Among the recombinant TSPOs, the BcTSPO.

Overexpression of recombinant Bacillus cereus TSPO (BcTSPO) in E. coli bacteria leads to its recovery with a bound hemin both in bacterial membrane (MB) and inclusion bodies (IB). Unlike mouse TSPO, BcTSPO purified in SDS detergent from IB is well structured and can bind various ligands such as high-affinity PK 11195, protoporphyrin IX (PPIX) and δ-aminolevulinic acid (ALA). For each of the three ligands, 1H-15N HSQC titration NMR experiments suggest that different amino acids of BcTSPO binding cavity are involved in the interaction. PPIX, an intermediate of heme biosynthesis, binds to the cavity of BcTSPO and its fluorescence can be significantly reduced in the presence of light and oxygen. The light irradiation leads to two products that have been isolated and characterized as photoporphyrins. They result from the addition of singlet oxygen to the two vinyl groups hence leading to the formation of hydroxyaldehydes. The involvement of water molecules, recently observed along with the binding of heme in Rhodobacter sphaeroides (RsTSPO) is highly probable. Altogether, these results raise the question of the role of TSPO in heme biosynthesis regulation as a possible scavenger of reactive intermediates.

Simultaneous Quantification of Trehalose and Trehalose 6-Phosphate by Hydrophilic Interaction Chromatography/Electrospray Accurate Mass Spectrometry with Application in Non-Targeted Metabolomics.

High-resolution mass spectrometry (HRMS) was coupled with ultra-high-performance liquid chromatography (UHPLC) to simultaneously quantify trehalose and trehalose 6-phosphate without derivatization or sample preparation. The use of full scan mode and exact mass analysis also makes it possible to carry out metabolomic analyses as well as semi-quantification. In addition, the use of different clusters in negative mode makes it possible to compensate for deficiencies in linearity and inerrant saturation at time-of-flight detectors. The method has been approved and validated for different matrices, yeasts, and bacteria, and has shown differentiation between bacteria as a function of growth temperatures.

Solid-state NMR study of structural heterogeneity of the apo WT mouse TSPO reconstituted in liposomes.

In the last decades, ligand binding to human TSPO has been largely used in clinical neuroimaging, but little is known about the interaction mechanism. Protein conformational mobility plays a key role in the ligand recognition and both, ligand-free and ligand-bound structures, are mandatory for characterizing the molecular binding mechanism. In the absence of crystals for mammalian TSPO, we have exploited solid-state nuclear magnetic resonance (ssNMR) spectroscopy under magic-angle spinning (MAS) to study the apo form of recombinant mouse TSPO (mTSPO) reconstituted in lipids. This environment has been previously described to permit binding of its high-affinity drug ligand PK11195 and appears therefore favourable for the study of molecular dynamics. We have optimized the physical conditions to get the best resolution for MAS ssNMR spectra of the ligand-free mTSPO. We have compared and combined various ssNMR spectra to get dynamical information either for the lipids or for the mTSPO. Partial assignment of residue types suggests few agreements with the published solution NMR assignment of the PK11195-bound mTSPO in DPC detergent. Moreover, we were able to observe some lateral chains of aromatic residues that were not assigned in solution. 13C double-quantum NMR spectroscopy shows remarkable dynamics for ligand-free mTSPO in lipids which may have significant implications on the recognition of the ligand and/or other protein partners.

Galleria mellonella as a Good Model to Study Acinetobacter baumannii Pathogenesis.

The invertebrate model, Galleria mellonella, has been widely used to study host-pathogen interactions due to its cheapness, ease of handling, and similar mammalian innate immune system. G. mellonella larvae have been proven to be useful and a reliable model for analyzing pathogenesis mechanisms of multidrug resistant Acinetobacter baumannii, an opportunistic pathogen difficult to kill. This review describes the detailed experimental design of G. mellonella/A. baumannii models, and provides a comprehensive comparison of various virulence factors and therapy strategies using the G. mellonella host. These investigations highlight the importance of this host-pathogen model for in vivo pathogen virulence studies. On the long term, further development of the G. mellonella/A. baumannii model will offer promising insights for clinical treatments of A. baumannii infection.

Colistin Treatment Affects Lipid Composition of Acinetobacter baumannii.

Multidrug-resistant Acinetobacter baumannii (A. baumannii) causes severe and often fatal healthcare-associated infections due partly to antibiotic resistance. There are no studies on A. baumannii lipidomics of susceptible and resistant strains grown at lethal and sublethal concentrations. Therefore, we analyzed the impact of colistin resistance on glycerolipids' content by using untargeted lipidomics on clinical isolate. Nine lipid sub-classes were annotated, including phosphatidylcholine, rarely detected in the bacterial membrane among 130 different lipid species. The other lipid sub-classes detected are phosphatidylethanolamine (PE), phosphatidylglycerol (PG), lysophosphatidylethanolamine, hemibismonoacylglycerophosphate, cardiolipin, monolysocardiolipin, diacylglycerol, and triacylglycerol. Under lethal and sublethal concentrations of colistin, significant reduction of PE was observed on the resistant and susceptible strain, respectively. Palmitic acid percentage was higher at colistin at low concentration but only for the susceptible strain. When looking at individual lipid species, the most abundant PE and PG species (PE 34:1 and PG 34:1) are significantly upregulated when the susceptible and the resistant strains are cultivated with colistin. This is, to date, the most exhaustive lipidomics data compilation of A. baumannii cultivated in the presence of colistin. This work is highlighting the plasma membrane plasticity used by this gram-negative bacterium to survive colistin treatment.

The Impact of Plasma Membrane Lipid Composition on Flagellum-Mediated Adhesion of Enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a major cause of foodborne gastrointestinal illness. The adhesion of EHEC to host tissues is the first step enabling bacterial colonization. Adhesins such as fimbriae and flagella mediate this process. Here, we studied the interaction of the bacterial flagellum with the host cell's plasma membrane using giant unilamellar vesicles (GUVs) as a biologically relevant model. Cultured cell lines contain many different molecular components, including proteins and glycoproteins. In contrast, with GUVs, we can characterize the bacterial mode of interaction solely with a defined lipid part of the cell membrane. Bacterial adhesion on GUVs was dependent on the presence of the flagellar filament and its motility. By testing different phospholipid head groups, the nature of the fatty acid chains, or the liposome curvature, we found that lipid packing is a key parameter to enable bacterial adhesion. Using HT-29 cells grown in the presence of polyunsaturated fatty acid (α-linolenic acid) or saturated fatty acid (palmitic acid), we found that α-linolenic acid reduced adhesion of wild-type EHEC but not of a nonflagellated mutant. Finally, our results reveal that the presence of flagella is advantageous for the bacteria to bind to lipid rafts. We speculate that polyunsaturated fatty acids prevent flagellar adhesion on membrane bilayers and play a clear role for optimal host colonization. Flagellum-mediated adhesion to plasma membranes has broad implications for host-pathogen interactions.IMPORTANCE Bacterial adhesion is a crucial step to allow bacteria to colonize their hosts, invade tissues, and form biofilm. Enterohemorrhagic Escherichia coli O157:H7 is a human pathogen and the causative agent of diarrhea and hemorrhagic colitis. Here, we use biomimetic membrane models and cell lines to decipher the impact of lipid content of the plasma membrane on enterohemorrhagic E. coli flagellum-mediated adhesion. Our findings provide evidence that polyunsaturated fatty acid (α-linolenic acid) inhibits E. coli flagellar adhesion to the plasma membrane in a mechanism separate from its antimicrobial and anti-inflammatory functions. In addition, we confirm that cholesterol-enriched lipid microdomains, often called lipid rafts, are important in bacterial adhesion. These findings demonstrate that plasma membrane adhesion via bacterial flagella play a significant role for an important human pathogen. This mechanism represents a promising target for the development of novel antiadhesion therapies.

Polydopamine-based molecularly imprinted thin films for electro-chemical sensing of nitro-explosives in aqueous solutions.

A sensitive electrochemical sensor was developed for the detection of nitro-explosives in aqueous solutions based on thin molecularly imprinted polydopamine films. Dopamine was identified in silico, based on DFT (density functional theory) calculations with the ωB97X-D/6-31G* basis set, as the best functional monomer and electropolymerized via cyclic voltammetry (CV) in the presence of carboxylic acid-based structural analogues ('dummy' templates) for two model nitro-explosives: TNT (2,4,6-trinitrotoluene) and RDX (Research Department eXplosive, 1,3,5-trinitroperhydro-1,3,5-triazine). This approach afforded a homogenous coverage of gold electrodes with imprinted films of tunable thickness. The electropolymerized molecularly imprinted polydopamine films allowed for a 105-fold sensitivity improvement over a bare gold electrode based on tracking the redox peaks of the targets by CV. This improved sensitivity is ascribed to the ability of the MIP to concentrate its target in proximity to the transduction element. The MIP films showed reproducible binding in phosphate buffer (10 mM, pH 7.4), with a dynamic range from 0.1 nM to 10 nM for both TNT and RDX and an increased selectivity over closely related structural analogues.

Insight into the Structural Features of TSPO: Implications for Drug Development.

The translocator protein (TSPO), an 18-kDa transmembrane protein primarily found in the outer mitochondrial membrane, is evolutionarily conserved and widely distributed across species. In mammals, TSPO has been described as a key member of a multiprotein complex involved in many putative functions and, over the years, several classes of ligand have been developed to modulate these functions. In this review, we consider the currently available atomic structures of mouse and bacterial TSPO and propose a rationale for the development of new ligands for the protein. We provide a review of TSPO monomeric and oligomeric states and their conformational flexibility, together with ligand-binding site and interaction mechanisms. These data are expected to help considerably the development of high-affinity ligands for TSPO-based therapies or diagnostics.

Solid-State NMR of Membrane Protein Reconstituted in Proteoliposomes, the Case of TSPO.

Structural studies of membrane proteins (MP) in a native or native-like environment remain a challenge. X-ray crystallography of three-dimensional crystals of MP in lipids and cryo-electron microscopy of two-dimensional crystals also in lipids have given atomic structures of several MP. Recent developments of solid-state NMR (ssNMR) provided structural data of MP in lipids and should give access to the dynamic behavior of MP's in a native-like environment. Preparation of samples for ssNMR is not trivial with overexpressed proteins since purified recombinant MP have to be reincorporated in proteoliposomes and concentrated in the small volume of the rotor used for ssNMR studies. We present here the protocol that we have used to study the recombinant mouse TSPO1, an integral membrane protein of 20 kDa mostly found in the outer membrane of mitochondria and overexpressed in E. coli bacteria.

Molecularly Imprinted Polymer Coated Quantum Dots for Multiplexed Cell Targeting and Imaging.

Advanced tools for cell imaging are of great interest for the detection, localization, and quantification of molecular biomarkers of cancer or infection. We describe a novel photopolymerization method to coat quantum dots (QDs) with polymer shells, in particular, molecularly imprinted polymers (MIPs), by using the visible light emitted from QDs excited by UV light. Fluorescent core-shell particles specifically recognizing glucuronic acid (GlcA) or N-acetylneuraminic acid (NANA) were prepared. Simultaneous multiplexed labeling of human keratinocytes with green QDs conjugated with MIP-GlcA and red QDs conjugated with MIP-NANA was demonstrated by fluorescence imaging. The specificity of binding was verified with a non-imprinted control polymer and by enzymatic cleavage of the terminal GlcA and NANA moieties. The coating strategy is potentially a generic method for the functionalization of QDs to address a much wider range of biocompatibility and biorecognition issues.

Plastic Antibodies for Cosmetics: Molecularly Imprinted Polymers Scavenge Precursors of Malodors.

Molecularly imprinted polymers (MIPs) are synthetic antibody mimics capable of specific molecular recognition. Advantageously, they are more stable, easy to tailor for a given application and less expensive than antibodies. These plastic antibodies are raising increasing interest and one relatively unexplored domain in which they could outplay these advantages particularly well is cosmetics. Here, we present the use of a MIP as an active ingredient of a cosmetic product, for suppressing body odors. In a dermo-cosmetic formulation, the MIP captures selectively the precursors of malodorous compounds, amidst a multitude of other molecules present in human sweat. These results pave the way to the fabrication of a novel generation of MIPs with improved selectivities in highly complex aqueous environments, and should be applicable to biotechnological and biomedical areas as well.

Deuterium MAS NMR studies of dynamics on multiple timescales: histidine and oxalic acid.

Deuterium ((2) H) magic-angle spinning (MAS) nuclear magnetic resonance is applied to monitor the dynamics of the exchanging labile deuterons of polycrystalline L-histidine hydrochloride monohydrate-d7 and α-oxalic acid dihydrate-d6 . Direct experimental evidence of fast dynamics is obtained from T1Z and T1Q measurements. Further motional information is extracted from two-dimensional single-quantum (SQ) and double-quantum (DQ) MAS spectra. Differences between the SQ and DQ linewidths clearly indicate the presence of motions on intermediate timescales for the carboxylic moiety and the D2 O in α-oxalic acid dihydrate, and for the amine group and the D2 O in L-histidine hydrochloride monohydrate. Comparison of the relaxation rate constants of Zeeman and quadrupolar order with the relaxation rate constants of the DQ coherences suggests the co-existence of fast and slow motional processes.

Cholesterol modulates the interaction of the islet amyloid polypeptide with membranes.

The deposition of insoluble amyloid fibrils resulting from the aggregation of the human islet amyloid polypeptide (hIAPP) within the islet of Langerhans is a pathological feature of type 2 diabetes mellitus (T2DM). Increasing evidence indicates that biological membranes play a key role in amyloid aggregation, modulating among others the kinetics of amyloid formation, and being the target of toxic species generated during amyloid formation. In T2DM patients, elevated levels of cholesterol, an important determinant of the physical state of biological membranes, are observed in ß-cells and are thought to directly impair ß-cell function and insulin secretion. However, it is not known whether cholesterol enhances membrane-interaction or membrane-insertion of hIAPP. In this study, we investigated the effect of cholesterol incorporated in zwitterionic and anionic membranes. Our circular dichroism and liquid state NMR data reveal that 10-30% of cholesterol slightly affects the aggregational and conformational behaviour of hIAPP. Additional fluorescence results indicate that 10 and 20% of cholesterol slightly slow down the kinetics of oligomer and fibril formation while anionic lipids accelerate this kinetics. This behavior might be caused by differences in membrane insertion and therefore in membrane binding of hIAPP. The membrane binding affinity was evaluated using (1)H NMR experiments and our results show that the affinity of hIAPP for membranes containing cholesterol is significantly smaller than that for membranes containing anionic lipids. Furthermore, we found that hIAPP-induced membrane damage is synchronized to fibril formation in the absence and in the presence of cholesterol.

Fast proton exchange in histidine: measurement of rate constants through indirect detection by NMR spectroscopy.

Owing to its imidazole side chain, histidine participates in various processes such as enzyme catalysis, pH regulation, metal binding, and phosphorylation. The determination of exchange rates of labile protons for such a system is important for understanding its functions. However, these rates are too fast to be measured directly in an aqueous solution by using NMR spectroscopy. We have obtained the exchange rates of the NH3(+) amino protons and the labile NH(ε2) and NH(δ1) protons of the imidazole ring by indirect detection through nitrogen-15 as a function of temperature (272 K

"On-the-fly" kinetics of enzymatic racemization using deuterium NMR in DNA-based chiral oriented media.

We report the in situ and real-time monitoring of the interconversion of L- and D-alanine-d3 by alanine racemase from Bacillus stearothermophilus directly observed by (2)H NMR spectroscopy in anisotropic phase. The enantiomers are distinguished by the difference of their (2)H quadrupolar splittings in a chiral liquid crystal containing short DNA fragments. The proof-of-principle, the reliability, and the robustness of this new method is demonstrated by the determination of the turnover rates of the enzyme using the Michaelis-Menten model.

Probing structural and motional features of the C-terminal part of the Human Centrin 2/P17-XPC microcrystalline complex by solid-state NMR spectroscopy.

Insight into structural and motional features of the C-terminal part of the Human Centrin 2 in complex with the peptide P17-XPC was obtained by using complementary solid-state NMR methods. We demonstrate that the experimental conditions and procedures of sample crystallization determine the quality of solid-state NMR spectra and the internal mobility of the protein. Two-dimensional (2D) (13)C-(13)C and (15)N-(15)N correlation spectra reveal intra- and inter-residue dipolar connectivities and provide partial, site-specific assignments of (13)C and (15)N resonance signals. The secondary structure of the C-ter HsCen2/P17-XPC complex in a microcrystalline state appears similar to that found in solution. Conformational flexibility is probed through relaxation-compensated measurements of dipolar order parameters that exploit the dynamics of cross-polarization in multidimensional experiments. The extracted dipolar coupling constants and relevant order parameters reveal increased backbone flexibility of the loops except for residues involved in coordination with the Ca(2+) cation that stabilizes the hydrophobic pocket containing the peptide P17-XPC.

Investigation of the interface in silica-encapsulated liposomes by combining solid state NMR and first principles calculations.

In the context of nanomedicine, liposils (liposomes and silica) have a strong potential for drug storage and release schemes: such materials combine the intrinsic properties of liposome (encapsulation) and silica (increased rigidity, protective coating, pH degradability). In this work, an original approach combining solid state NMR, molecular dynamics, first principles geometry optimization, and NMR parameters calculation allows the building of a precise representation of the organic/inorganic interface in liposils. {(1)H-(29)Si}(1)H and {(1)H-(31)P}(1)H Double Cross-Polarization (CP) MAS NMR experiments were implemented in order to explore the proton chemical environments around the silica and the phospholipids, respectively. Using VASP (Vienna Ab Initio Simulation Package), DFT calculations including molecular dynamics, and geometry optimization lead to the determination of energetically favorable configurations of a DPPC (dipalmitoylphosphatidylcholine) headgroup adsorbed onto a hydroxylated silica surface that corresponds to a realistic model of an amorphous silica slab. These data combined with first principles NMR parameters calculations by GIPAW (Gauge Included Projected Augmented Wave) show that the phosphate moieties are not directly interacting with silanols. The stabilization of the interface is achieved through the presence of water molecules located in-between the head groups of the phospholipids and the silica surface forming an interfacial H-bonded water layer. A detailed study of the (31)P chemical shift anisotropy (CSA) parameters allows us to interpret the local dynamics of DPPC in liposils. Finally, the VASP/solid state NMR/GIPAW combined approach can be extended to a large variety of organic-inorganic hybrid interfaces.

Determination of the antisymmetric part of the chemical shift anisotropy tensor via spin relaxation in nuclear magnetic resonance.

Relaxation processes induced by the antisymmetric part of the chemical shift anisotropy tensor (henceforth called anti-CSA) are usually neglected in NMR relaxation studies. It is shown here that anti-CSA components contribute to longitudinal relaxation rates of the indole (15)N nucleus in tryptophan in solution at different magnetic fields and temperatures. To determine the parameters of several models for rotational diffusion and internal dynamics, we measured the longitudinal relaxation rates R(1)=1/T(1) of (15)N, the (15)N-(1)H dipole-dipole (DD) cross-relaxation rates (Overhauser effects), and the cross-correlated CSA/DD relaxation rates involving the second-rank symmetric part of the CSA tensor of (15)N at four magnetic fields B(0)=9.4, 14.1, 18.8, and 22.3 T (400, 600, 800, and 950 MHz for protons) over a temperature range of 270

Revealing molecular self-assembly and geometry of non-covalent halogen bonding by solid-state NMR spectroscopy.

We report a new spectroscopic fingerprint of intermolecular contacts in halogen bond-driven self-assembling aggregates and a precise determination of intermolecular NI distances in microcrystalline samples.

Improving resolution in single-scan 2D spectroscopy.

New schemes are introduced that allow one to improve the resolution in the indirect dimension of single-scan 'ultrafast' two-dimensional NMR spectra. The methods combine undersampling with band-selective pulses to recover signals that lie outside the detection bandwidth. The efficiency is illustrated for homonuclear total correlation spectroscopy (TOCSY) of quinidine.