

Highlights: •The loop between S0 and S1 of BK channel was overexpressed and purified in DPC. •NMR studies indicated BK-IS1 contained two helices connected by a flexible loop. •Mg{sup 2+} titration of BK-IS1 indicated two possible binding sites of divalent ions. -- Abstract: The BK channel, a tetrameric potassium channel with very high conductance, has a central role in numerous physiological functions. The BK channel can be activated by intracellular Ca{sup 2+} and Mg{sup 2+}, as well as by membrane depolarization. Unlike other tetrameric potassium channels, the BK channel has seven transmembrane helices (S0–S6) including an extra helix S0. The intracellular segment between S0 and S1 (BK-IS1) is essential to BK channel functions and Asp99 in BK-IS1 is reported to be responsible for Mg{sup 2+} coordination. In this study, BK-IS1 (44–113) was over-expressed using a bacterial system and purified in the presence of detergent micelles for multidimensional heteronuclear nuclear magnetic resonance (NMR) structural studies. Backbone resonance assignment and secondary structure analysis showed that BK-IS1 contains two amphipathic helices connected by a 36-residue loop. Amide {sup 1}H–{sup 15}N heteronuclear NOE analysis indicated that the loop is very flexible, while the two amphipathic helices are possibly stabilized through interaction with the membrane. A solution NMR-based titration assay of BK-IS1 was performed with various concentrations of Mg{sup 2+}. Two residues (Thr45 and Leu46) with chemical shift changes were observed but no, or very minor, chemical shift difference was observed for Asp99, indicating a possible site for binding divalent ions or other modulation partners.

60 APPLIED LIFE SCIENCES↗

Structural comparison of four different antibodies interacting with human papillomavirus 16 and mechanisms of neutralization

Cryo-electron microscopy (cryo-EM) was used to solve the structures of human papillomavirus type 16 (HPV16) complexed with fragments of antibody (Fab) from three different neutralizing monoclonals (mAbs): H16.1A, H16.14J, and H263.A2. The structure-function analysis revealed predominantly monovalent binding of each Fab with capsid interactions that involved multiple loops from symmetry related copies of the major capsid protein. The residues identified in each Fab-virus interface map to a conformational groove on the surface of the capsomer. In addition to the known involvement of the FG and HI loops, the DE loop was also found to constitute the core of each epitope. Surprisingly, the epitope mapping also identified minor contributions by EF and BC loops. Complementary immunological assays included mAb and Fab neutralization. The specific binding characteristics of mAbs correlated with different neutralizing behaviors in pre- and post-attachment neutralization assays. - Highlights: • We present HPV16-Fab complexes from neutralizing mAbs: H16.1A, H16.14J, and H263.A2. • The structure-function analysis revealed predominantly monovalent binding of each mAb. • Capsid–Fab interactions involved multiple loops from symmetry related L1 proteins. • Besides the known FG and HI loops, epitope mapping also identified DE, EF, and BC loops. • Neutralizing assays complement the structures to show multiple neutralization mechanisms.

60 APPLIED LIFE SCIENCES↗

A Study of the Interaction, Morphology, and Structure in Trypsin-Epigallocatechin-3-Gallate Complexes

Understanding the interaction between proteins and polyphenols is of significance to food industries. The aim of this research was to investigate the mode of aggregation for trypsin-EGCG (Epigallocatechin-3-gallate) complexes. For this, the complex was characterized by fluorescence spectroscopy, circular dichroism (CD) spectra, small-angel X-ray scattering (SAXS), and atomic force microscope (AFM) techniques. The results showed that the fluorescence intensity of trypsin-EGCG complexes decreased with increasing the concentration of EGCG, indicating that the interaction between trypsin and EGCG resulted in changes in the microenvironment around fluorescent amino acid residues. The results of CD analysis showed conformational changes in trypsin after binding with EGCG. The results from SAXS analysis showed that the addition of EGCG results in the formation of aggregates of trypsin-EGCG complexes, and increasing the concentration of EGCG resulted in larger aggregates. AFM images showed that the trypsin-EGCG complex formed aggregates of irregular ellipsoidal shapes with the size of about 200 × 400 × 200 nm, with EGCG interconnecting the trypsin particles. Overall, according to these results, it was concluded that the large aggregates of trypsin-EGCG complexes are formed from several small aggregates that are interconnected. The results of this study shed some light on the interaction between digestive enzymes and EGCG.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

A general non-Abelian density matrix renormalization group algorithm with application to the C{sub 2} dimer

We extend our previous work [S. Sharma and G. K.-L. Chan, J. Chem. Phys. 136, 124121 (2012)], which described a spin-adapted (SU(2) symmetry) density matrix renormalization group algorithm, to additionally utilize general non-Abelian point group symmetries. A key strength of the present formulation is that the requisite tensor operators are not hard-coded for each symmetry group, but are instead generated on the fly using the appropriate Clebsch-Gordan coefficients. This allows our single implementation to easily enable (or disable) any non-Abelian point group symmetry (including SU(2) spin symmetry). We use our implementation to compute the ground state potential energy curve of the C{sub 2} dimer in the cc-pVQZ basis set (with a frozen-core), corresponding to a Hilbert space dimension of 10{sup 12} many-body states. While our calculated energy lies within the 0.3 mE{sub h} error bound of previous initiator full configuration interaction quantum Monte Carlo and correlation energy extrapolation by intrinsic scaling calculations, our estimated residual error is only 0.01 mE{sub h}, much more accurate than these previous estimates. Due to the additional efficiency afforded by the algorithm, the excitation energies (T{sub e}) of eight lowest lying excited states: a{sup 3}Π{sub u}, b{sup 3}Σ{sub g}{sup −}, A{sup 1}Π{sub u}, c{sup 3}Σ{sub u}{sup +}, B{sup 1}Δ{sub g}, B{sup ′1}Σ{sub g}{sup +}, d{sup 3}Π{sub g}, and C{sup 1}Π{sub g} are calculated, which agree with experimentally derived values to better than 0.06 eV. In addition, we also compute the potential energy curves of twelve states: the three lowest levels for each of the irreducible representations {sup 1}Σ{sub g}{sup +}, {sup 1}Σ{sub u}{sup +}, {sup 1}Σ{sub g}{sup −}, and {sup 1}Σ{sub u}{sup −}, to an estimated accuracy of 0.1 mE{sub h} of the exact result in this basis.

71 CLASSICAL AND QUANTUM MECHANICS, GENERAL PHYSIC↗

Crystal structure of importin-α3 bound to the nuclear localization signal of Ran-binding protein 3

Ran-binding protein 3 (RanBP3) is a primarily nuclear Ran-binding protein that functions as an accessory factor in the Ran GTPase system. RanBP3 associates with Ran-specific nucleotide exchange factor RCC1 and enhances its catalytic activity towards Ran. RanBP3 also promotes CRM1-mediated nuclear export as well as CRM1-independent nuclear export of β-catenin, Smad2, and Smad3. Nuclear import of RanBP3 is dependent on the nuclear import adaptor protein importin-α and, RanBP3 is imported more efficiently by importin-α3 than by other members of the importin-α family. Protein kinase signaling pathways control nucleocytoplasmic transport through phosphorylation of RanBP3 at Ser58, immediately C-terminal to the nuclear localization signal (NLS) in the N-terminal region of RanBP3. Here we report the crystal structure of human importin-α3 bound to an N-terminal fragment of human RanBP3 containing the NLS sequence that is necessary and sufficient for nuclear import. The structure reveals that RanBP3 binds to importin-α3 residues that are strictly conserved in all seven isoforms of human importin-α at the major NLS-binding site, indicating that the region of importin-α outside the NLS-binding site, possibly the autoinhibotory importin-β1-binding domain, may be the key determinant for the preferential binding of RanBP3 to importin-α3. Computational docking simulation indicates that phosphorylation of RanBP3 at Ser58 could potentially stabilize the association of RanBP3 with importin-α through interactions between the phosphate moiety of phospho-Ser58 of RanBP3 and a cluster of basic residues (Arg96 and Lys97 in importin-α3) on armadillo repeat 1 of importin-α. - Highlights: • Ran-binding protein 3 (RanBP3) is an accessory factor in the Ran GTPase system. • The N-terminal region of RanBP3 contains a nuclear localization signal (NLS). • Crystal structure of importin-α3 bound to the NLS of RanBP3 is solved. • Phosphorylation of RanBP3 at Ser58 may modulate binding affinity to importin-α.

60 APPLIED LIFE SCIENCES↗

Environmental Impacts of Wind Power Development on the Population Biology of Greater Prairie-Chickens

1. We investigated the impacts of wind power development on the demography, movements, and population genetics of Greater Prairie-Chickens (Tympanuchus cupido) at three sites in northcentral and eastern Kansas for a 7-year period. Only 1 of 3 sites was developed for wind power, the 201MW Meridan Way Wind Power Facility at the Smoky Hills site in northcentral Kansas. Our project report is based on population data for prairie chickens collected during a 2-year preconstruction period (2007-2008), a 3-year postconstruction period (2009-2011) and one final year of lek surveys (2012). Where relevant, we present preconstruction data from our field studies at reference sites in the northern Flint Hills (2007-2009) and southern Flint Hills (2006-2008). 2. We addressed seven potential impacts of wind power development on prairie chickens: lek attendance, mating behavior, use of breeding habitat, fecundity rates, natal dispersal, survival rates, and population numbers. Our analyses of pre- and postconstruction impacts are based on an analysis of covariance design where we modeled population performance as a function of treatment period, distance to eventual or actual site of the nearest wind turbine, and the interaction of these factors. Our demographic and movement data from the 6-year study period at the Smoky Hills site included 23 lek sites, 251 radio-marked females monitored for 287 bird-years, and 264 nesting attempts. Our genetic data were based on genotypes of 1,760 females, males and chicks that were screened with a set of 27 microsatellite markers that were optimized in the lab. 3. In our analyses of lek attendance, the annual probability of lek persistence during the preconstruction period was ~0.9. During the postconstruction period, distance to nearest turbine did not have a significant effect on the probability of lek persistence. However, the probability of lek persistence increased from 0.69 at 0 m to 0.89 at 30 km from turbines, and most abandoned lek sites were located <5 km from turbines. Probability of lek persistence was significantly related to habitat and number of males. Leks had a higher probability of persistence in grasslands than agricultural fields, and increased from ~0.2 for leks of 5 males, to >0.9 for leks of 10 or more males. Large leks in grasslands should be a higher priority for conservation. Overall, wind power development had a weak effect on the annual probability of lek persistence. 3. We used molecular methods to investigate the mating behavior of prairie chickens. The prevailing view for lek-mating grouse is that females mate once to fertilize the clutch and that conspecific nest parasitism is rare. We found evidence that females mate multiple times to fertilize the clutch (8-18% of broods, 4-38% of chicks) and will parasitize nests of other females during egg-laying (~17% of nests). Variable rates of parentage were highest in the fragmented landscapes at the Smoky Hills field site, and were lower at the Flint Hills field site. Comparisons of the pre- and postconstruction periods showed that wind energy development did not affect the mating behaviors of prairie chickens. 4. We examined use of breeding habitats by radio-marked females and conducted separate analyses for nest site selection, and movements of females not attending nests or broods. The landscape was a mix of native prairie and agricultural habitats, and nest site selection was not random because females preferred to nest in grasslands. Nests tended to be closer to turbines during the postconstruction period and there was no evidence of behavioral avoidance of turbines by females during nest site selection. Movements of females not attending nests or broods showed that females crossed the site of the wind power development at higher rates during the preconstruction period (20%) than the postconstruction period (11%), and that movements away from turbines were more frequent during the postconstruction period. Thus, wind power development appears to affect movements in breeding habitats but not nest site selection of female prairie chickens during the breeding season. 5. We tested the effects of wind power development on five components of female fecundity: timing of clutch initiation, clutch size of first nests and renests, nest survival, and hatchability of eggs. Average date of clutch initiation was 26 April, clutch size was 12.7 and 10.6 eggs for first nests and renests, probability of nest survival was low at 0.18, but egg hatchability was high at 0.79. Wind power development had no impact on reproductive effort or nesting success, and all five components of fecundity were not related to treatment period or distance to turbine. Nest survival was the main factor limiting reproductive output of female prairie chickens and most losses were due to predation. Daily nest survival was strongly related to vegetative cover at the nest. Changes to rangeland management practices that would double nesting cover from 2.5 to 5 dm would triple the probability of nest survival from 0.17 to 0.52. Grass and forb cover had weak positive effects on daily nest survival whereas shrub cover, proximity to woodlands, and recent rainfall had negative effects. Reproductive performance of prairie chickens is low in managed rangelands in northcentral Kansas and efforts to improve range conditions and reduce predator activity would aid recovery of prairie chicken populations. 6. We used molecular methods to investigate patterns of natal dispersal in prairie chickens. High rates of nest failure limited the number of young that we could sample. Direct detections of natal dispersal were limited because survival of newly hatched chicks to become adults were low and because we were unable to detect dispersal distances outside of our study area. Direct observations of natal movements were limited and were inadequate to make conclusions about the potential impacts of wind energy development on natal dispersal. Spatial correlograms of genetic distance among males at leks were a more sensitive measure of population structure, and indicated a weak effect of wind energy development on the spatial genetic structure of prairie chickens. 7. We tested the effects of wind power development on female survival with time-to-event models, and on residual body mass of males with analyses of covariance. Distance to turbine and the interaction of distance and treatment period had no effect on female survival. Contrary to predictions of negative impacts of wind power development, the probability of female was lowest during the preconstruction period (0.274) and increased significantly during the postconstruction period (0.543). Inspection of hazard functions indicated that the difference in annual survival could be attributed to a higher risk of mortality during the lekking season in the preconstruction period. We suggest that wind power development may have improved ecological conditions for prairie chickens by disrupting the foraging behavior of diurnal raptors that kill prairie chickens at lek sites. In support of this idea, raptor kills tended to be farther from turbines during the postconstruction period whereas mammalian kills were closer. Analyses of the major causes of mortality did not support our hypothesis because the odds of raptor predation were greater after development was completed. Most mortality losses of radio-marked females were due to predation, and losses to collision mortality or harvest were rare events. Low rates of natural mortality during fall and winter imply that harvest is likely to be additive mortality in prairie chickens. Wind energy development reduced the residual body mass of male Greater Prairie-Chickens at lek sites near turbines. Low values of residual body mass could have a negative impact on individual survival or fecundity rates, or may be related to predation risk and flight performance of males displaying at open lek sites. 8. We tested for impacts of wind power development on population numbers of prairie chickens with monitoring of male numbers at leks, and with genetic measures of population structure. Lek counts indicated that wind power development did not affect the population size of prairie chickens. Peak counts of males at leks were recorded the first year after construction was completed and the highest rates of population change were observed during the interval when the wind power facility was constructed. Population numbers of prairie chickens near and distant from turbines appeared to covary in parallel, probably because bird numbers were linked by dispersal movements. Estimates of population viability based on genetic diversity, effective population size and rates of population exchange did not show annual changes and were unaffected by wind development during our study. Estimates of relatedness among males at the same and different leks suggested that wind power development has either reduced dispersal rates or changed settlement patterns, leading to higher rates of relatedness among males displaying at the same lek site. 9. Greater Prairie-Chickens were not strongly affected by wind power development in Kansas. Negative impacts of wind power development included a trend for reductions in lek persistence near turbines, behavioral avoidance of turbines by females during their breeding season movements, and changes in the genetic structure of males at leks that were consistent with reduced dispersal or recruitment rates. We found no impacts of wind power development on nest site selection, female reproductive effort or nesting success, or population numbers. Positive impacts of wind power development included an increase in female survival rates. We hypothesized that the unexpected increase in female survival was related to changes in trophic interactions and disruption of the foraging behavior of raptors that kill prairie chickens at lek site. 10. Research funding for this project included a grant from the 20% Wind by 2030 Program of the Department of Energy (this final report), and grants from the Kansas Department of Wildlife, Parks and Tourism, the National Fish and Wildlife Federation, and initial funding from the National Wind Coordinating Collaborative. Research products from data collected during the preconstruction period have included five peer-reviewed research articles and two PhD dissertations at Kansas State University. Additional manuscripts are in review for possible publication in 2013-2014.

17 WIND ENERGY↗

The N‐terminal intrinsically disordered region of Ncb5or docks with the cytochrome b 5 core to form a helical motif that is of ancient origin

Abstract NADH cytochromeb 5 oxidoreductase (Ncb5or) is a cytosolic ferric reductase implicated in diabetes and neurological conditions. Ncb5or comprises cytochromeb 5 (b 5 ) and cytochromeb 5 reductase (b 5 R) domains separated by a CHORD‐Sgt1 (CS) linker domain. Ncb5or redox activity depends on proper inter‐domain interactions to mediate electron transfer from NADH or NADPH via FAD to heme. While full‐length human Ncb5or has proven resistant to crystallization, we have succeeded in obtaining high‐resolution atomic structures of theb 5 domain and a construct containing the CS andb 5 R domains (CS/b 5 R). Ncb5or also contains an N‐terminal intrinsically disordered region of 50 residues that has no homologs in other protein families in animals but features a distinctive, conserved L 34 MDWIRL 40 motif also present in reduced lateral root formation (RLF) protein in rice and increased recombination center 21 in baker's yeast, all attaching to ab 5 domain. After unsuccessful attempts at crystallizing a human Ncb5or construct comprising the N‐terminal region naturally fused to theb 5 domain, we were able to obtain a high‐resolution atomic structure of a recombinant rice RLF construct corresponding to residues 25–129 of human Ncb5or (52% sequence identity; 74% similarity). The structure reveals Trp 120 (corresponding to invariant Trp 37 in Ncb5or) to be part of an 11‐residue α‐helix (S 116 QMDWLKLTRT 126 ) packing against two of the four helices in theb 5 domain that surround heme (α2 and α5). The Trp 120 side chain forms a network of interactions with the side chains of four highly conserved residues corresponding to Tyr 85 and Tyr 88 (α2), Cys 124 (α5), and Leu 47 in Ncb5or. Circular dichroism measurements of human Ncb5or fragments further support a key role of Trp 37 in nucleating the formation of the N‐terminal helix, whose location in the N/b 5 module suggests a role in regulating the function of this multi‐domain redox enzyme. This study revealed for the first time an ancient origin of a helical motif in the N/b 5 module as reflected by its existence in a class of cytochromeb 5 proteins from three kingdoms among eukaryotes.

Biochemistry & Molecular Biology↗

Identification of Small-Molecule Inhibitors of Fibroblast Growth Factor 23 Signaling via In Silico Hot Spot Prediction and Molecular Docking to α-Klotho

Fibroblast growth factor 23 (FGF23) is a therapeutic target for treating hereditary and acquired hypophosphatemic disorders, such as X-linked hypophosphatemic (XLH) rickets and tumor-induced osteomalacia (TIO), respectively. FGF23-induced hypophosphatemia is mediated by signaling through a ternary complex formed by FGF23, the FGF receptor (FGFR), and α-Klotho. Currently, disorders of excess FGF23 are treated with an FGF23-blocking antibody, burosumab. Small-molecule drugs that disrupt protein/protein interactions necessary for the ternary complex formation offer an alternative to disrupting FGF23 signaling. In this study, the FGF23:α-Klotho interface was targeted to identify small-molecule protein/protein interaction inhibitors since it was computationally predicted to have a large fraction of hot spots and two druggable residues on α-Klotho. We further identified Tyr433 on the KL1 domain of α-Klotho as a promising hot spot and α-Klotho as an appropriate drug-binding target at this interface. Subsequently, we performed in silico docking of ~5.5 million compounds from the ZINC database to the interface region of α-Klotho from the ternary crystal structure. Following docking, 24 and 20 compounds were in the final list based on the lowest binding free energies to α-Klotho and the largest number of contacts with Tyr433, respectively. Herein, five compounds were assessed experimentally by their FGF23-mediated extracellular signal-regulated kinase (ERK) activities in vitro, and two of these reduced activities significantly. Both these compounds were predicted to have favorable binding affinities to α-Klotho but not have a large number of contacts with the hot spot Tyr433. ZINC12409120 was found experimentally to disrupt FGF23:α-Klotho interaction to reduce FGF23-mediated ERK activities by 70% and have a half maximal inhibitory concentration (IC50) of 5.0 ± 0.23 μM. Molecular dynamics (MD) simulations of the ZINC12409120:α-Klotho complex starting from in silico docking poses reveal that the ligand exhibits contacts with residues on the KL1 domain, the KL1–KL2 linker, and the KL2 domain of α-Klotho simultaneously, thereby possibly disrupting the regular function of α-Klotho and impeding FGF23:α-Klotho interaction. ZINC12409120 is a candidate for lead optimization.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗