The Use of MALDI-TOF-MS and In Silico Studies for Determination of Antimicrobial Peptides’ Affinity to Bacterial Cells

Several methods have been proposed for determining the binding affinity of antimicrobial peptides (AMPs) to bacterial cells. Here the utilization of MALDI-TOF-MS was proposed as a reliable and efficient method for high throughput AMP screening. The major advantage of the technique consists of finding AMPs that are selective and specific to a wide range of Gram-negative and -positive bacteria, providing a simple reliable screening tool to determine the potential candidates for broad spectrum antimicrobial drugs. As a prototype, amp-1 and -2 were used, showing highest activity toward Gram-negative and -positive membranes respectively. In addition, in silico molecular docking studies with both peptides were carried out for the membranes. In silico results indicated that both peptides presented affinity for DPPG and DPPE phospholipids, constructed in order to emulate an in vivo membrane bilayer. As a result, amp-1 showed a higher complementary surface for Gram-negative while amp-2 showed higher affinity to Gram-positive membranes, corroborating MS analyses. In summary, results here obtained suggested that in vitro methodology using MALDI-TOF-MS in addition to theoretical studies may be able to improve AMP screening quality.     

Can the Effective Potential of a Linear Quadrupole be Extended to Values of the Mathieu Parameter q Up to 0.90?

The motion of ions in a linear quadrupole is usually described by solutions to the Mathieu equation. A simplifying approximation to this theory that is widely used for low values of the Mathieu parameters a and q describes ion motion in an effective potential. In this work, we have calculated the effective potential for any q from displacements of calculated ion trajectories caused by a dipole DC electric field. It is assumed that the dipole DC electric field at the center of the displaced trajectory is countered by an “effective” electric field. For all q values, the effective electric field is found to increase linearly with the distance from the center of the quadrupole. The trapping forces probed in this way increase continuously with q up to the first stability region boundary at q=0.908. The well depth (D) at any q can be described by $ D=q\frac{V_{rf}}{c} $ , where c=3.955±0.005, very similar to the standard effective potential model with c=4.000.

Conceptualization and Synthesis of the First Inosito-Inositol (Decahydroxydecalin,DHD): In silico Binding to β-Amyloid Protein

Previously unknown entities in the form of 1,2,3,4,5,6,7,8,9,10-decahydroxydecalins (DHDs) have been conceptualized and the first member of this class, an inosito-inositol, has been synthesized from aromatic hydrocarbon naphthalene following a flexible strategy that is amenable to diversity creation. The DHD accessed here has been subjected to preliminary in silico evaluation with Aβ and may hold some promise in Alzheimer's disease therapeutics.     

Can the nonadiabatic photodynamics of aminopyrimidine be a model for the ultrafast deactivation of adenine?

The reaction path for the ultrafast deactivation of 6-aminopyrimidine (6AP) has been investigated by means of ab initio surface-hopping dynamics. The electronic vertical excitation spectrum, excited-state S1 minima, and minima on the crossing seam of 6AP resemble well those found for adenine. The deactivation from the S1 to the S0 state takes place at the ultrafast time scale of 400 fs and involves the out-of-plane ring deformation of the C4 atom, a position that is sterically restricted in adenine by the imidazole ring. Mechanical restrictions have been used to simulate in a simple way the role of the imidazole group in adenine. As a result, deactivation via out-of-plane ring deformation of the C2 and N3 atoms are observed in good agreement with predictions for adenine. These dynamics results show that the previously suggested ring puckering deactivation paths really exist at a time scale, which is compatible with experimentally observed life times. The electronic structure of the crossing seam has been shown to have the same nature as those of simple biradicaloid systems, a feature which seems to be valid for any cyclic system.     

Mechanism of GTP: Can all of the available data be accommodated?

Group transfer polymerization (GTP)^a of acrylic monomers is a living system mediated by a trialkylsilyl capped growing chain end. The fact that it operates at temperatures as high as 100° C differentiates GTP from living anionic polymerization, which at best operates at 25° C for hindered methacrylates. To accommodate all of the mechanistic data available it appears that at least two mechanistic pathways are required: an associative process for mild nucleophilic catalysts and a dissociative process for strong nucleophilic catalysts.     

Can the speed of sound be used for detecting critical states of fluid mixtures?

The phenomenology of sound speeds in fluid mixtures is examined near and across critical lines. Using literature data for binary and ternary mixtures, it is shown that the ultrasound speed along an isotherm-isopleth passes through a minimum value in the form of an angular (or V-shaped) point at critical states. The relation between critical and pseudo-critical coordinates is discussed. For nonazeotropic fixed-composition fluid mixtures, pseudo-critical temperatures and pressures are found to be lower than the corresponding critical temperatures and pressures. The analysis shows that unstable pseudo-critical states cannot be detected using acoustic methods. The thermodynamic link between sound speeds and isochoric heat capacities is formulated and discussed in terms of p-Vm-T derivatives capable of being calculated using cubic equations of state. Based on the Griffiths-Wheeler theory of critical phenomena, a new specific link between critical sound speeds and critical isochoric heat capacities is deduced in terms of the rate of change of critical pressures and critical temperatures along the p-T projection of the critical locus of binary fluid mixtures. It is shown that the latter link can be used to obtain estimates of critical isochoric heat capacities from the experimental determination of critical speeds of sound. The applicability domain of the new link does not include binary systems at compositions along the critical line for which the rate of change in pressure with temperature changes sign. The new equation is combined with thermodynamic data to provide approximate numerical estimates for the speed of sound in two mixtures of carbon dioxide and ethane at different temperatures along their critical isochores. A clear decrease in the sound speed is found at critical points. A similar behavior is suggested by available critical heat capacity data for several binary fluid mixtures. Using an acoustic technique, the critical temperature and pressure were determined for three different mixtures of methane and propane, and compared with literature data obtained using conventional methods. It is concluded that acoustic-based techniques are reliable to determine, for the most part, critical surfaces of fluid mixtures. The remaining few cases where the present analysis cannot be applied could be tested by the thermodynamic calculation of critical sound speeds using crossover equations of state in conjunction with experimentally determined critical isochoric heat capacities.     

Can free energy calculations be fast and accurate at the same time? Binding of low-affinity,non-peptide inhibitors to the SH2 domain of the src protein

The usefulness of free-energy calculations in non-academic environments, in general, and in the pharmaceutical industry, in particular, is a long-time debated issue, often considered from the angle of cost/performance criteria. In the context of the rational drug design of low-affinity, non-peptide inhibitors to the SH2 domain of the (pp60)src tyrosine kinase, the continuing difficulties encountered in an attempt to obtain accurate free-energy estimates are addressed. free-energy calculations can provide a convincing answer, assuming that two key-requirements are fulfilled: (i) thorough sampling of the configurational space is necessary to minimize the statistical error, hence raising the question: to which extent can we sacrifice the computational effort, yet without jeopardizing the precision of the free-energy calculation? (ii) the sensitivity of binding free-energies to the parameters utilized imposes an appropriate parametrization of the potential energy function, especially for non-peptide molecules that are usually poorly described by multipurpose macromolecular force fields. Employing the free-energy perturbation method, accurate ranking, within +/-0.7 kcal/mol, is obtained in the case of four non-peptide mimes of a sequence recognized by the (pp60)src SH2 domain.     

Can simple enones be useful partners for the catalytic stereoselective alkylation of indoles?

A new catalytic system for the first example of enantioselective Friedel-Crafts-type (FC) addition of indoles to simple enones is described. The use of an equimolar amount of chiral [Al(salen)Cl] and 2,6-lutidine (10 mol %) was found to be effective in promoting the conjugate addition of indoles to (E)-arylcrotyl ketones, furnishing the corresponding beta-indolyl ketones in excellent yield and high enantioselectivity (ee up to 89%). The role of the base was investigated through spectroscopic as well as computational analyses, which suggested that in situ formation of a new chiral (base.[Al(salen)]) complex was operating under our reaction conditions. In particular, a stable cationic [Al(salen)] hexacoordinate trans complex with the additive base and the enone is suggested as being responsible for the stereocontrolled reaction. Finally, detailed monitoring of the reaction course was carried out showing that a conventional FC pathway induced by [Al(salen)Cl] acting as a Lewis acid is operating.     

Time to Face the Fats: What Can Mass Spectrometry Reveal about the Structure of Lipids and Their Interactions with Proteins?

Since the 1950s, X-ray crystallography has been the mainstay of structural biology, providing detailed atomic-level structures that continue to revolutionize our understanding of protein function. From recent advances in this discipline, a picture has emerged of intimate and specific interactions between lipids and proteins that has driven renewed interest in the structure of lipids themselves and raised intriguing questions as to the specificity and stoichiometry in lipid-protein complexes. Herein we demonstrate some of the limitations of crystallography in resolving critical structural features of ligated lipids and thus determining how these motifs impact protein binding. As a consequence, mass spectrometry must play an important and complementary role in unraveling the complexities of lipid-protein interactions. We evaluate recent advances and highlight ongoing challenges towards the twin goals of (1) complete structure elucidation of low, abundant, and structurally diverse lipids by mass spectrometry alone, and (2) assignment of stoichiometry and specificity of lipid interactions within protein complexes.     

Competition of Ligands and the 18-mer Binding Domain of the RHAU Helicase for G-Quadruplexes: Orthosteric or Allosteric Binding Mechanism?

Stabilizing the DNA and RNA structures known as G-quadruplexes (G4s) using specific ligands is a strategy that has been proposed to fight cancer. However, although G-quadruplex:ligand (G4:L) interactions have often been investigated, whether or not ligands are able to disrupt G-quadruplex:protein (G4:P) interactions remains poorly studied. In this study, using native mass spectrometry, we have investigated ternary G4:L:P complexes formed by G4s, some of the highest affinity ligands, and the binding domain of the RHAU helicase. Our results suggest that RHAU binds not only preferentially to parallel G4s, but also to free external G-quartets. We also found that, depending on the G4, ligands could prevent the binding of the peptide, either by direct competition for the binding sites (orthosteric inhibition) or by inducing conformational changes (allosteric inhibition). Notably, the ligand Cu–ttpy (ttpy=4′-tolyl-2,2′:6′,2′′-terpyridine) induced a conformational change that increased the binding of the peptide. This study illustrates that it is important to not only characterize drug–target interactions, but also how the binding to other partners is affected.     

A question of policy: should tests for the self-disproportionation of enantiomers (SDE) be mandatory for reports involving scalemates?

The self-disproportionation of enantiomers (SDE) is a phenomenon that can lead to the perturbation of the enantiomeric excess (ee) in fractions obtained from a scalemic sample that has been subjected to a physical process. While fractional crystallization is widely appreciated as a means to effect enantiopurification, processes that are potentially able to give rise to the SDE phenomenon, notably chromatography, are greatly underappreciated in this regard. In this exposition we question if sufficient care is being taken by workers to avoid the erroneous reporting of stereochemical outcomes in asymmetric synthesis, natural products work, and other chiral-based areas of study due to ignorance of the SDE phenomenon and recommend the incorporation of SDE tests via sublimation and achiral chromatography as outlined herein to check for the occurrence of the SDE phenomenon in the applied methodology and routine experimental work.     

Can the bis(diboranyl) structure of B(4)H(10) be observed? The story continues

Pathways for the conversion of the unknown bis(diboranyl) isomer of tetraborane(10) (B(4)H(10)) to the known arachno isomer have been determined for the first time with the use of an electron correlation ab initio quantum chemical method and without the use of constraints in determination of the transition structures. Two isomers of tetraborane(10), one new, with a pentacoordinated boron atom have been found on the theoretical potential energy surface. Several other pathways for molecular rearrangement of tetraborane(10) have also been characterized. The theoretical method was MP2 theory with the 6-31G(d,p) basis set. The most likely pathway for the conversion of the bis(diboranyl) isomer of tetraborane(10) to the arachno isomer is a concerted pathway with two pentacoordinated intermediates. The highest energy transition state for this pathway lies 27.7 kcal/mol above the bis(diboranyl) isomer. At the same level of the theory, the bis(diboranyl) isomer lies 9.2 kcal/mol above the known arachno isomer. The two isomers with a pentacoordinated boron atom lie 12.5 and 13.1 kcal/mol above the arachno isomer.     

Calibration of the X‐Ray Photoelectron Spectroscopy Binding Energy Scale for the Characterization of Heterogeneous Catalysts: Is Everything Really under Control?

Investigations of X‐ray photoelectron spectra from solid samples need corrections for the surface charging effect. For powder samples such as heterogeneous catalysts and their supports, the C ? (C,H) component of the C 1s peak is often used as an internal standard for the calibration of the binding energy scale. Although this method is widely recognized as suitable for the study of heterogeneous catalysts, we show that a significant calibration bias can be encountered upon comparing samples with different bulk composition. In this paper, a series of SiO₂–Al₂O₃ supports and Pd/SiO₂–Al₂O₃ catalysts with various Si/Al ratios were studied. The spectra issued from these samples were processed with the classical calibration method on the basis of the carbon peak. Important discrepancies in the relative position of the photoelectron peaks were noticed. After systematically discarding instrument‐related issues, a true chemical influence of the bulk matrix on the analyzed surface species was evidenced. The extent of this chemical effect was dependent on the composition of the sample and more precisely on its ionicity. Two possible mechanisms for this chemical effect were proposed and discussed. Finally, an alternative calibration method was offered.     

Calculations of the effect of tunneling on the Swain-Schaad exponents (SSEs) for the 1,5-hydrogen shift in 5-methyl-1,3-cyclopentadiene. Can SSEs be used to diagnose the occurrence of tunneling?

MPW1K density functional calculations, carried out with the 6-31+G(d,p) basis set, have been combined with canonical variational transition state theory (CVT) and small-curvature tunneling (SCT) corrections in order to compute the primary kinetic isotope effects for rearrangement of 5-methyl-1,3-cyclopentadiene (1) to 1-methyl-1,3-cyclopentadiene (2). The Swain-Schaad exponents, SSE = ln(kH/kT)/ln(kD/kT), for this reaction have been computed over the temperature range 100-600 K. Tunneling results in both large positive and large negative deviations from the value of SSE = 3.26, expected from consideration of only the effect of the isotopic mass on passage over the reaction barrier. In the rearrangement of 1 to 2, SSE approximately 3.26, not only at temperatures >400 K, where tunneling is relatively unimportant, but also around 170 K, where tunneling by both H and D is the dominant mode of reaction. Thus, from an experimental finding that SSE approximately 3.26 at a single temperature, it cannot be rigorously concluded that tunneling is unimportant. Measurement of SSEs over a broad temperature range is advisable; but measurement of the temperature dependence of just kH/kD can be used to establish more unequivocally whether tunneling is important, without the necessity of measuring kT.     

Binding of proteins to the minor groove of DNA: What are the structural and energetic determinants for kinking a basepair step?

The structural and energetic determinants for kinking a basepair step by minor groove-insertion of the protein side chains of PurR, LacI, LEF-1, IHF, Sac7d, and Sso7d, have been calculated by molecular dynamics/potential of mean force simulations. The structural determinants of the kinked structures are: two contiguous furanose rings achieve different conformations, in the region of C3'endo (A-DNA) and C2'endo (B-DNA); the chi torsion angle always takes values characteristic of the C2'endo conformation of B-DNA, independently of sugar puckering; and protein side chain insertion increases slide (from negative to positive values), rise, and roll, and decreases twist. The energetic determinants of DNA kinking are: the conformational transition of the sugar-phosphate backbone is not energetically demanding; the relative importance of the interbase parameters in the free energy penalty is slide, followed by twist and rise, and concluding with shift and roll; and the characteristic increase of roll and decrease of twist, upon side chain insertion, tends to stabilize the process of DNA kinking.     

Can Dynamics Be Responsible for the Complex Multipeak Infrared Spectra of NO Adsorbed to Copper(II) Sites in Zeolites?

Copper‐exchanged SSZ‐13 is a very efficient material in the selective catalytic reduction of NO_x using ammonia (deNO_x‐SCR) and characterizing the underlying distribution of copper sites in the material is of prime importance to understand its activity. The IR spectrum of NO adsorbed to divalent copper sites are modeled using ab initio molecular dynamics simulations. For most sites, complex multi‐peak spectra induced by the thermal motion of the cation as well as the adsorbate are found. A finite temperature spectrum for a specific catalyst was constructed, which shows excellent agreement with previously reported data. Additionally these findings allow active and inactive species in deNO_x‐SCR to be identified. To the best of our knowledge, this is the first time such complex spectra for single molecules adsorbed to single active centers have been reported in heterogeneous catalysis, and we expect similar effects to be important in a large number of systems with mobile active centers.     

Characterization of the Cysteine Content in Proteins Utilizing Cysteine Selenylation with 266 nm Ultraviolet Photodissociation (UVPD)

Characterization of the cysteine content of proteins is a key aspect of proteomics. By defining both the total number of cysteines and their bound/unbound state, the number of candidate proteins considered in database searches is significantly constrained. Herein we present a methodology that utilizes 266 nm UVPD to count the number of free and bound cysteines in intact proteins. In order to attain this goal, proteins were derivatized with N-(phenylseleno)phthalimide (NPSP) to install a selectively cleavable Se–S bond upon 266 UVPD. The number of Se–S bonds cleaved upon UVPD, a process that releases SePh moieties, corresponds to the number of cysteine residues per protein.

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Graphical Abstract ?

     

Exploring the Structural Details of Cu(I) Binding to α-Synuclein by NMR Spectroscopy

The aggregation of α-synuclein (AS) is selectively enhanced by copper in vitro, and the interaction is proposed to play a potential role in vivo. In this work, we report the structural, residue-specific characterization of Cu(I) binding to AS and demonstrate that the protein is able to bind Cu(I) with relatively high affinity in a coordination environment that involves the participation of Met1 and Met5 residues. This knowledge is a key to understanding the structural-aggregation basis of the copper-catalyzed oxidation of AS.     

How Low Can You Go? Low Densities of Poly(ethylene glycol) Surfactants Attract Stealth Proteins

It is now well‐established that the surface chemistry and “stealth” surface functionalities such as poly(ethylene glycol) (PEG) chains of nanocarriers play an important role to decrease unspecific protein adsorption of opsonizing proteins, to increase the enrichment of specific stealth proteins, and to prolong the circulation times of the nanocarriers. At the same time, PEG chains are used to provide colloidal stability for the nanoparticles. However, it is not clear how the chain length and density influence the unspecific and specific protein adsorption keeping at the same time the stability of the nanoparticles in a biological environment. Therefore, this study aims at characterizing the protein adsorption patterns depending on PEG chain length and density to define limits for the amount of PEG needed for a stealth effect by selective protein adsorption as well as colloidal stability during cell experiments. PEG chains are introduced using the PEGylated Lutensol AT surfactants, which allow easy modification of the nanoparticle surface. These findings indicate that a specific enrichment of stealth proteins already occurs at low PEG concentrations; for the decrease of unspecific protein adsorption and finally the colloidal stability a full surface coverage is advised.     

Chemo-enzymatic Synthesis and RAFT Polymerization of 6-O-Methacryloyl Mannose: A Suitable Glycopolymer for Binding to the Tetrameric Lectin Concanavalin A?

Summary: The chemo-enzymatic synthesis of 6-O-methacryloyl mannose (MaM) glycomonomer was successfully performed for the first time. Subsequent aqueous RAFT polymerization of the monomer yielded well-defined, linear poly(6-O-methacryloyl mannose) (PMaM) glycopolymers without the need for protecting and deprotecting group chemistry. As well as investigating the RAFT polymerization kinetics of this monomer using various initial monomer to chain transfer agent concentration ratios, the protein binding ability of the generated glycopolymer was tested using concanavalin A, a known mannose-residue binding lectin.