Analyzing slowly exchanging protein conformations by ion mobility mass spectrometry: study of the dynamic equilibrium of prolyl oligopeptidase

Ion mobility mass spectrometry (IMMS) is a biophysical technique that allows the separation of isobaric species on the basis of their size and shape. The high separation capacity, sensitivity and relatively fast time scale measurements confer IMMS great potential for the study of proteins in slow (µs–ms) conformational equilibrium in solution. However, the use of this technique for examining dynamic proteins is still not generalized. One of the major limitations is the instability of protein ions in the gas phase, which raises the question as to what extent the structures detected reflect those in solution. Here, we addressed this issue by analyzing the conformational landscape of prolyl oligopeptidase (POP) – a model of a large dynamic enzyme in the µs–ms range – by native IMMS and compared the results obtained in the gas phase with those obtained in solution. In order to interpret the experimental results, we used theoretical simulations. In addition, the stability of POP gaseous ions was explored by charge reduction and collision‐induced unfolding experiments. Our experiments disclosed two species of POP in the gas phase, which correlated well with the open and closed conformations in equilibrium in solution; moreover, a gas‐phase collapsed form of POP was also detected. Therefore, our findings not only support the potential of IMMS for the study of multiple co‐existing conformations of large proteins in slow dynamic equilibrium in solution but also stress the need for careful data analysis to avoid artifacts. Copyright © 2016 John Wiley & Sons, Ltd.     

Organosulfate Formation through the Heterogeneous Reaction of Sulfur Dioxide with Unsaturated Fatty Acids and Long‐Chain Alkenes

The heterogeneous reaction between SO₂ and unsaturated compounds results in the efficient production of organosulfates for several fatty acids and long‐chain alkenes. The presence of an acid group, the physical state of the reactants (solid or liquid), the nature of the double bond (cis, trans, terminal), and the use of light irradiation all have an impact on the reaction rate. The reaction was investigated using different set‐ups (coated flow tube, aerosol flow tube, and diffuse reflectance infrared Fourier transform cell). The reaction products were identified by high‐resolution mass spectrometry and the impact of this reaction on organosulfate formation in the atmosphere is discussed.     

Zero‐Valent Amino‐Olefin Cobalt Complexes as Catalysts for Oxygen Atom Transfer Reactions from Nitrous Oxide

The synthesis and characterization of several zero‐valent cobalt complexes with a bis(olefin)‐amino ligand is presented. Some of these complexes proved to be efficient catalysts for the selective oxidation of secondary and allylic phosphanes, as well as diphosphanes, even with a direct P?P bond. With 5 mol % catalyst loadings the oxidations proceed under mild conditions (25–70 °C, 7–22 h, 2 bar N₂O) and afford good to excellent yields (65–98 %). In this process, the greenhouse gas N₂O is catalytically converted into benign N₂ and added‐value organophosphorus compounds, some of which are difficult to obtain otherwise.     

On minimum metric dimension of honeycomb networks

A minimum metric basis is a minimum set W of vertices of a graph G(V,E) such that for every pair of vertices u and v of G, there exists a vertex wW with the condition that the length of a shortest path from u to w is different from the length of a shortest path from v to w. The honeycomb and hexagonal networks are popular mesh-derived parallel architectures. Using the duality of these networks we determine minimum metric bases for hexagonal and honeycomb networks.     

A Porous Gelatin Methacrylate-Based Material for 3D Cell-Laden Constructs

3D constructs are fundamental in tissue engineering and cancer modeling, generating a demand for tailored materials creating a suitable cell culture microenvironment and amenable to be bioprinted. Gelatin methacrylate (GelMA) is a well-known functionalized natural polymer with good printability and binding motifs allowing cell adhesion; however, its tight micropores induce encapsulated cells to retain a non-physiological spherical shape. To overcome this problem, blended GelMa is here blended with Pluronic F-127 (PLU) to modify the hydrogel internal porosity by inducing the formation of larger mesoscale pores. The change in porosity also leads to increased swelling and a slight decrease in Young's modulus. All blends form stable hydrogels both when cast in annular molds and bioprinted in complex structures. Embedded cells maintain high viability, and while Neuroblastoma cancer cells typically aggregate inside the mesoscale pores, Mesenchymal Stem Cells stretch in all three dimensions, forming cell–cell and cell–ECM interactions. The results of this work prove that the combination of tailored porous materials with bioprinting techniques enables to control both the micro and macro architecture of cell-laden constructs, a fundamental aspect for the development of clinically relevant in vitro constructs.     

Application of pulsed field gradient NMR with high gradient strength for studies of self-diffusion in lipid membranes on the nanoscale

This work demonstrates the feasibility of noninvasive studies of lipid self-diffusion in model lipid membranes on the nanoscale using proton pulsed field gradient (PFG) NMR spectroscopy with high (up to 35 T/m) gradient amplitudes. Application of high gradients affords for the use of sufficiently small diffusion times under the conditions when the width of the gradient pulses is much smaller than the diffusion time. As a result, PFG NMR studies of partially restricted or anomalous diffusion in lipid bilayers become possible over length scales as small as 100 nm. This length scale is important because it is comparable to the size of membrane domains, or lipid rafts, which are believed to exist in biomembranes. In this work, high-gradient PFG NMR has been applied to study lipid self-diffusion in three-component planar-supported multibilayers (1,2-dioleoyl- sn-glycerol-3-phosphocholine/sphingomyelin/cholesterol). The degree of lipid orientation in the bilayers was determined with (31)P NMR. A special insert was designed to mechanically align the multibilayer stack at the magic angle with respect to the direction of the constant magnetic field to address the detrimental effects of proton dipole-dipole interactions on the NMR signal. This insert is an alternative to the conventional method of magic angle orientation of lipid membranes, the goniometer probe, which is not compatible with commercial high-gradient coils because of the lack of space in the magnet bore. Macroscopic orientation of the multibilayer stacks using the insert was confirmed with (1)H NMR spectroscopic studies and the comparison of results obtained from identical experiments using a goniometer probe for orientation. Diffusion studies were carried out at three different constant magnetic field strengths ( B 0) over a range of temperatures and diffusion times. The measured diffusivities were found to be in agreement with the data obtained previously by techniques that are limited to much larger length scales of diffusion observation than high-gradient PFG NMR.     

Supramolecular sensing with phosphonate cavitands

Phosphonate cavitands are an emerging class of synthetic receptors for supramolecular sensing. The molecular recognition properties of the third-generation tetraphosphonate cavitands toward alcohols and water at the gas-solid interface have been evaluated by means of three complementary techniques and compared to those of the parent mono- and diphosphonate cavitands. The combined use of ESI-MS and X-ray crystallography defined precisely the host-guest association at the interface in terms of type, number, strength, and geometry of interactions. Quartz crystal microbalance (QCM) measurements then validated the predictive value of such information for sensing applications. The importance of energetically equivalent multiple interactions on sensor selectivity and sensitivity has been demonstrated by comparing the molecular recognition properties of tetraphosphonate cavitands with those of their mono- and diphosphonate counterparts.     

Determination of peanut allergens in cereal-chocolate-based snacks: metal-tag inductively coupled plasma mass spectrometry immunoassay versus liquid chromatography/electrospray ionization tandem mass spectrometry

A comparison of two methods for the identification and determination of peanut allergens based on europium (Eu)-tagged inductively coupled plasma mass spectrometry (ICP-MS) immunoassay and on liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) with a triple quadrupole mass analyzer was carried out on a complex food matrix like a chocolate rice crispy-based snack. The LC/MS/MS method was based on the determination of four different peptide biomarkers selective for the Ara h2 and Ara h3/4 peanut proteins. The performance of this method was compared with that of a non-competitive sandwich enzyme-linked immunosorbent assay (ELISA) method with ICP-MS detection of the metal used to tag the antibody for the quantitative peanut protein analysis in food. The limit of detection (LOD) and quantitation of the ICP-MS immunoassay were 2.2 and 5 microg peanuts g(-1) matrix, respectively, the recovery ranged from 86 +/- 18% to 110 +/- 4% and linearity was proved in the 5-50 microg g(-1) range. The LC/MS/MS method allowed us to obtain LODs of 1 and 5 microg protein g(-1) matrix for Ara h3/4 and Ara h2, respectively, thus obtaining significantly higher values with respect to the ELISA ICP-MS method, taking into account the different expression for concentrations. Linearity was established in the 10-200 microg g(-1) range of peanut proteins in the food matrix investigated and good precision (RSD <10%) was demonstrated. Both the two approaches, used for screening or confirmative purposes, showed the power of mass spectrometry when used as a very selective detector in difficult matrices even if some limitations still exist, i.e. matrix suppression in the LC/ESI-MS/MS procedure and the change of the Ag/Ab binding with matrix in the ICP-MS method.     

Solvent-free, microwave assisted 1,3-cycloaddition of nitrones with vinyl nucleobases for the synthesis of N,O-nucleosides

A number of 4′-aza-2′,3′-dideoxy nucleosides have been synthesized in good yields and short reaction times by straightforward microwave assisted 1,3-cycloaddition of vinyl nucleobases with nitrones, in the absence of solvent. The vinyl nucleobases are used, for the first time, in their unprotected form.     

A new class of luminescent tricarbonyl rhenium(I) complexes containing bridging 1,2-diazine ligands: electrochemical, photophysical, and computational characterization

A novel class of luminescent tricarbonyl rhenium(I) complexes of general formula [Re2(mu-X)2(CO)6(mu-diaz)] (X=halogen and diaz=1,2-diazine) was prepared by reacting [ReX(CO)5] with 0.5 equiv of diazine (seven different ligands were used). The bridging coordination of the diazine in these dinuclear complexes was confirmed by single-crystal X-ray analysis. Cyclic voltammetry in acetonitrile showed for all the complexes (but the phthalazine derivative) a chemically and electrochemically reversible ligand-centered reduction, as well as a reversible metal-centered bielectronic oxidation. With respect to the prototypical luminescent [ReCl(CO)3(bpy)] complex, the oxidation is more difficult and the reduction easier (about +0.3 V), so that a similar highest occupied molecular orbital-lowest unoccupied molecular orbital gap is observed. All of the complexes exhibit photoluminescence at room temperature in solution, with broad unstructured emission from metal-to-ligand charge-transfer states, at lambda in the range 579-620 nm. Lifetimes (tau=20-2200 ns) and quantum yields (Phi up to 0.12) dramatically change upon varying the bridging ligand X and the diazine substituents: in particular, quantum yields decrease in the series Cl, Br, and I and in the presence of substituents at the alpha positions of the pyridazine ring. A combined density functional and time-dependent density functional study of the geometry, relative stability, electronic structure, and photophysical properties of all the pyridazine derivatives was performed. The nature of the excited states involved in the electronic absorption spectra was ascertained, and trends in the energy of the highest occupied and lowest unoccupied molecular orbitals upon changing the pyridazine substituents and the bridging halogen ligands were discussed. The observed emission properties of these complexes were shown to be related to a combination of steric and electronic factors affecting their ground-state geometry and their stability.