Detecting Protein–Glycolipid Interactions Using Glycomicelles and CaR-ESI-MS

This study reports on the use of the catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS) assay, combined with glycomicelles, as a method for detecting specific interactions between water-soluble proteins and glycolipids (GLs) in aqueous solution. The B subunit homopentamers of cholera toxin (CTB₅) and Shiga toxin type 1 B (Stx1B₅) and the gangliosides GM1, GM2, GM3, GD1a, GD1b, GT1b, and GD2 served as model systems for this study. The CTB₅ exhibits broad specificity for gangliosides and binds to GM1, GM2, GM3, GD1a, GD1b, and GT1b; Stx1B₅ does not recognize gangliosides. The CaR-ESI-MS assay was used to analyze solutions of CTB₅ or Stx1B₅ and individual gangliosides (GM1, GM2, GM3, GD1a, GD1b, GT1b, and GD2) or mixtures thereof. The high affinity interaction of CTB₅ with GM1 was successfully detected. However, the apparent affinity, as determined from the mass spectra, is significantly lower than that of the corresponding pentasaccharide or when GM1 is presented in model membranes such as nanodiscs. Interactions between CTB₅ and the low affinity gangliosides GD1a, GD1b, and GT1b, as well as GD2, which served as a negative control, were detected; no binding of CTB₅ to GM2 or GM3 was observed. The CaR-ESI-MS results obtained for Stx1B₅ reveal that nonspecific protein-ganglioside binding can occur during the ESI process, although the extent of binding varies between gangliosides. Consequently, interactions detected for CTB₅ with GD1a, GD1b, and GT1b are likely nonspecific in origin. Taken together, these results reveal that the CaR-ESI-MS/glycomicelle approach for detecting protein–GL interactions is prone to false positives and false negatives and must be used with caution.

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Endonuclease-based Method for Detecting the Sequence Specific DNA Binding Protein on Double-stranded DNA Microarray

The double-stranded DNA (dsDNA) probe contains two different protein binding sites. One is for DNA- binding proteins to be detected and the other is for a DNA restriction enzyme. The two sites were arranged together with no base interval. The working principle of the capturing dsDNA probe is described as follows: the capturing probe can be cut with the DNA restriction enzyme (such as EcoR I) to cause a sticky terminal, if the probe is not bound with a target protein, and the sticky terminal can be extended and labeled with Cy3-dUTP by DNA polymerase. When the probe is bound with a target protein, the probe is not capable to be cut by the restriction enzyme because of space obstruction. The amount of the target DNA binding proteins can be measured according to the variations of fluorescent signals of the corresponding probes.     

Polyelectrolyte Polyethylenimine–DNA Complexes in the Composition of Voltammetric Sensors for Detecting DNA Damage

Journal of Analytical Chemistry - A voltammetric DNA sensor based on the polyelectrolyte complexes of polyethylenimine and DNA obtained by self-assembly from solutions of components on a glassy...     

Counterion effects in the preparation of aldehyde–bisulfite adducts

The identification and development of an aldehyde–bisulfite adduct as an isolable starting material in the synthesis of the CETP inhibitor Evacetrapib are described. The physical properties of the sodium and potassium analogs are compared, and the extension of the scope of this study to include an investigation into the solid state properties of a range of sodium and potassium bisulfite adducts of commonly encountered aldehydes is discussed.     

Copper–Oxygen Dynamics in the Tyrosinase Mechanism

The dinuclear copper enzyme, tyrosinase, activates O₂ to form a (μ-η²:η²-peroxido)dicopper(II) species, which hydroxylates phenols to catechols. However, the exact mechanism of phenolase reaction in the catalytic site of tyrosinase is still under debate. We herein report the near atomic resolution X-ray crystal structures of the active tyrosinases with substrate l -tyrosine. At their catalytic sites, CuA moved toward l -tyrosine (CuA1 → CuA2), whose phenol oxygen directly coordinates to CuA2, involving the movement of CuB (CuB1 → CuB2). The crystal structures and spectroscopic analyses of the dioxygen-bound tyrosinases demonstrated that the peroxide ligand rotated, spontaneously weakening its O−O bond. Thus, the copper migration induced by the substrate-binding is accompanied by rearrangement of the bound peroxide species so as to provide one of the peroxide oxygen atoms with access to the phenol substrate's ϵ carbon atom.     

Protein–Protein Binding Affinities in Solution Determined by Electrospray Mass Spectrometry

Electrospray ionization (ESI) allows the transfer of multi-protein complexes into the gas phase, thereby providing a simple approach for monitoring the stoichiometry of these noncovalent assemblies by mass spectrometry (MS). It remains unclear, however, whether the measured ion abundance ratios of free and bound species are suitable for determining solution-phase binding affinities (K _d values). Many types of mass spectrometers employ rf-only quadrupoles as ion guides. This work demonstrates that the settings used for these devices are a key factor for ensuring uniform transmission behavior, which is a prerequisite for meaningful affinity measurements. Using bovine β-lactoglobulin and hemoglobin as model systems, it is demonstrated that under carefully adjusted conditions the “direct” ESI-MS approach is capable of providing K _d values that are in good agreement with previously published solution-phase data. Of the several ion sources tested, a regular ESI emitter operated with pressure-driven flow at 1 μL min^–1 provided the most favorable results. Potential problems in these experiments include conformationally-induced differences in ionization efficiencies, inadvertent collision-induced dissociation, and ESI-induced clustering artifacts. A number of simple tests can be conducted to assess whether or not these factors are prevalent under the conditions used. In addition, the fidelity of the method can be scrutinized by performing measurements over a wide concentration range. Overall, this work supports the viability of the direct ESI-MS approach for determining binding affinities of protein–protein complexes in solution.     

Solid-Phase Synthesis and Purification of Protein–DNA Origami Nanostructures

We present a facile method for the combined synthesis and purification of protein-decorated DNA origami nanostructures (DONs). DONs bearing reductively cleavable biotin groups in addition to ligands for ligation of recombinant proteins are bound to magnetic beads. Protein immobilization is conducted with a large protein excess to achieve high ligation yields. Subsequent to cleavage from the solid support, pure sample solutions are obtained which are suitable for direct AFM analysis of occupation patterns. We demonstrate the method's utility using three different orthogonal ligation methods, the “halo-based oligonucleotide binder” (HOB), a variant of Halo-tag, the “SpyTag/SpyCatcher” (ST/SC) system, and the enzymatic “ybbR tag” coupling. We find surprisingly low efficiency for ST/SC ligation, presumably due to electrostatic repulsion and steric hindrance, whereas the ybbR method, despite its ternary nature, shows good ligation yields. Our method is particularly useful for the development of novel ligation methods and the synthesis of mechanically fragile DONs that present protein patterns for surface-based cell assays.     

Protein–polysaccharide interactions and aggregates in food formulations

The protein–polysaccharide combinations that lead to electrostatic complex and coacervate formation are the object of extensive research using both layer-by-layer and mixed emulsion approaches. The protein–polysaccharide conjugates demonstrated interesting physicochemical properties as stabilizers and emulsifiers, as well as texture modifiers in food products. Furthermore, they are potential optimal nutrient delivery systems. Their complex behavior due to several factors such as pH, ionic strength, concentration, heat, and mechanical treatments is the main reason behind the continuous growth of the research field. The review is reporting some recent advances on the topic, along with an overview of the possible interactions between protein and polysaccharide, from Maillard reaction to enzymatic cross-linking passing through coacervates.     

Metal–base pairing in DNA

The use of DNA as a molecular wire in nanoscale electronic architectures would greatly benefit from its capability of sequence-specific self-assembly. Although single electrons and positive charges have been shown to be transmitted by natural DNA over a distance of several base pairs, the high ohmic resistance of unmodified oligonucleotides imposes a serious obstacle. Exchanging some or all of the Watson–Crick base pairs in DNA by metal complexes may solve this problem and evolve DNA-like materials with superior conductivity for future nano-electronic applications. The so-called metal–base pairs are formed from suitable transition metal ions and ligand-like nucleosides which are introduced into both of the two pairing strands by automated DNA synthesis. This review illustrates the basic concepts of metal–base pairing and highlights recent developments in the field.     

Stable Isotope Labeling Strategy for Protein–Ligand Binding Analysis in Multi-Component Protein Mixtures

Described here is a stable isotope labeling protocol that can be used with a chemical modification- and mass spectrometry-based protein–ligand binding assay for detecting and quantifying both the direct and indirect binding events that result from protein–ligand binding interactions. The protocol utilizes an H₂¹⁶O₂ and H₂¹⁸O₂ labeling strategy to evaluate the chemical denaturant dependence of methionine oxidation in proteins both in the presence and absence of a target ligand. The differential denaturant dependence to the oxidation reactions performed in the presence and absence of ligand provides a measure of the protein stability changes that occur as a result of direct interactions of proteins with the target ligand and/or as a result of indirect interactions involving other protein–ligand interactions that are either induced or disrupted by the ligand. The described protocol utilizes the ¹⁸O/¹⁶O ratio in the oxidized protein samples to quantify the ligand-induced protein stability changes. The ratio is determined using the isotopic distributions observed for the methionine-containing peptides used for protein identification in the LC-MS-based proteomics readout. The strategy is applied to a multi-component protein mixture in this proof-of-principle experiment, which was designed to evaluate the technique’s ability to detect and quantify the direct binding interaction between cyclosporin A and cyclophilin A and to detect the indirect binding interaction between cyclosporin A and calcineurin (i.e., the protein–protein interaction between cyclophilin A and calcineurin that is induced by cyclosporin A binding to cyclophilin A).     

Competitive Reactions in Three-Component System Cationic Colloid–Anionic Liposome–Protein

The formation of complexes of anionic liposomes (50 nm) and polymer microspheres with grafted polycationic chains with a diameter of 240 nm (spherical polycationic brushes) in a physiological solution at a NaCl concentration of 0.15 mol/L is investigated. Liposomes are quantitatively adsorbed on the surface of brushes; every brush can bind up to 24 intact liposomes. The saturated brush–liposome complex is able to additionally bind negatively charged protein albumin; the excess of protein does not displace liposomes from the complex with brushes. The obtained results are important for understanding the mechanism of formation and functioning of electrostatic multiliposomal containers in biological media containing a high amount of protein.     

Interactions in the Protein–Zinc Salt–Organic Tanning Agent System

Russian Journal of General Chemistry - The suitability of a combined tanning system comprising zinc salts and an organic tanning agent as an alternative to chromium compounds was considered. The...     

Association of Chromosomal Telomere DNA With Nuclear Matrix in HeLa Cell

Using Electron Spectroscopic Imaging (ESI), we visualized the in situ binding of nucleic acids to nuclear matrix and 3H-thymidine incorporation which indicates that a small partial DNA bound to nuclear matrix tightly. Furthermore we found that chromosomal telomere DNA could bind to nuclear matrix specifically by the dot and Southern hybridization. The result of the Southwestern blot suggests that telomere DNA has high affinity to lamin B, vimentin and some nuclear matrix proteins. Therefore, the nuclear matrix and lamina of HeLa cell are possibly associated with spatial organization and action of chromosome.     

Detecting Protein–Glycolipid Interactions Using CaR-ESI-MS and Model Membranes: Comparison of Pre-loaded and Passively Loaded Picodiscs

Catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS), implemented using model membranes (MMs), is a promising approach for the discovery of glycolipid ligands of glycan-binding proteins (GBPs). Picodiscs (PDs), which are lipid-transporting complexes composed of the human sphingolipid activator protein saposin A and phospholipids, have proven to be useful MMs for such studies. The present work compares the use of conventional (pre-loaded) PDs with passively loaded PDs (^PLPDs) for CaR-ESI-MS screening of glycolipids against cholera toxin B subunit homopentamer (CTB₅). The pre-loaded PDs were prepared from a mixture of purified glycolipid and phospholipid or a mixture of lipids extracted from tissue, while the ^PLPDs were prepared by incubating PDs containing only phospholipid with glycolipid-containing lipid mixtures in aqueous solution. Time-dependent changes in the composition of the ^PLPDs produced by incubation with glycomicelles of the ganglioside GM1 were monitored using collision-induced dissociation of the gaseous PD ions and from the extent of ganglioside binding to CTB₅ measured by ESI-MS. GM1 incorporation into PDs was evident within a few hours of incubation. At incubation times ≥ 10 days, GM1 binding to CTB₅ was indistinguishable from that observed with pre-loaded PDs produced directly from GM1 at the same concentration. Comparison of ganglioside binding to CTB₅ measured for pre-loaded PDs and ^PLPDs prepared from glycolipids extracted from pig and mouse brain revealed that the ^PLPDs allow for the detection of a greater number of ganglioside ligands. Together, the results of this study suggest ^PLPDs may have advantages over conventionally prepared PDs for screening glycolipids against GBPs using CaR-ESI-MS.

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Protein–small molecule docking with receptor flexibility in iMOLSDOCK

We have earlier reported the iMOLSDOCK technique to perform ‘induced-fit’ peptide–protein docking. iMOLSDOCK uses the mutually orthogonal Latin squares (MOLSs) technique to sample the conformation and the docking pose of the small molecule ligand and also the flexible residues of the receptor protein, and arrive at the optimum pose and conformation. In this paper we report the extension carried out in iMOLSDOCK to dock nonpeptide small molecule ligands to receptor proteins. We have benchmarked and validated iMOLSDOCK with a dataset of 34 protein–ligand complexes as well as with Astex Diverse dataset, with nonpeptide small molecules as ligands. We have also compared iMOLSDOCK with other flexible receptor docking tools GOLD v5.2.1 and AutoDock Vina. The results obtained show that the method works better than these two algorithms, though it consumes more computer time. The source code and binary of MOLS 2.0 (under a GNU Lesser General Public License) are freely available for download at https://sourceforge.net/projects/mols2-0/files/.     

Photochemical Reactions in the Synthesis of Protein–Drug Conjugates

The ability to modify biologically active molecules such as antibodies with drug molecules, fluorophores or radionuclides is crucial in drug discovery and target identification. Classic chemistry used for protein functionalisation relies almost exclusively on thermochemically mediated reactions. Our recent experiments have begun to explore the use of photochemistry to effect rapid and efficient protein functionalisation. This article introduces some of the principles and objectives of using photochemically activated reagents for protein ligation. The concept of simultaneous photoradiosynthesis of radiolabelled antibodies for use in molecular imaging is introduced as a working example. Notably, the goal of producing functionalised proteins in the absence of pre-association (non-covalent ligand-protein binding) introduces requirements that are distinct from the more regular use of photoactive groups in photoaffinity labelling. With this in mind, the chemistry of thirteen different classes of photoactivatable reagents that react through the formation of intermediate carbenes, electrophiles, dienes, or radicals, is assessed.     

Investigating Protein–Protein Allosteric Network using Current-Flow Scheme

Protein dynamic network analysis provides a powerful tool for investigating protein allosteric regulation. We recently developed a current-flow betweenness scheme for protein network analysis and demonstrated that this method, that is, using current-flow betweenness as edge weights, is more appropriate and more robust for investigating the signal transmission between two predefined protein residues or domains as compared with direct usage of correlation scores as edge weights. Here we seek to expand the current-flow scheme to study allosteric regulations involving protein–protein binding. Specifically, we investigated three gain-of-function mutations located at the binding interface of ALK2 (also known as ACVR1) kinase and its inhibitory protein FKBP12. We first searched for the optimal smoothing function for contact network construction and then calculated the subnetwork between FKBP12 protein and the kinase ATP binding site using current-flow betweenness. By comparing the networks between the wild-type and three mutants, we have identified statistically significant changes in the protein–protein networks that are common among all three mutants that allosterically shift the kinase toward a catalytically competent configuration. © 2019 Wiley Periodicals, Inc.     

On Restraints in End-Point Protein–Ligand Binding Free Energy Calculations

The impact of harmonic restraints on protein heavy atoms and ligand atoms on end-point free energy calculations is systematically characterized for 54 protein–ligand complexes. We observe that stronger restraints reduce the equilibration time and statistical inefficiency, suppress conformational sampling, influence correlation with experiment, and monotonically decrease the estimated loss of entropy upon binding, leading to stronger estimated binding free energies in most systems. A statistical estimator that reweights for the biasing potential and includes data prior to the estimated equilibration time has the highest correlation with experiment. A spring constant of 20 cal mol⁻¹ Å⁻² maintains a near-native energy landscape and suppresses artifactual energy minima while minimally limiting thermal fluctuations about the crystal structure. © 2019 Wiley Periodicals, Inc.     

Fragment-Based Stabilizers of Protein–Protein Interactions through Imine-Based Tethering

Small-molecule stabilization of protein–protein interactions (PPIs) is a promising concept in drug discovery, however the question how to identify or design chemical starting points in a “bottom-up” approach is largely unanswered. We report a novel concept for identifying initial chemical matter for PPI stabilization based on imine-forming fragments. The imine bond offers a covalent anchor for site-directed fragment targeting, whereas its transient nature enables efficient analysis of structure–activity relationships. This bond enables fragment identification and optimisation using protein crystallography. We report novel fragments that bind specifically to a lysine at the PPI interface of the p65-subunit-derived peptide of NF-κB with the adapter protein 14-3-3. Those fragments that subsequently establish contacts with the p65-derived peptide, rather than with 14-3-3, efficiently stabilize the 14-3-3/p65 complex and offer novel starting points for molecular glues.