Thermotropic phase transition in soluble nanoscale lipid bilayers

The role of lipid domain size and protein-lipid interfaces in the thermotropic phase transition of dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) bilayers in Nanodiscs was studied using small-angle X-ray scattering (SAXS), differential scanning calorimetry (DSC), and generalized polarization (GP) of the lipophilic probe Laurdan. Nanodiscs are water-soluble, monodisperse, self-assembled lipid bilayers encompassed by a helical membrane scaffold protein (MSP). MSPs of different lengths were used to define the diameter of the Nanodisc lipid bilayer from 76 to 108 A and the number of DPPC molecules from 164 to 335 per discoidal structure. In Nanodiscs of all sizes, the phase transitions were broader and shifted to higher temperatures relative to those observed in vesicle preparations. The size dependences of the transition enthalpies and structural parameters of Nanodiscs reveal the presence of a boundary lipid layer in contact with the scaffold protein encircling the perimeter of the disc. The thickness of this annular layer was estimated to be approximately 15 A, or two lipid molecules. SAXS was used to measure the lateral thermal expansion of Nanodiscs, and a steep decrease of bilayer thickness during the main lipid phase transition was observed. These results provide the basis for the quantitative understanding of cooperative phase transitions in membrane bilayers in confined geometries at the nanoscale.     

Calculations on the rate of the ion-molecule reaction between NH3+ and H2

The rate coefficient for the ion-molecule reaction NH3(+) + H2 --> NH4(+) + H has been calculated as a function of temperature with the use of the statistical phase space approach. The potential surface and reaction complex and transition state parameters used in the calculation have been taken from ab initio quantum chemical calculations. The calculated rate coefficient has been found to mimic the unusual temperature dependence measured in the laboratory, in which the rate coefficient decreases with decreasing temperature until 50-100 K and then increases at still lower temperatures. Quantitative agreement between experimental and theoretical rate coefficients is satisfactory given the uncertainties in the ab initio results and in the dynamics calculations. The rate coefficient for the unusual three-body process NH3(+) + H2 + He --> NH4(+) + H + He has also been calculated as a function of temperature and the result found to agree well with a previous laboratory determination.     

How do azoles inhibit cytochrome P450 enzymes? A density functional study

To examine how azole inhibitors interact with the heme active site of the cytochrome P450 enzymes, we have performed a series of density functional theory studies on azole binding. These are the first density functional studies on azole interactions with a heme center and give fundamental insight into how azoles inhibit the catalytic function of P450 enzymes. Since azoles come in many varieties, we tested three typical azole motifs representing a broad range of azole and azole-type inhibitors: methylimidazolate, methyltriazolate, and pyridine. These structural motifs represent typical azoles, such as econazole, fluconazole, and metyrapone. The calculations show that azole binding is a stepwise mechanism whereby first the water molecule from the resting state of P450 is released from the sixth binding site of the heme to create a pentacoordinated active site followed by coordination of the azole nitrogen to the heme iron. This process leads to the breaking of a hydrogen bond between the resting state water molecule and the approaching inhibitor molecule. Although, formally, the water molecule is released in the first step of the reaction mechanism and a pentacoordinated heme is created, this does not lead to an observed spin state crossing. Thus, we show that release of a water molecule from the resting state of P450 enzymes to create a pentacoordinated heme will lead to a doublet to quartet spin state crossing at an Fe-OH(2) distance of approximately 3.0 A, while the azole substitution process takes place at shorter distances. Azoles bind heme with significantly stronger binding energies than a water molecule, so that these inhibitors block the catalytic cycle of the enzyme and prevent oxygen binding and the catalysis of substrate oxidation. Perturbations within the active site (e.g., a polarized environment) have little effect on the relative energies of azole binding. Studies with an extra hydrogen-bonded ethanol molecule in the model, mimicking the active site of the CYP121 P450, show that the resting state and azole binding structures are close in energy, which may lead to chemical equilibrium between the two structures, as indeed observed with recent protein structural studies that have demonstrated two distinct azole binding mechanisms to P450 heme.     

Determination of ion mobility collision cross sections for unresolved isomeric mixtures using tandem mass spectrometry and chemometric deconvolution

Ion mobility (IM) is an important analytical technique for determining ion collision cross section (CCS) values in the gas-phase and gaining insight into molecular structures and conformations. However, limited instrument resolving powers for IM may restrict adequate characterization of conformationally similar ions, such as structural isomers, and reduce the accuracy of IM-based CCS calculations. Recently, we introduced an automated technique for extracting “pure” IM and collision-induced dissociation (CID) mass spectra of IM overlapping species using chemometric deconvolution of post-IM/CID mass spectrometry (MS) data [J. Am. Soc. Mass Spectrom., 2014, 25, 1810–1819]. Here we extend those capabilities to demonstrate how extracted IM profiles can be used to calculate accurate CCS values of peptide isomer ions which are not fully resolved by IM. We show that CCS values obtained from deconvoluted IM spectra match with CCS values measured from the individually analyzed corresponding peptides on uniform field IM instrumentation. We introduce an approach that utilizes experimentally determined IM arrival time (AT) “shift factors” to compensate for ion acceleration variations during post-IM/CID and significantly improve the accuracy of the calculated CCS values. Also, we discuss details of this IM deconvolution approach and compare empirical CCS values from traveling wave (TW)IM-MS and drift tube (DT)IM-MS with theoretically calculated CCS values using the projected superposition approximation (PSA). For example, experimentally measured deconvoluted TWIM-MS mean CCS values for doubly-protonated RYGGFM, RMFGYG, MFRYGG, and FRMYGG peptide isomers were 288.₈ Å², 295.₁ Å², 296.₈ Å², and 300.₁ Å²; all four of these CCS values were within 1.5% of independently measured DTIM-MS values.     

Diagnosing laser-driven,shock-heated foam target with Al absorption spectroscopy on OMEGA EP

Results on diagnoses of laser-driven, shock-heated foam plasma with time-resolved Al 1s-2p absorption spectroscopy are reported. Experiments were carried out to produce a platform for the study of relativistic electron transport. In cone-guided Fast Ignition (FI), relativistic electrons generated by a high-intensity, short-pulse igniter beam must be transported through a cone tip to an imploded core. Transport of the energetic electrons could be significantly affected by the temperature-dependent resistivity of background plasmas. The experiment was conducted using four UV beams of the OMEGA EP laser at the Laboratory For Laser Energetics. One UV beam (1.2 kJ, 3.5 ns square) was used to launch a shock wave into a foam package target, consisting of 200 mg/cm³ CH foam with aluminum dopant and a solid plastic container surrounding the foam layer. The other three UV beams with the total energy of 3.2 kJ in 2.5 ns pulse duration were tightly focused onto a Sm dot target to produce a point X-ray source in the energy range of 1.4–1.6 keV. The quasi-continuous X ray signal was transmitted through the shock-heated Al-doped, foam layer and recorded with an X-ray streak camera. The measured 1s-2p Al absorption features were analyzed using an atomic physics code FLYCHK. Electron temperature of 40 eV inferred from the spectral analysis is consistent with 2-D DRACO Radiation-hydrodynamic simulations.     

Multi‐Component Kinetic Modeling for Controlling Local Compositions in Thermosensitive Polymers

Summary: An explicit terminal copolymerization kinetic model accounting for the copolymerization of up to four different comonomers is developed and applied to model the conversion profiles and local compositional gradients in functionalized PNIPAM‐based polymer and hydrogel systems. The kinetics of the functional comonomer(s) have a large influence on both the mole fraction and chain distribution of functional groups in polymers. Strategies are developed to synthesize polymers with uniform compositions by applying semi‐batch techniques or via copolymerization of multiple monomers with the same target functionality but with divergent reactivities relative to NIPAM. Synthetic protocols are also designed to maximize the compositional uniformity and randomness of ampholytic polymers.

Instantaneous mole fractions of monomers in polymers as a function of the overall monomer conversion for the copolymerizations of NIPAM, MBA, and two functional monomers: MMA and acrylamide.     

Global intercompany assessment of ICIEF platform comparability for the characterization of therapeutic proteins

An international team spanning 19 sites across 18 biopharmaceutical and in vitro diagnostics companies in the United States, Europe, and China, along with one regulatory agency, was formed to compare the precision and robustness of imaged CIEF (ICIEF) for the charge heterogeneity analysis of the National Institute of Standards and Technology (NIST) mAb and a rhPD-L1-Fc fusion protein on the iCE3 and the Maurice instruments. This information has been requested to help companies better understand how these instruments compare and how to transition ICIEF methods from iCE3 to the Maurice instrument. The different laboratories performed ICIEF on the NIST mAb and rhPD-L1-Fc with both the iCE3 and Maurice using analytical methods specifically developed for each of the molecules. After processing the electropherograms, statistical evaluation of the data was performed to determine consistencies within and between laboratory and outlying information. The apparent isoelectric point (pI) data generated, based on two-point calibration, for the main isoform of the NIST mAb showed high precision between laboratories, with RSD values of less than 0.3% on both instruments. The SDs for the NIST mAb and the rhPD-L1-Fc charged variants percent peak area values for both instruments are less than 1.02% across different laboratories. These results validate the appropriate use of both the iCE3 and Maurice for ICIEF in the biopharmaceutical industry in support of process development and regulatory submissions of biotherapeutic molecules. Further, the data comparability between the iCE3 and Maurice illustrates that the Maurice platform is a next-generation replacement for the iCE3 that provides comparable data.