Structural Microheterogeneity Research Articles

Multiple Isomers of Photolumazine V Bind MR1 and Differentially Activate MAIT Cells.

In response to microbial infection, the nonclassical Ag-presenting molecule MHC class I-related protein 1 (MR1) presents secondary microbial metabolites to mucosal-associated invariant T (MAIT) cells. In this study, we further characterize the repertoire of ligands captured by MR1 produced in Hi5 (Trichoplusia ni) cells from Mycobacterium smegmatis via mass spectrometry. We describe the (to our knowledge) novel MR1 ligand photolumazine (PL)V, a hydroxyindolyl-ribityllumazine with four isomers differing in the positioning of a hydroxyl group. We show that all four isomers are produced by M. smegmatis in culture and that at least three can induce MR1 surface translocation. Furthermore, human MAIT cell clones expressing distinct TCR β-chains differentially responded to the PLV isomers, demonstrating that the subtle positioning of a single hydroxyl group modulates TCR recognition. This study emphasizes structural microheterogeneity within the MR1 Ag repertoire and the remarkable selectivity of MAIT cell TCRs.

Dissecting the Molecular Origin of g-Tensor Heterogeneity and Strain in Nitroxide Radicals in Water: Electron Paramagnetic Resonance Experiment versus Theory.

Nitroxides are common EPR sensors of microenvironmental properties such as polarity, numbers of H-bonds, pH, and so forth. Their solvation in an aqueous environment is facilitated by their high propensity to form H-bonds with the surrounding water molecules. Their g- and A-tensor elements are key parameters to extracting the properties of their microenvironment. In particular, the gxx value of nitroxides is rich in information. It is known to be characterized by discrete values representing nitroxide populations previously assigned to have different H-bonds with the surrounding waters. Additionally, there is a large g-strain, that is, a broadening of g-values associated with it, which is generally correlated with environmental and structural micro-heterogeneities. The g-strain is responsible for the frequency dependence of the apparent line width of the EPR spectra, which becomes evident at high field/frequency. Here, we address the molecular origin of the gxx heterogeneity and of the g-strain of a nitroxide moiety (HMI: 2,2,3,4,5,5-hexamethylimidazolidin-1-oxyl, C9H19N2O) in water. To treat the solvation effect on the g-strain, we combined a multi-frequency experimental approach with ab initio molecular dynamics simulations for structural sampling and quantum chemical EPR property calculations at the highest realistically affordable level, including an explicitly micro-solvated HMI ensemble and the embedded cluster reference interaction site model. We could clearly identify the distinct populations of the H-bonded nitroxides responsible for the gxx heterogeneity experimentally observed, and we dissected the role of the solvation shell, H-bond formation, and structural deformation of the nitroxide in the creation of the g-strain associated with each nitroxide subensemble. Two contributions to the g-strain were identified in this study. The first contribution depends on the number of hydrogen bonds formed between the nitroxide and the solvent because this has a large and well-understood effect on the gxx-shift. This contribution can only be resolved at high resonance frequencies, where it leads to distinct peaks in the gxx region. The second contribution arises from configurational fluctuations of the nitroxide that necessarily lead to g-shift heterogeneity. These contributions cannot be resolved experimentally as distinct resonances but add to the line broadening. They can be quantitatively analyzed by studying the apparent line width as a function of microwave frequency. Interestingly, both theory and experiment confirm that this contribution is independent of the number of H-bonds. Perhaps even more surprisingly, the theoretical analysis suggests that the configurational fluctuation broadening is not induced by the solvent but is inherently present even in the gas phase. Moreover, the calculations predict that this broadening decreases upon solvation of the nitroxide.

Structural microheterogeneity and hydrogen bonding properties in the mixtures of two ionic liquids with a common imidazolium cation

A recent focus in the ionic liquid field is to mix two (or more) ionic liquids to expand the available properties. In this work, the interactions and structural properties of 1-ethyl-3-methylimidazolium bis(trifluoromethyl)sulfonylimide (EMIMTFSI) and 1-ethyl-3-methylimidazolium dicyanamide (EMIMDCA) mixtures were investigated by employing multiple experimental methods and DFT calculations. Microheterogeneity of the liquid mixtures was characterized in detail and four complexes or clusters were identified, i.e. 2EMIMTFSI, 2EMIMDCA, 2EMIMTFSI-[DCA]− and 2EMIMDCA-[TFSI]−. Hydrogen bonding interactions between cations and anions were found to be ubiquitous but all in the category of weak interactions. The main interaction sites for [EMIM]+, [TFSI]−, and [DCA]− were found to be C2−H, O/N atoms, and N (in C≡N). This work adds value to an in-depth understanding of the microstructures and molecular interactions in the specified ionic liquid mixtures and would shed light to the general understanding of the ionic liquid systems as a whole.

A combined QTAIM, DFT and molecular dynamics study on the nanoscale dynamical and structural organization of imidazolium-based dicationic ionic liquids

In this work, the effects of linkage alkyl chain length on the dynamical and structural micro-heterogeneities of some imidazolium-based dicationic ionic liquids (DILs) i.e. [Cn(mim)2][BF4]2, (n = 3, 6, 9, and 12) were studied via quantum mechanical calculations and also classical molecular dynamics (MD) simulations. Hydrogen bondings and also the interaction energies between the studied ions have been calculated using the density functional theory (DFT) calculations and also the quantum theory of atoms in molecules (QTAIM) method. The results showed that with increasing the linkage alkyl chain length of DILs, the interaction energies and electron densities are decreased, because the electrostatic interactions in DILs are weakened with increasing the –CH2 groups in the linkage chain. Different kinds of combined distribution functions (CDFs) were computed to better understand the orientations of ions in the studied DILs. Although, two rings of cation are almost perpendicular with respect to each other in all studied DILs, the orientation of linkage chain with respect to the rings is different in short- and long-linkage chain DILs. To understand the arrangement of the nearest neighboring ions, neighborhood analysis and spatial distribution functions (SDFs) of ions were analyzed. Furthermore, the mean square displacement (MSD), non-Gaussian parameter, van Hove correlation of cation and anions and also the ion pair, ion cage, hydrogen bond and reorientation dynamics were computed. DILs with longer alkyl chain length have more deviation in non-Gaussian parameter and Gaussian behavior of distribution of particle displacements, probably due to weakening the electrostatic interactions. Also, they have higher ion cage/ion pair stabilities than DILs with shorter linkage chain. Altogether, the linkage chain length in DILs has significant influences on their organizations in such a way that with increasing linkage chain length, their dynamical and structural micro-heterogeneities increase.

Microheterogeneity-Induced Vibrational Spectral Dynamics of Aqueous 1-Alkyl-3-methylimidazolium Tetrafluoroborate Ionic Liquids of Different Cationic Chain Lengths.

We have monitored the impacts of an increment in the alkyl chain length of the imidazolium-based tetrafluoroborate ionic liquids on the local deuteroxyl probe modes of interest. For this study, we have taken 1-ethyl-3-methylimidazolium tetrafluoroborate [EMIm][BF4], 1-butyl-3-methylimidazolium tetrafluoroborate [BMIm][BF4], 1-octyl-3-methylimidazolium tetrafluoroborate [OMIm][BF4], and 1-decyl-3-methylimidazolium tetrafluoroborate [DMIm][BF4] ionic liquid solutions with 5% HOD in H2O as the vibrational reporter of the associated ultrafast system dynamics. Classical molecular dynamics (MD) simulations were employed to determine molecular structure and dynamic properties, while the spectral profiles were derived by applying the wavelet analysis of classical trajectories. Spatial distribution functions reveal the heterogeneity within the molecular structures of the ionic liquids (ILs) with varying alkyl chain lengths. The intense position of the spectral peak, the frequency corresponding to the shoulder peak, and the spectral linewidth of the O-D stretch distribution are not influenced by the increment in the cationic chain length. In addition, the ionic liquid (IL) [BMIm][BF4] exhibits a notable trend; the dynamic timescales are longer than the other studied systems. Therefore, we have performed the Voronoi decomposition analysis of the ionic and the polar-apolar domains, symmetrically increasing the length of alkyl chains on the IL cations. Domain analysis reveals structural microheterogeneity; the anions form discrete domains, and the ionic liquid constituting cations form continuous domains irrespective of the alkyl chain length on the imidazolium cations. Therefore, this computational ultrafast spectroscopy study aids in forming a molecular-level picture of the ionic liquid cations and anions in the liquid phase, providing a detailed interpretation of the spectral properties of the probe stretching vibrations.

Native Ion Mobility-Mass Spectrometry-Enabled Fast Structural Interrogation of Labile Protein Surface Modifications at the Intact Protein Level.

Protein sialylation has been closely linked to many diseases including Alzheimer's disease (AD). It is also broadly implicated in therapeutics operating in a pattern-dependent (e.g., Neu5Ac vs Neu5Gc) manner. However, how the sialylation pattern affects the AD-associated, transferrin-assisted iron/Aβ cellular uptake process remains largely ill-defined. Herein, we report the use of native ion mobility-mass spectrometry (IM-MS)-based fast structural probing methodology, enabling well-controlled, synergistic, and in situ manipulation of mature glycoproteins and attached sialic acids. IM-MS-centered experiments enable the combinatorial interrogation of sialylation effects on Aβ cytotoxicity and the chemical, conformational, and topological stabilities of transferrin. Cell viability experiments suggest that Neu5Gc replacement enhances the transferrin-assisted, iron loading-associated Aβ cytotoxicity. Native gel electrophoresis and IM-MS reveal that sialylation stabilizes transferrin conformation but inhibits its dimerization. Collectively, IM-MS is adapted to capture key sialylation intermediates involved in fine-tuning AD-associated glycoprotein structural microheterogeneity. Our results provide the molecular basis for the importance of sustaining moderate TF sialylation levels, especially Neu5Ac, in promoting iron cellular transportation and rescuing iron-enhanced Aβ cytotoxicity.

Mass Spectrometric Methods for the Analysis of Heparin and Heparan Sulfate.

Glycosaminoglycans like heparin and heparan sulfate exhibit a high degree of structural microheterogeneity. This structural heterogeneity results from the biosynthetic process that produces these linear polysaccharides in cells and tissues. Heparin and heparan sulfate play critical roles in normal physiology and pathophysiology, hence it is important to understand how their structural features may influence overall activity. Therefore, high-resolution techniques like mass spectrometry represent a key part of the suite of methodologies available to probe the fine structural details of heparin and heparan sulfate. This chapter outlines the application of techniques like LC-MS and LC-MS/MS to study the composition of these polysaccharides, and techniques like GPC-MS that allow for an analysis of oligosaccharide fragments in these mixtures.

Effect of the initial austenitization temperature on the structural microheterogeneity during drawing of carbon steel with a pearlite structure

During wire production, strain fields can be distributed inhomogeneously over the section during drawing and cause structural micro-inhomogeneity, which significantly affects the stability of the process. However, during plastic deformation of carbon steel with a pearlite structure, the interlamellar spacing in the ferrite-carbide mixture and the size of pearlite colonies, which determine the deformation behavior of steel, are of great importance. In addition, in the wire manufacturing technology, heat treatment operations are used with heating the steel to the austenitic state, the temperature of which significantly affects the formation of the structure and properties of the steel. The paper investigates the effect of the austenitization temperature on the structural microheterogeneity of a wire made of carbon steel with a pearlite structure after drawing. The results of studying the microstructure, determining the interlamellar spacing, the anisotropy coefficient of pearlite colonies, as well as the distribution of microhardness over the cross section of the sample during drawing after different temperatures of preliminary austenitization are presented. It is shown that after preliminary austenitization at temperatures of 900, 950 and 1000 °C in a wire made of carbon steel with a pearlite structure, microstructural inhomogeneity in the dispersion of the ferrite-pearlite mixture is observed. It manifests itself as a difference of the interlamellar spacing in pearlite at the surface and in the center of the sample cross section and is retained during subsequent drawing with a total reduction of 8 to 15%. It has been established that the temperature of preliminary austenitization has practically no effect on the anisotropy coeffi cient of pearlite colonies in the initial state after austenitization, and it does not change over the cross section of the sample. However, with subsequent drawing with an increase in the total reduction, the anisotropy coefficient increases, while it increases from the surface to the center of the sample. It is revealed that with an increase in the preliminary austenitization temperature from 900 to 1000 °C, the microstructural inhomogeneity in the drawn wire is manifested to a greater extent, which can be associated with an increase in the grain size of the initial austenite, the size of pearlite colonies, and the interlamellar spacing in pearlite. Microstructural inhomogeneity is confirmed by the nature of the distribution of microhardness over the cross section of the sample. The research was carried out with the financial support of the Russian Foundation for Basic Research and DNT within the framework of the scientific project No. 18-58-45008 IND_a.

Shallow sampling by multi-shot laser ablation and its application within U-Pb zircon geochronology

The interaction of incident laser radiation and sample substrate is complex and difficult to predict. Natural zircons are often both structurally and chemically heterogeneous in 3-dimensional space. Encountering growth-related, structural micro-heterogeneities, inclusions and chemical complexities is almost inevitable during a static ablation of several tens of seconds. A multi-shot approach to laser ablation described here implements a minimal sample exposure time to incident laser radiation by applying multiple 1 Hz shots to a single sample location in delayed succession. This process can be conceptualised as a “slowing down” of a high-frequency (5–20 Hz) static laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) analysis until each laser pulse is distinct albeit transient. The ability to integrate and collate signal pulses for a small number of consecutive laser shots, as opposed to continuously pulsing the laser, produces precise age determinations (~1% reproducibility, 2σ level) on small sample volumes (704 ± 23 μm3 on 91500 zircon standard). The multi-shot LA-ICP-MS protocol employed here significantly reduces the effect of ‘downhole’ fractionation as the resultant craters are extremely shallow (as shallow as 0.56 ± 0.02 μm on 91500 zircon standard) and maintain an aspect ratio of ≪1. Further benefits include a reduced probability of thermally induced effects (e.g., substrate melting), plasma loading, and the potential for signal mixing (with depth) in a heterogeneous sample.

Molecular Mechanism of Water Reorientation Dynamics in Dimethyl Sulfoxide Aqueous Mixtures.

Nonmonotonic composition dependence is often observed for numerous properties in the aqueous mixtures of small amphiphilic molecules. The molecular picture underlying this structure-activity relationship, however, remains largely elusive. We herein studied water reorientation dynamics in the aqueous mixture of dimethyl sulfoxide (DMSO), which has a significant nonmonotonic composition dependence, using molecular dynamic simulation and an extended molecular jump model. The analysis indicates that this nonideal behavior is driven by the collective frame diffusion component of water reorientation, which decelerates in the water-rich regime because of the strengthened hydrogen bonds and accelerates in the water-poor regime as the hydrogen bonding network is broken into smaller aggregates. The current work therefore connects the microheterogeneity in the solvation structure of DMSO-water with its nonmonotonic hydration dynamics and sheds new light on how microsegregation leads to the multiscale hydration nonideality in general.

Linear Synthesis of De novo Oligo‐Iduronic Acid

L‐Iduronic acid (IdoA) plays a pivotal role in glycosaminoglycan (GAG) protein interactions. However, the structural microheterogeneity of GAG appears to impede the systematic investigation of IdoA functions. Under such conditions, oligo‐Iduronic acid (Oligo‐IdoA) are ideal and straightforward heparin mimetics to unravel the relationship between IdoA structure and functions. Herein, we report for the first‐time linear synthesis of rare oligo‐IdoA precursor utilizing anhydrous β‐L‐idopyranosyl and IdoA thiophenol building block. After screening various synthetic strategies, we have Installed successive IdoA by 5 step reactions with 25–26 % overall yield. These oligo‐IdoA are expected to be excellent probes to understand conformational plasticity of IdoA and fine tune carbohydrate–protein interactions.

Effects of microscopic pore structure heterogeneity on the distribution and morphology of remaining oil

Abstract Waterflooding experiments were performed using Micro-CT on four cores of different pore structures from Donghe sandstone reservoirs in the Tarim Basin. The water, oil and grains were accurately separated by the advanced image processing technology, the pore network model was established, and parameters such as the number of throats and the throat size distribution were calculated to characterize the microscopic heterogeneity of pore structure, the flow of oil phase during displacement, and the morphology and distribution of remaining oil after displacement. The cores with the same macroscopic porosity-permeability have great differences in microscopic heterogeneity of pore structure. Both macro porosity-permeability and micro heterogeneity of pore structure have an influence on the migration of oil phase and the morphology and distribution of remaining oil. When the heterogeneity is strong, the water phase will preferentially flow through the dominant paths and the remaining oil clusters will be formed in the small pores. The more the number of oil clusters (droplets) formed during displacement process, the smaller the average volume of cluster is, and the remaining oil is dominated by the cluster continuous phase with high saturation. The weaker the heterogeneity, the higher the pore sweep efficiency is, and the remaining oil clusters are mainly trapped in the form of non-continuous phase. The distribution and morphology of micro remaining oil are related to the absolute permeability, capillary number and micro-heterogeneity. So, the identification plate of microscopic residual oil continuity distribution established on this basis can describe the relationship between these three factors and distribution of remaining oil and identify the continuity of the remaining oil distribution accurately.

Comprehensive Proteoform Characterization of Plasma Complement Component C8\u03b1\u03b2\u03b3 by Hybrid Mass Spectrometry Approaches

The human complement hetero-trimeric C8αβγ (C8) protein assembly (~ 150 kDa) is an important component of the membrane attack complex (MAC). C8 initiates membrane penetration and coordinates MAC pore formation. Here, we charted in detail the structural micro-heterogeneity within C8, purified from human plasma, combining high-resolution native mass spectrometry and (glyco)peptide-centric proteomics. The intact C8 proteoform profile revealed at least ~ 20 co-occurring MS signals. Additionally, we employed ion exchange chromatography to separate purified C8 into four distinct fractions. Their native MS analysis revealed even more detailed structural micro-heterogeneity on C8. Subsequent peptide-centric analysis, by proteolytic digestion of C8 and LC-MS/MS, provided site-specific quantitative profiles of different types of C8 glycosylation. Combining all this data provides a detailed specification of co-occurring C8 proteoforms, including experimental evidence on N-glycosylation, C-mannosylation, and O-glycosylation. In addition to the known N-glycosylation sites, two more N-glycosylation sites were detected on C8. Additionally, we elucidated the stoichiometry of all C-mannosylation sites in all the thrombospondin-like (TSP) domains of C8α and C8β. Lastly, our data contain the first experimental evidence of O-linked glycans located on C8γ. Albeit low abundant, these O-glycans are the first PTMs ever detected on this subunit. By placing the observed PTMs in structural models of free C8 and C8 embedded in the MAC, it may be speculated that some of the newly identified modifications may play a role in the MAC formation.Graphical ᅟ

Characterization of therapeutic protein AvidinOX by an integrated analytical approach.

AvidinOX, the oxidized derivative of Avidin, is a chemically modified glycoprotein, being currently under clinical investigation for targeted delivery of radioactive biotin to inoperable tumors. AvidinOX is produced by 4-hydroxyazobenzene-2-carboxylic acid (HABA)-assisted sodium periodate oxidation of Avidin. The peculiar property of the periodate-generated glycol-split carbohydrate moieties to form Schiff's bases with amino groups of the tissue proteins allows to achieve a tissue half-life of 2weeks compared to 2h of native Avidin. Carbohydrate oxidation, along with possible minor amino acid modifications, introduces additional microheterogeneity in the glycoprotein structure, making its characterization even more demanding than for native glycoproteins. Aiming at the elucidation of the effects of oxidation conditions on the AvidinOX protein backbone and sugars, this microheterogeneous glycoprotein derivative was characterized for the first time using a combination of different analytical methods, including colorimetric methods, mass spectrometry, hollow-fiber flow field-flow fractionation with UV and multi-angle laser scattering detection (HF5-UV-MALS), and NMR. The proposed integrated approach reveals structural features of AvidinOX relevant for its biological activity, e.g., oxidized sites within both carbohydrate moieties and protein backbone and conformational stability, and will be considered as an analytical tool for AvidinOX industrial preparations. It is worth noting that this study enriches also the structural data of native Avidin published up-to-date (e.g., glycan structure and distribution, peptide fingerprint, etc.). Graphical abstract Scheme of phenylacetic hydrazide/MALDI-TOF approach for quantification of aldehydes in AvidinOX based on the determination of the number of hydrazone adducts between hydrazide reagent and aldehyde groups of protein.

Conventional and unconventional secretory proteins expressed with silkworm bombyxin signal peptide display functional fidelity

Growth factors are signaling molecules which orchestrate cell growth, proliferation and differentiation. The majority are secreted proteins, exported through the classical endoplasmic reticulum (ER)/Golgi-dependent pathway, but a few are released by unconventional ER/Golgi-independent means. Human fibroblast growth factor 2 (FGF2) and insulin-like growth factor 1 (IGF1), are canonical prototypes secreted by the unconventional and conventional pathway, respectively. We herein examined whether expression of these two growth factors in the Bombyx mori nucleopolyhedrovirus (BmNPV)-based silkworm expression system with its innate signal peptide, bombyxin, secures structural homogeneity at the signal peptide cleavage site regardless of the native secretory route. Proteomic analysis mapped structural microheterogeneity of signal peptide cleavage at the amino terminus of FGF2, whereas IGF1 displayed homogeneous amino-terminal cleavage with complete removal of the bombyxin signal peptide. A cell proliferation assay revealed potent functional activity of both FGF2 and IGF1, suggesting that FGF2 amino-terminal microheterogeneity does not alter mitogenic activity. These findings demonstrate that the occurrence of amino-terminal structural homogeneity may be associated with the original secretion mechanism of a particular growth factor. Furthermore, our results highlight the bombyxin signal peptide as a reliable secretion sequence applicable to mass production of functionally active secretory proteins in a silkworm-based expression platform.

Proteoform Profile Mapping of the Human Serum Complement Component C9 Revealing Unexpected New Features of N-, O-, and C-Glycosylation.

The human complement C9 protein (∼65 kDa) is a member of the complement pathway. It plays an essential role in the membrane attack complex (MAC), which forms a lethal pore on the cellular surface of pathogenic bacteria. Here, we charted in detail the structural microheterogeneity of C9 purified from human blood serum, using an integrative workflow combining high-resolution native mass spectrometry and (glyco)peptide-centric proteomics. The proteoform profile of C9 was acquired by high-resolution native mass spectrometry, which revealed the co-occurrence of ∼50 distinct mass spectrometry (MS) signals. Subsequent peptide-centric analysis, through proteolytic digestion of C9 and liquid chromatography (LC)-tandem mass spectrometry (MS/MS) measurements of the resulting peptide mixtures, provided site-specific quantitative profiles of three different types of C9 glycosylation and validation of the native MS data. Our study provides a detailed specification, validation, and quantification of 15 co-occurring C9 proteoforms and the first direct experimental evidence of O-linked glycans in the N-terminal region. Additionally, next to the two known glycosylation sites, a third novel, albeit low abundant, N-glycosylation site on C9 is identified, which surprisingly does not possess the canonical N-glycosylation sequence N-X-S/T. Our data also reveal a binding of up to two Ca2+ ions to C9. Mapping all detected and validated sites of modifications on a structural model of C9, as present in the MAC, hints at their putative roles in pore formation or receptor interactions. The applied methods herein represent a powerful tool for the unbiased in-depth analysis of plasma proteins and may advance biomarker discovery, as aberrant glycosylation profiles may be indicative of the pathophysiological state of the patients.

Biosynthesis of the Methylthioxylose Capping Motif of Lipoarabinomannan in Mycobacterium tuberculosis.

Lipoarabinomannan (LAM) is a lipoglycan found in abundant quantities in the cell envelope of all mycobacteria. The nonreducing arabinan termini of LAM display species-specific structural microheterogeneity that impacts the biological activity of the entire molecule. Mycobacterium tuberculosis, for instance, produces mannoside caps made of one to three α-(1 → 2)-Manp-linked residues that may be further substituted with an α-(1 → 4)-linked methylthio-d-xylose (MTX) residue. While the biological functions and catalytic steps leading to the formation of the mannoside caps of M. tuberculosis LAM have been well established, the biosynthetic origin and biological relevance of the MTX motif remain elusive. We here report on the discovery of a five-gene cluster dedicated to the biosynthesis of the MTX capping motif of M. tuberculosis LAM, and on the functional characterization of two glycosyltransferases, MtxS and MtxT, responsible, respectively, for the production of decaprenyl-phospho-MTX (DP-MTX) and the transfer of MTX from DP-MTX to the mannoside caps of LAM. Collectively, our NMR spectroscopic and mass spectrometric analyses of mtxS and mtxT overexpressors and knockout mutants support a biosynthetic model wherein the conversion of 5'-methylthioadenosine, which is a ubiquitous byproduct of spermidine biosynthesis, into 5'-methylthioribose-1-phosphate precedes the formation of a 5'-methylthioribose nucleotide sugar, followed by the epimerization at C-3 of the ribose residue, and the transfer of MTX from the nucleotide sugar to decaprenyl-phosphate yielding the substrate for transfer onto LAM. The conservation of the MTX biosynthetic genes in a number of Actinomycetes suggests that this discrete glycosyl substituent may be more widespread in prokaryotes than originally thought.

Structure, Function and Biological Activity of Lipopolysaccharide Lipid A

Bacterial lipopolysaccharides (LPS) are the major outer surface membrane components present in almost all gram-negative bacteria. It consists of poly- or oligosaccharide region that is anchored in the outer membrane by a specific lipid moiety termed lipid A. Recent studies have shown that it is only the lipid A of LPS that has the function of endotoxin. Despite its general structural conservation, lipid A also has considerable structural microheterogeneity which can vary depending on diverse factors including bacterial adaptation to changing environment and external stimuli, incomplete biosynthesis, and breakdown products and/or chemical modifications. Therefore it is more appropriate to consider lipid A as a family of structurally related molecular species with different acylation and phosphorylation patterns rather than as an individual, homogeneous molecule. The studies of structure-function relationship of lipid A, which has the typical structure of E. coli type lipid A backbone, demonstrated that activities differed depending on: 1) the number of phosphoryl and acyl residues, 2) the substituted site of phosphoryl and acyl residues, 3) the chain length of acyl residues, 4) lipid A conformation. Current investigations showed that lipid A and also the integral outer membrane proteins responsible for the final stage of LPS transport are the pinpoints in solving the problem of bacterial drug resistance. The identification of inhibitors that specifically target LPS transport in vitro and more importantly in vivo have a significant potential for the development of novel drugs against multi-drug resistant pathogenic bacteria.

Hybrid mass spectrometry approaches in glycoprotein analysis and their usage in scoring biosimilarity.

Many biopharmaceutical products exhibit extensive structural micro-heterogeneity due to an array of co-occurring post-translational modifications. These modifications often effect the functionality of the product and therefore need to be characterized in detail. Here, we present an integrative approach, combining two advanced mass spectrometry-based methods, high-resolution native mass spectrometry and middle-down proteomics, to analyse this micro-heterogeneity. Taking human erythropoietin and the human plasma properdin as model systems, we demonstrate that this strategy bridges the gap between peptide- and protein-based mass spectrometry platforms, providing the most complete profiling of glycoproteins. Integration of the two methods enabled the discovery of three undescribed C-glycosylation sites on properdin, and revealed in addition unexpected heterogeneity in occupancies of C-mannosylation. Furthermore, using various sources of erythropoietin we define and demonstrate the usage of a biosimilarity score to quantitatively assess structural similarity, which would also be beneficial for profiling other therapeutic proteins and even plasma protein biomarkers.

Influence of peritectic transformation on the hardening, structure, and properties of steel castings

The influence of peritectic transformation on the deposition of solid phase over the cross section of pearlitic steel ingots with 0.2–0.4% C is studied. To prevent structural and chemical microheterogeneity, measures ensuring dense and uniform metal over the casting cross section are proposed.