Dynamics of ABC Transporter P-glycoprotein in Three Conformational States.

We used hydrogen-deuterium exchange mass spectrometry (HDX-MS) to obtain a comprehensive view of transporter dynamics (85.8% sequence coverage) occurring throughout the multidrug efflux transporter P-glycoprotein (P-gp) in three distinct conformational states: predominantly inward-facing apo P-gp, pre-hydrolytic (E552Q/E1197Q) P-gp bound to Mg+2-ATP, and outward-facing P-gp bound to Mg+2-ADP-VO4-3. Nucleotide affinity was measured with bio-layer interferometry (BLI), which yielded kinetics data that fit a two Mg+2-ATP binding-site model. This model has one high affinity site (3.2 ± 0.3 µM) and one low affinity site (209 ± 25 µM). Comparison of deuterium incorporation profiles revealed asymmetry between the changes undergone at the critical interfaces where nucleotide binding domains (NBDs) contact intracellular helices (ICHs). In the pre-hydrolytic state, both interfaces between ICHs and NBDs decreased exchange to similar extents relative to inward-facing P-gp. In the outward-facing state, the ICH-NBD1 interface showed decreased exchange, while the ICH-NBD2 interface showed less of an effect. The extracellular loops (ECLs) showed reduced deuterium uptake in the pre-hydrolytic state, consistent with an occluded conformation. While in the outward-facing state, increased ECL exchange corresponding to EC domain opening was observed. These findings point toward asymmetry between both NBDs, and they suggest that pre-hydrolytic P-gp occupies an occluded conformation

A Born-Oppenheimer photolysis model of N_2O fractionation

The isotopically light N_2O produced by microbial activity is thought to be balanced by the return of heavy stratospheric nitrous oxide. The Yung and Miller [1997] method that first explained these trends yields photolytic fractionation factors ∼half those observed by experiment or predicted quantum mechanically, however. To address these issues, we present here a Born-Oppenheimer photolysis model that uses only commonly available spectroscopic data. The predicted fractionations quantitatively reproduce laboratory data, and have been incorporated into zonally averaged atmospheric simulations. Like McLinden et al. [2003] , who employ a three-dimensional chemical transport model with cross sections scaled to match laboratory data, we find excellent agreement between predictions and stratospheric measurements; additional processes that contribute to the mass independent anomaly in N_2O can only account for a fraction of its global budget

Oxygen isotopic composition of carbon dioxide in the middle atmosphere

The isotopic composition of long-lived trace molecules provides a window into atmospheric transport and chemistry. Carbon dioxide is a particularly powerful tracer, because its abundance remains >100 parts per million by volume (ppmv) in the mesosphere. Here, we successfully reproduce the isotopic composition of CO2 in the middle atmosphere, which has not been previously reported. The mass-independent fractionation of oxygen in CO2 can be satisfactorily explained by the exchange reaction with O(1D). In the stratosphere, the major source of O(1D) is O3 photolysis. Higher in the mesosphere, we discover that the photolysis of 16O17O and 16O18O by solar Lyman-{alpha} radiation yields O(1D) 10–100 times more enriched in 17O and 18O than that from ozone photodissociation at lower altitudes. This latter source of heavy O(1D) has not been considered in atmospheric simulations, yet it may potentially affect the "anomalous" oxygen signature in tropospheric CO2 that should reflect the gross carbon fluxes between the atmosphere and terrestrial biosphere. Additional laboratory and atmospheric measurements are therefore proposed to test our model and validate the use of CO2 isotopic fractionation as a tracer of atmospheric chemical and dynamical processes

Evidence for O-atom exchange in the O(^1D) + N_2O reaction as the source of mass-independent isotopic fractionation in atmospheric N_2O

Recent experiments have shown that in the oxygen isotopic exchange reaction for O(^1D) + CO_2 the elastic channel is approximately 50% that of the inelastic channel [Perri et al., 2003]. We propose an analogous oxygen atom exchange reaction for the isoelectronic O(^1D) + N_2O system to explain the mass-independent isotopic fractionation (MIF) in atmospheric N_2O. We apply quantum chemical methods to compute the energetics of the potential energy surfaces on which the O(^1D) + N_2O reaction occurs. Preliminary modeling results indicate that oxygen isotopic exchange via O(^1D) + N_2O can account for the MIF oxygen anomaly if the oxygen atom isotopic exchange rate is 30–50% that of the total rate for the reactive channels

Reply to comment by Röckmann and Kaiser on "Evidence for O-atom exchange in the O(^1D) + N_2O reaction as the source of mass-independent isotopic fractionation in atmospheric N_2O"

Based upon the authors’ questioning of the existence of the C_(2v) intermediate, we have reviewed our evidence for the existence of this state. It now appears that this state was in fact an artifact of our calculation [Yung et al., 2004], and was a saddle point rather than a true minimum. Our desire to provide a timely response to this criticism has kept us from determining exactly what minimum structure will be obtained by a full minimization at the level of theory employed. However, it is clear that the C_(2v) symmetry of the compound is broken in such a way that the two N-O bonds are no longer equivalent. We are grateful to the authors for helping us resolve this issue

Excitation of nightside magnetosonic waves observedby Van Allen Probes

Abstract During the recovery phase of the geomagnetic storm on 30-31 March 2013, Van Allen Probe A detected enhanced magnetosonic (MS) waves in a broad range of L = 1.8-4.7 and magnetic local time (MLT) = 17-22 h, with a frequency range ∼10-100 Hz. In the meanwhile, distinct proton ring distributions with peaks at energies of ∼10 keV, were also observed in L = 3.2-4.6 and L = 5.0-5.6. Using a subtracted bi-Maxwellian distribution to model the observed proton ring distribution, we perform three-dimensional ray tracing to investigate the instability, propagation, and spatial distribution of MS waves. Numerical results show that nightside MS waves are produced by proton ring distribution and grow rapidly from the source location L = 5.6 to the location L = 5.0 but remain nearly stable at locations L \u3c 5.0. Moreover, waves launched toward lower L shells with different initial azimuthal angles propagate across different MLT regions with divergent paths at first, then gradually turn back toward higher L shells and propagate across different MLT regions with convergent paths. The current results further reveal that MS waves are generated by a ring distribution of ∼10 keV proton and proton ring in one region can contribute to the MS wave power in another region. Key Points: Correlated Van Allen Probe data of MS wave and proton ringGrowth rates are peaked at the harmonics of the proton gyrofrequencyMS waves propagate inward divergently and outward convergently

Designer Reagents for Mass Spectrometry-Based Proteomics: Clickable Cross-Linkers for Elucidation of Protein Structures and Interactions

We present novel homobifunctional amine-reactive clickable cross-linkers (CXLs) for investigation of three-dimensional protein structures and protein–protein interactions (PPIs). CXLs afford consolidated advantages not previously available in a simple cross-linker, including (1) their small size and cationic nature at physiological pH, resulting in good water solubility and cell-permeability, (2) an alkyne group for bio-orthogonal conjugation to affinity tags via the click reaction for enrichment of cross-linked peptides, (3) a nucleophilic displacement reaction involving the 1,2,3-triazole ring formed in the click reaction, yielding a lock-mass reporter ion for only clicked peptides, and (4) higher charge states of cross-linked peptides in the gas-phase for augmented electron transfer dissociation (ETD) yields. Ubiquitin, a lysine-abundant protein, is used as a model system to demonstrate structural studies using CXLs. To validate the sensitivity of our approach, biotin-azide labeling and subsequent enrichment of cross-linked peptides are performed for cross-linked ubiquitin digests mixed with yeast cell lysates. Cross-linked peptides are detected and identified by collision induced dissociation (CID) and ETD with linear quadrupole ion trap (LTQ)-Fourier transform ion cyclotron resonance (FTICR) and LTQ-Orbitrap mass spectrometers. The application of CXLs to more complex systems (e.g., in vivo cross-linking) is illustrated by Western blot detection of Cul1 complexes including known binders, Cand1 and Skp2, in HEK 293 cells, confirming good water solubility and cell-permeability

Isotopic composition of stratospheric ozone

We present a kinetic calculation for the isotopic composition of stratospheric ozone. The calculated enrichments of ^(49)O_3 and ^(50)O_3 are in agreement with atmospheric measurements made at midlatitudes. Integrating the kinetic fractionation processes in the formation and photolysis of ozone, we obtain enrichments of ∼7.5–10.5 and ∼7.5–12.5% (referenced to atmospheric O_2) for δ^(49)O_3 and δ^(50)O_3, respectively, at altitudes between 20 and 35 km; the photolysis in the Hartley band of ozone is responsible for the observed altitude variation. The overall magnitude of the ozone enrichments (∼10%) is large compared with that commonly known in atmospheric chemistry and geochemistry. The heavy oxygen atom in ozone is therefore useful as a tracer of chemical species and pathways that involve ozone or its derived products. For example, the mass anomalies of oxygen in two greenhouse gases, CO_2 and N_2O, are likely the consequences of the transfer of heavy oxygen atoms from ozone

Seasonal cycle of C^(16)O^(16)O, C^(16)O^(17)O, and C^(16)O^(18)O in the middle atmosphere: Implications for mesospheric dynamics and biogeochemical sources and sinks of CO_2

The isotopic anomaly of oxygen in atmospheric CO_2 is caused by exchange reactions with isotopically anomalous O(^1D) in the middle atmosphere. In the stratosphere, the major source of O(^1D) is O_3 photolysis; O_3 is known to possess mass-independent isotopic composition, with δ^(49)O_3 ≈ δ^(50)O_3 ≈ 100‰ relative to atmospheric O_2. Higher in the mesosphere, Lyman α-driven photodissociation of O2 provides a more important source of heavy O(^1D) than O_3 photolysis. Here we present a two-dimensional simulation of the isotopic composition of CO_2 from the surface to an altitude of ∼130 km that adequately reproduce the observed seasonal cycle of CO_2 in the upper troposphere and the age of air in the stratosphere. Our model results suggest that stratospheric-tropospheric exchange not only modifies the level of heavy CO_2 in the troposphere, but also influences its seasonal cycle. Thus the isotopic composition of CO_2 in the troposphere/biosphere could be affected by the downwelling air from the stratosphere. The predicted size of the effect is detectable by current instrumentation. Implications for the use of the isotopic composition of CO_2 to constrain the gross carbon flux between the atmosphere and terrestrial biosphere and the dynamics in the remote mesosphere are discussed

House dust mites possess a polymorphic, single domain putative peptidoglycan d,l endopeptidase belonging to the NlpC/P60 Superfamily

AbstractA 14kDa protein homologous to the γ-d-glutamyl-l-diamino acid endopeptidase members of the NlpC/P60 Superfamily has been described in Dermatophagoides pteronyssinus and Dermatophagoides farinae but it is not clear whether other species produce homologues. Bioinformatics revealed homologous genes in other Sarcopteformes mite species (Psoroptes ovis and Blomia tropicalis) but not in Tetranychus urticae and Metaseiulus occidentalis. The degrees of identity (similarity) between the D. pteronyssinus mature protein and those from D. farinae, P. ovis and B. tropicalis were 82% (96%), 77% (93%) and 61% (82%), respectively. Phylogenetic studies showed the mite proteins were monophyletic and shared a common ancestor with both actinomycetes and ascomycetes. The gene encoding the D. pteronyssinus protein was polymorphic and intronless in contrast to that reported for D. farinae. Homology studies suggest that the mite, ascomycete and actinomycete proteins are involved in the catalysis of stem peptide attached to peptidoglycan. The finding of a gene encoding a P60 family member in the D. pteronyssinus genome together with the presence of a bacterial promotor suggests an evolutionary link to one or more prokaryotic endosymbionts