Showing papers in "Journal of Molecular Recognition in 2010"
TL;DR: A new approach to design sequence modifications for a TCR recognizing the human leukocyte antigen (HLA)‐A2 restricted tumor epitope NY‐ESO‐1, a cancer testis antigen expressed not only in melanoma, but also on several other types of cancers, is developed.
Abstract: Recognition by the T-cell receptor (TCR) of immunogenic peptides (p) presented by class I major histocompatibility complexes (MHC) is the key event in the immune response against virus infected cells or tumor cells. The major determinant of T cell activation is the affinity of the TCR for the peptide-MHC complex, though kinetic parameters are also important. A study of the 2C TCR/SIYR/H-2Kb system using a binding free energy decomposition (BFED) based on the MM–GBSA approach had been performed to assess the performance of the approach on this system. The results showed that the TCR-p-MHC BFED including entropic terms provides a detailed and reliable description of the energetics of the interaction (Zoete and Michielin, 2007). Based on these results, we have developed a new approach to design sequence modifications for a TCR recognizing the human leukocyte antigen (HLA)-A2 restricted tumor epitope NY-ESO-1. NY-ESO-1 is a cancer testis antigen expressed not only in melanoma, but also on several other types of cancers. It has been observed at high frequencies in melanoma patients with unusually positive clinical outcome and, therefore, represents an interesting target for adoptive transfer with modified TCR. Sequence modifications of TCR potentially increasing the affinity for this epitope have been proposed and tested in vitro. T cells expressing some of the proposed TCR mutants showed better T cell functionality, with improved killing of peptide-loaded T2 cells and better proliferative capacity compared to the wild type TCR expressing cells. These results open the door of rational TCR design for adoptive transfer cancer therapy. Copyright © 2010 John Wiley & Sons, Ltd.
88 citations
TL;DR: The solution structure of the nucleocapsid‐binding domain of the measles virus phosphoprotein (XD) is described and the NTAIL resonance behavior in the titration experiments is consistent with a complex binding model with more than two states.
Abstract: In this report, the solution structure of the nucleocapsid-binding domain of the measles virus phosphoprotein (XD, aa 459-507) is described. A dynamic description of the interaction between XD and the disordered C-terminal domain of the nucleocapsid protein, (N(TAIL), aa 401-525), is also presented. XD is an all alpha protein consisting of a three-helix bundle with an up-down-up arrangement of the helices. The solution structure of XD is very similar to the crystal structures of both the free and bound form of XD. One exception is the presence of a highly dynamic loop encompassing XD residues 489-491, which is involved in the embedding of the alpha-helical XD-binding region of N(TAIL). Secondary chemical shift values for full-length N(TAIL) were used to define the precise boundaries of a transient helical segment that coincides with the XD-binding domain, thus shedding light on the pre-recognition state of N(TAIL). Titration experiments with unlabeled XD showed that the transient alpha-helical conformation of N(TAIL) is stabilized upon binding. Lineshape analysis of NMR resonances revealed that residues 483-506 of N(TAIL) are in intermediate exchange with XD, while the 475-482 and 507-525 regions are in fast exchange. The N(TAIL) resonance behavior in the titration experiments is consistent with a complex binding model with more than two states.
86 citations
TL;DR: This review highlights some of the particularly interesting reports from 2008 employing ITC, with a particular focus on protein interactions with other proteins, nucleic acids, lipids and drugs.
Abstract: Isothermal titration calorimetry (ITC) is a fast, accurate and label-free method for measuring the thermodynamics and binding affinities of molecular associations in solution. Because the method will measure any reaction that results in a heat change, it is applicable to many different fields of research from biomolecular science, to drug design and materials engineering, and can be used to measure binding events between essentially any type of biological or chemical ligand. ITC is the only method that can directly measure binding energetics including Gibbs free energy, enthalpy, entropy and heat capacity changes. Not only binding thermodynamics but also catalytic reactions, conformational rearrangements, changes in protonation and molecular dissociations can be readily quantified by performing only a small number of ITC experiments. In this review, we highlight some of the particularly interesting reports from 2008 employing ITC, with a particular focus on protein interactions with other proteins, nucleic acids, lipids and drugs. As is tradition in these reviews we have not attempted a comprehensive analysis of all 500 papers using ITC, but emphasize those reports that particularly captured our interest and that included more thorough discussions we consider exemplify the power of the technique and might serve to inspire other users.
73 citations
TL;DR: A novel magnetic support based on gum Arabic (GA) coated iron oxide magnetic nanoparticles (MNP) has been endowed with affinity properties towards immunoglobulin G (IgG) molecules and the success of the in situ triazine ligand synthesis was confirmed by fluorescence assays.
Abstract: A novel magnetic support based on gum Arabic (GA) coated iron oxide magnetic nanoparticles (MNP) has been endowed with affinity properties towards immunoglobulin G (IgG) molecules The success of the in situ triazine ligand synthesis was confirmed by fluorescence assays Two synthetic ligands previously developed for binding to IgG, named as ligand 22/8 (artificial Protein A) and ligand 8/7 (artificial Protein L) were immobilized on to MNPs coated with GA (MNP_GA) The dimension of the particles core was not affected by the surface functionalization with GA and triazine ligands The hydrodynamic diameters of the magnetic supports indicate that the coupling of GA leads to the formation of larger agglomerates of particles with about 1 microm, but the introduction of the triazine ligands leads to a decrease on MNPs size The non-functionalized MNP_GA bound 28 mg IgG/g, two times less than bare MNP (60 mg IgG/g) MNP_GA modified with ligand 22/8 bound 133 mg IgG/g support, twice higher than the value obtained for ligand 8/7 magnetic adsorbents (65 mg/g) Supports modified with ligand 22/8 were selected to study the adsorption and the elution of IgG The adsorption of human IgG on this support followed a Langmuir behavior with a Q(max) of 344 mg IgG/g support and K(a) of 15 x 10(5) M The studies on different elution conditions indicated that although the 005 M citrate buffer (pH 3) presented good recovery yields (elution 64% of bound protein), there was occurrence of iron leaching at this acidic pH Therefore, a potential alternative would be to elute bound protein with a 005 M glycine-NaOH (pH 11) buffer
73 citations
TL;DR: The evaluation of the amino acid–nucleotide recognition has been investigated analysing datasets for predicting the association preferences and the geometry that favours the interaction.
Abstract: In this review, the protein–DNA interactions are discussed considering different perspectives, and the biological occurrence of this interaction is explained at atomic level. The evaluation of the amino acid–nucleotide recognition has been investigated analysing datasets for predicting the association preferences and the geometry that favours the interaction.
Based on this knowledge, an affinity chromatographic method was developed also exploiting this biological favoured contact. In fact, the implementation of this technique brings the possibility to apply the concept of molecular interactions to the development of new purification methodologies. In addition, the integration of the information recovered by all the different perspectives can bring new insights about some biological mechanisms, though not totally clarified. Copyright © 2010 John Wiley & Sons, Ltd.
42 citations
TL;DR: N nano‐mapping of VE‐cadherin was performed using simultaneous topography and recognition imaging (TREC) technique onto microvascular endothelial cells from mouse myocardium and revealed prominent ‘dark’ spots which were found to be collocalized with cytoskeletal filaments supporting the hypothesis that VE-cadhersin is linked to actin filaments.
Abstract: Vascular endothelial (VE)-cadherin is predominantly responsible for the mechanical linkage between endothelial cells, where VE-cadherin molecules are clustered and linked through their cytoplasmic domain to the actin-based cytoskeleton. Clustering and linkage of VE-cadherin to actin filaments is a dynamic process and changes according to the functional state of the cells. Here nano-mapping of VE-cadherin was performed using simultaneous topography and recognition imaging (TREC) technique onto microvascular endothelial cells from mouse myocardium (MyEnd). The recognition maps revealed prominent ‘dark’ spots (domains or clusters) with the sizes from 10 to 250 nm. These spots arose from a decrease of oscillation amplitude during specific binding between VE-cadherin cis-dimers. They were assigned to characteristic structures of the topography images. After treatment with nocodazole so as to depolymerize microtubules, VE-cadherin domains with a typical ellipsoidal form were still found to be collocalized with cytoskeletal filaments supporting the hypothesis that VE-cadherin is linked to actin filaments. Compared to other conventional techniques such as immunochemistry or single molecule optical microscopy, TREC represents an alternative method to quickly obtain the local distribution of receptors on cell surface with an unprecedented lateral resolution of several nanometers. Copyright © 2010 John Wiley & Sons, Ltd.
42 citations
TL;DR: This bioelectrode (aDNA‐Glu‐PANI‐CNT/ITO) exhibits specificity toward N. gonorrhoeae species and shows negative response with non‐Neisseria gonor Rhoeae Neisseria species (NgNS) and other gram negative bacteria (GNB).
Abstract: Polyaniline/carbon nanotubes composite (PANI-CNT) electrochemically deposited onto indium-tin-oxide (ITO) coated glass plate has been utilized for Neisseria gonorrhoeae detection by immobilizing 5'-amino-labeled Neisseria gonorrhoeae probe (aDNA) using glutaraldehyde as a cross-linker. PANI-CNT/ITO and aDNA-Glu-PANI-CNT/ITO electrodes have been characterized using scanning electron microscopy (SEM), Fourier Transform Infrared (FT-IR) spectroscopy, cyclic voltammetry (CV), and differential pulse voltammetry (DPV). This bioelectrode can be used to detect N. gonorrhoeae using methylene blue (MB) as redox indicator with response time of 60 s and stability of about 75 days when stored under refrigerated conditions. DPV studies reveal that this bioelectrode can detect complementary DNA concentration from 1 x 10(-6) M to 1 x 10(-17) M with detection limit of 1.2 x 10(-17) M. Further, this bioelectrode (aDNA-Glu-PANI-CNT/ITO) exhibits specificity toward N. gonorrhoeae species and shows negative response with non-Neisseria gonorrhoeae Neisseria species (NgNS) and other gram negative bacteria (GNB).
41 citations
TL;DR: The review represents a particularly useful resource for anyone intending to design a screening library for FBDD and use SPR to detect and quantify binding and other classes of ‘pathological’ (non-ideal) binding behaviour.
Abstract: I am writing in connection with the excellent review article by Rebecca Rich and David Myszka (2010) that was recently published in this journal. As described in a recent publication (Perspicace et al., 2009), Surface Plasmon Resonance (SPR) is a particularly appropriate technology for detection and quantification of ligand binding in fragment based drug discovery (FBDD) (Erlanson et al., 2004; Practical Fragments: http:// practicalfragments.blogspot.com; Fragment Based Drug Discovery Literature: http://fbdd-lit.blogspot.com). Furthermore, SPR can be used to characterize (Giannetti et al., 2008) promiscuous inhibition and other classes of ‘pathological’ (non-ideal) binding behaviour. The review represents a particularly useful resource for anyone intending to design (Blomberg et al., 2009) a screening library for FBDD and use SPR to detect and quantify binding.
36 citations
TL;DR: The RNA extraction procedure was optimized, and a high yield was obtained in the separation of sRNA and ribosomal RNA (rRNA) from total RNA (RNAt), which takes advantage of its simplicity and significant cost reduction.
Abstract: 6S RNA is an abundant non-coding RNA in Escherichia coli (E coli), but its function has not been discovered until recently The first advance on 6S RNA function was the demonstration of its ability to bind the σ70-holoenzyme form of RNA polymerase, inhibiting its activity and consequently the transcription process The growing interest in the investigation of non-coding small RNAs (sRNA) calls for the development of new methods for isolation and purification of RNA This work presents an optimized RNA extraction procedure and describes a new affinity chromatography method using a histidine support to specifically purify 6S RNA from other E coli sRNA species The RNA extraction procedure was optimized, and a high yield was obtained in the separation of sRNA and ribosomal RNA (rRNA) from total RNA (RNAt) This improved method takes advantage of its simplicity and significant cost reduction, since some complex operations have been eliminated A purification strategy was also developed to separate 6S RNA from an sRNA mixture Pure RNA can be advantageously obtained using the histidine-affinity chromatography method, aiming at its application to structural or functional studies Copyright © 2010 John Wiley & Sons, Ltd
29 citations
TL;DR: The implementation of the Fast Analytical Continuum Treatment of Solvation (FACTS) as an implicit solvation model in small molecules docking calculations has been assessed using the EADock docking program, and results strongly support the use of FACTS for docking.
Abstract: Protein-ligand docking has made important progress during the last decade and has become a powerful tool for drug development, opening the way to virtual high throughput screening and in silico structure-based ligand design. Despite the flattering picture that has been drawn, recent publications have shown that the docking problem is far from being solved, and that more developments are still needed to achieve high successful prediction rates and accuracy. Introducing an accurate description of the solvation effect upon binding is thought to be essential to achieve this goal. In particular, EADock uses the Generalized Born Molecular Volume 2 (GBMV2) solvent model, which has been shown to reproduce accurately the desolvation energies calculated by solving the Poisson equation. Here, the implementation of the Fast Analytical Continuum Treatment of Solvation (FACTS) as an implicit solvation model in small molecules docking calculations has been assessed using the EADock docking program. Our results strongly support the use of FACTS for docking. The success rates of EADock/FACTS and EADock/GBMV2 are similar, i.e. around 75% for local docking and 65% for blind docking. However, these results come at a much lower computational cost: FACTS is 10 times faster than GBMV2 in calculating the total electrostatic energy, and allows a speed up of EADock by a factor of 4. This study also supports the EADock development strategy relying on the CHARMM package for energy calculations, which enables straightforward implementation and testing of the latest developments in the field of Molecular Modeling.
29 citations
TL;DR: This work investigated the feasibility of using phenyl boronate (PB) chromatography for the direct capture of monoclonal antibodies from a CHO cell supernatant and the most promising results were obtained using 20 mM 4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic acid at pH 8.5.
Abstract: In this work, we investigated the feasibility of using phenyl boronate (PB) chromatography for the direct capture of monoclonal antibodies from a CHO cell supernatant. Preliminary results, using pure protein solutions have shown that PB media can bind to human antibodies, not only at strong alkaline conditions but also at acidic pH values. In fact, antibodies have been found to bind in the pH range 5.5–8.5. On the other hand, insulin and human serum albumin did not bind at alkaline pH but at lower pH, which reflects the importance of non-specific interactions with the matrix. Different binding and eluting buffers were evaluated for the capture of immunoglobulin G (IgG) from a CHO cell supernatant and the most promising results were obtained using 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid at pH 8.5 as binding buffer and 1.5 M Tris–HCl as eluting buffer. Using a step elution, all IgG was recovered in the elution pool with a maximum purification factor of 56. A gradient elution allowed a further increase of the final purity, yet achieving a slightly lower yield. IgG recovery was around 85% and the purification factor was 76. The highest purity was obtained when the pH of the cell supernatant feed was previously adjusted to 8.5. Starting from an initial protein purity of 1.1% and high-performance liquid chromatography (HPLC) purity of 2.2%, after PB adsorption, a final protein purity of 85% and a HPLC purity of 88% was achieved. Copyright © 2010 John Wiley & Sons, Ltd.
TL;DR: It is demonstrated that deletion into the first Ca2+‐binding loop of the dockerin module results in an efficient and robust affinity tag that can be generally applied for protein purification.
Abstract: Cellulose, a major component of plant matter, is degraded by a cell surface multiprotein complex called the cellulosome produced by several anaerobic bacteria. This complex coordinates the assembly of different glycoside hydrolases, via a high-affinity Ca2+-dependent interaction between the enzyme-borne dockerin and the scaffoldin-borne cohesin modules. In this study, we characterized a new protein affinity tag, ΔDoc, a truncated version (48 residues) of the Clostridium thermocellum Cel48S dockerin. The truncated dockerin tag has a binding affinity (KA) of 7.7 × 108 M−1, calculated by a competitive enzyme-linked assay system. In order to examine whether the tag can be used for general application in affinity chromatography, it was fused to a range of target proteins, including Aequorea victoria green fluorescent protein (GFP), C. thermocellum β-glucosidase, Escherichia coli thioesterase/protease I (TEP1), and the antibody-binding ZZ-domain from Staphylococcus aureus protein A. The results of this study significantly extend initial studies performed using the Geobacillus stearothermophilus xylanase T-6 as a model system. In addition, the enzymatic activity of a C. thermocellum β-glucosidase, purified using this approach, was tested and found to be similar to that of a β-glucosidase preparation (without the ΔDoc tag) purified using the standard His-tag. The truncated dockerin derivative functioned as an effective affinity tag through specific interaction with a cognate cohesin, and highly purified target proteins were obtained in a single step directly from crude cell extracts. The relatively inexpensive beaded cellulose-based affinity column was reusable and maintained high capacity after each cycle. This study demonstrates that deletion into the first Ca2+-binding loop of the dockerin module results in an efficient and robust affinity tag that can be generally applied for protein purification. Copyright © 2010 John Wiley & Sons, Ltd.
TL;DR: Warfarin's capacity to adapt its structure as a function of environment in conjunction with the fluorescence behaviours of the various isomers together provide an environment‐dependent molecular switch with reporter properties, which allows for the simultaneous detection of warfarin in different states with lifetimes spanning the range of 0.10–5.5 ns.
Abstract: The complex nature of the structure of the anticoagulant warfarin is reflected in the diversity of binding modes observed in warfarin-protein recognition systems. A series of theoretical, (1)H-NMR and steady state and time resolved fluorescence spectroscopic studies, have been used to establish correlations between the molecular environment provided by various solvent systems and the relative concentrations of the various members of warfarin's ensemble of isomers. A consequence of these observations is that the judicious choice of solvent system or molecular environment of warfarin allows for manipulation of the position of the equilibrium between isomeric structures such as the hemiacetal and open phenol-keto forms, the latter even possible in a deprotonated form, where in each case unique spectroscopic properties are exhibited by the respective structures. Collectively, warfarin's capacity to adapt its structure as a function of environment in conjunction with the fluorescence behaviours of the various isomers together provide an environment-dependent molecular switch with reporter properties, which allows for the simultaneous detection of warfarin in different states with lifetimes spanning the range < 0.10-5.5 ns. These characteristics are here used to examine warfarin binding domains in a series of materials (solvents, protein, inorganic matrix and synthetic polymer). Moreover, these studies demonstrate the potential for using warfarin, or other switchable analogues thereof, as a tool for studying molecular level characteristics, for example local dielectricity.
TL;DR: Using different physicochemical methods of antibody analysis it is shown that the immune system of multiple sclerosis patients generates a variety of anti‐DNA abzymes of different type and with different catalytic properties, which can play an important role in multiple sclerosis pathogenesis.
Abstract: We present the first evidence demonstrating that small fractions of IgGs of all four subclasses (IgG1-IgG4) are catalytically active in the hydrolysis of DNA and on average their relative activity (nM supercoiled DNA/1mg IgG/1 h) increases in the order: IgG1 (0.58) < IgG2 (0.94) < IgG3 (1.4) < IgG4 (4.1), while their approximate relative contribution to the total activity of abzymes increases in the order: IgG1 (6.9%) < IgG3 (9.3%) < IgG2 (18.2%) < IgG4 (65.6%). On average IgGs containing light chains of the lambda-type are severalfold more active in the hydrolysis of DNA than IgGs with light chains of the kappa-type. Using different physicochemical methods of antibody analysis we have shown that the immune system of multiple sclerosis patients generates a variety of anti-DNA abzymes of different type and with different catalytic properties, which can play an important role in multiple sclerosis pathogenesis.
TL;DR: Novel hydrolytic activity of the anti‐histone H1 antibodies (Ab) toward hist one H1 and myelin basic protein (MBP) was shown and intrinsic activity was confirmed to be an intrinsic property of the IgG molecule.
Abstract: Novel hydrolytic activity of the anti-histone H1 antibodies (Ab) toward histone H1 and myelin basic protein (MBP) was shown. Blood serum of ten patients with clinically diagnosed systemic lupus erythematosus (SLE), and nine healthy donors (control) were screened for the anti-histone H1 antibody- and anti-MBP antibody-mediated specific proteolytic activity. IgGs were isolated by chromatography on Protein G-Sepharose, and four of ten SLE patients appeared to possess IgGs that were capable of cleaving both histone H1 and MBP. Such activity was confirmed to be an intrinsic property of the IgG molecule, since it was preserved at gel filtration at alkaline and acidic pH. At the same time, proteolytic activity was absent in the sera-derived Ab of all healthy donors under control. Anti-histone IgGs were purified by the affinity chromatography on histone H1-Sepharose. Their cross-reactivity toward cationic proteins (histones, lysozyme, and MBP) and their capability of hydrolyzing histone H1 and MBP were detected. However, these IgGs were not cleaving core histones, lysozyme, or albumin. Capability of cleaving histone H1 and MBP was preserved after additional purification of anti-histone H1 IgGs by the HPLC gel filtration. The protease activity of anti-histone H1 IgG Ab was inhibited by serine protease inhibitors. Copyright © 2010 John Wiley & Sons, Ltd.
TL;DR: New insight is provided on the recognition of ribosome by sparsomycin and the challenges in calculations of absolute binding free energies in these systems are highlighted.
Abstract: The standard (absolute) binding free energy of the antibiotic sparsomycin with the 50S bacteria ribosomal subunit is calculated using molecular dynamics (MD) free energy perturbation (FEP) simulations with restraining potentials developed by Wang et al. [Biophys. J. 91:2798–2814 (2006)]. In the simulation protocol, restraining potentials are activated for the orientational and translational movements of the ligand relative to the binding site when it is decoupled from the binding pocket, and then released once the ligand fully interacts with the rest of the system. A reduced system is simulated to decrease the computational cost of the FEP/MD calculations and the effects of the surrounding atoms are incorporated using the generalized solvent boundary potential (GSBP) method. The loss of conformational freedom of the ligand upon binding is characterized using the potential of mean force (PMF) as a function of the root-mean-square deviation (RMSD) relative to the bound conformation. The number of water molecules in the binding pocket is allowed to fluctuate dynamically in response to the ligand during the calculations by combining FEP/MD with grand canonical Monte Carlo (GCMC) simulations. The calculated binding free energy is about −6 kcal/mol, which is in reasonable agreement with the experimental value. The information gleaned from this study provides new insight on the recognition of ribosome by sparsomycin and highlights the challenges in calculations of absolute binding free energies in these systems. Copyright © 2010 John Wiley & Sons, Ltd.
TL;DR: 2 1 0 21 0 2 10 2 1 0 two 1 02 1 1 0 1 0 0 2 2 1 1 2 1 3 0 2 3 1 0 3 0 0 0 1 1 10 1 0 4 0 2.1 0.1 1 0.5 0.6 0.7 0.4 0.3 0.2 0.9 0.8 0.
Abstract: 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 1 0 2 Dynamical aspects of molecular recognition
TL;DR: Results of the single molecule force spectroscopy study of specific interactions between ribonuclease barnase and its inhibitor barstar are presented and the measured value of specific interaction does not depend on the NaCl concentration.
Abstract: Results of the single molecule force spectroscopy study of specific interactions between ribonuclease barnase and its inhibitor barstar are presented. Experimental data obtained for the force loading rate ranging 2-70 nN/s are well approximated by a single straight line, from which the dissociation barrier of the width of 0.12 nm and height of 0.75-0.85X10(-19) J can be inferred. The measured value of specific interaction does not depend on the NaCl concentration. This apparently contradicts the well-known dependence of the binding energy of this pair on the salt concentration, but such a "contradiction" is explained by the insensitivity of the force spectroscopy data to the relatively long-range electrostatic interaction. The latter essentially contributes to the value of barnase-barstar binding energy revealed by biochemical measurements, and it is exactly this electrostatic interaction which is influenced by the salt concentration. Copyright (C) 2010 John Wiley & Sons, Ltd.
TL;DR: The binding free energy, essential dynamics and hydration analysis have been conducted to characterize the dynamics' properties associated with the binding recognition between each set of antibiotics and the RNA duplex and it is found that the hydration sites with long residence time identified between the ring III of two 4,6‐linked antibiotics and phosphate oxygen atoms of G1405/U1406 may be worthy of further exploration for rational drug design.
Abstract: A molecular dynamics (MD) simulation has been performed for two sets of aminoglycoside antibiotics bound with an RNA duplex corresponding to the aminoacyl-tRNA decoding site of the 16S rRNA to characterize the energetics and dynamics of binding for several aminoglycosides. The binding free energy, essential dynamics and hydration analysis have been conducted to characterize the dynamics' properties associated with the binding recognition between each set of antibiotics and the RNA duplex. We have built several dynamic models with reasonable binding free energies showing good correlation with the experimental data. We have also conducted a hydration analysis on some long residency water molecules detected as W8 and W49 sites around the U1406 . U1495 pair and which are found to be important in binding recognition and in causing some apparent stretch variations of this pair during the dynamic studies. In addition, we also find that the hydration sites with long residence time identified between the ring III of two 4,6-linked antibiotics (tobramycin and kanamycin) and phosphate oxygen atoms of G1405/U1406 may be worthy of further exploration for rational drug design.
TL;DR: The tableaux provide a database, derived from the world‐wide protein data bank, mineable in studies of protein architecture, and the basis for a complete enumeration of possible protein folding patterns, which can be compared with the corpus of known structures.
Abstract: Comparing and classifying protein folding patterns allows organizing the known structures, structure search and retrieval, and investigation of general principles of protein architecture. We have been developing a concise tableau representation of protein folding patterns, based on the order and contact patterns of elements of secondary structure: helices and strands of sheet (Lesk, 1995; Kamat and Lesk, 2007; Konagurthu et al., 2008). The tableaux provide a database, derived from the world-wide protein data bank, mineable in studies of protein architecture, including: (i) determination of statistical properties of secondary structure contacts in an unbiased set of protein domains, (ii) investigations of the range of, and relationships among, protein topologies, (iii) investigation of the relationship between local structure of proteins and the complete folding topology, (iv) potential for fold identification from amino acid sequence, and (v) the basis for a complete enumeration of possible protein folding patterns, which can be compared with the corpus of known structures.
TL;DR: The possibility to reveal the presence of low‐affinity antibodies and to determine the affinities of both categories of antibodies as well as the ratio of their concentrations is demonstrated.
Abstract: Traditionally used methods of antibody affinity determination either by ELISA or by the surface plasmon resonance technique do not allow detection of the presence of low-affinity antibodies in samples of high-affinity antibodies. In this paper we demonstrate the possibility to reveal their presence and to determine the affinities of both categories of antibodies as well as the ratio of their concentrations. This is especially important since by using traditional methods for antibody affinity evaluation the admixture of low-affinity antibodies in a sample diminishes the accuracy in determination of specific antibody affinity. In addition, the presence of an admixture of low-affinity antibodies may be an important biological characteristic of the system under study; their revelation and the evaluation of their binding parameters may be valuable in many cases for obtaining a more complete characterization of the binding properties of the multiple antibodies generated in an immune response.
TL;DR: In vitro assays demonstrated that cells from different origins are affected differently by the presence of the nanoparticles, and the methods followed for GA coating of MNPs endow distinct surface characteristics that probably underlie the observed differences when in contact with the cells.
Abstract: Iron oxide magnetic nanoparticles (MNPs) were synthesized by the chemical co-precipitation method and coated with gum arabic (GA) by physical adsorption and covalent attachment. Cultures of mammalian cell lines (HEK293, CHO and TE671) were grown in the presence of uncoated and GA-coated MNPs. Cellular growth was followed by optical microscopy in order to assess the proportion of cells with particles, alterations in cellular density and the presence of debris. The in vitro assays demonstrated that cells from different origins are affected differently by the presence of the nanoparticles. Also, the methods followed for GA coating of MNPs endow distinct surface characteristics that probably underlie the observed differences when in contact with the cells. In general, the nanoparticles to which the GA was adsorbed had a smaller ability to attach to the cells' surface and to compromise the viability of the cultures.
TL;DR: Investigation of chromatographic protein purification in a holistic manner that includes ion‐exchange, HIC, gel filtration and affinity separations for the first time is investigated.
Abstract: The rational selection of optimal protein purification sequences, as well as mathematical models that simulate and allow optimization of chromatographic protein purification processes have been developed for purification procedures such as ion-exchange, hydrophobic interaction and gel filtration chromatography. This paper investigates the extension of such analysis to affinity chromatography both in the selection of chromatographic processes and in the use of the rate model for mathematical modelling and simulation. Two affinity systems were used: Blue Sepharose and Protein A. The extension of the theory developed previously for ion-exchange and HIC chromatography to affinity separations is analyzed in this paper. For the selection of operations two algorithms are used. In the first, the value of η, which corresponds to the efficiency (resolution) of the actual chromatography and, Σ, which determines the amount of a particular contaminant eliminated after each separation step, which determines the purity, have to be determined. It was found that the value of both these parameters is not generic for affinity separations but will depend on the type of affinity system used and will have to be determined on a case by case basis. With Blue Sepharose a salt gradient was used and with Protein A, a pH gradient. Parameters were determined with individual proteins and simulations of the protein mixtures were done. This approach allows investigation of chromatographic protein purification in a holistic manner that includes ion-exchange, HIC, gel filtration and affinity separations for the first time.
TL;DR: In this paper, the binding mode of anti-gastrin17 (G17, pyroEGPWLEEEEEAYGWMDF-NH(2)) monoclonal antibodies and their CDR-derived synthetic binders (SBs) with G17 was analyzed.
Abstract: We present data that reveal crucial differences between the binding mode of anti-gastrin17 (G17, pyroEGPWLEEEEEAYGWMDF-NH(2)) monoclonal antibodies (mAbs) and their CDR-derived synthetic binders (SBs) with G17. The mAbs recognize the N-terminal sequence of G17 (pyroEGPWL) with nanomolar affinity and high sequence selectivity. Molecular simulations suggest that G17 recognition is based primarily on a multitude of weak antibody-ligand interactions (H-bonding, van der Waals, etc.) inside a structurally well-defined cleft-like binding pocket. Relatively small structural changes (e.g. G-2 to A for G17) have a drastic impact on affinity, which is characteristic for antibody-like binding. In contrast, SBs recognize various sequences, including G17-unrelated targets with affinities of 1:1 complexes estimated in the 0.1-1.0 mM range. In most cases however, the G17/SB complex stoichiometries are not well-defined, giving rise to multimer aggregate formation with high apparent complex stabilities. Mutational studies on both G17 and SBs reveal the importance of positively charged (K/R) and aromatic residues (W/Y/F) for G17/SB complex formation. We propose that the synthetic binders use combinations of electrostatic, hydrophobic, and/or cation-π interactions in a variety of ways due to their intrinsic flexibility. This may also be the reason for their relatively low target specificity. We speculate that our findings are of general relevance, in showing that high-affinity mAbs do not necessarily provide the optimal basis for functional mimics design.
TL;DR: Molecular imprinting proved to be an effective means toward increase in the yield of large‐ring CDs of a specific size in the biocatalytic production of these interesting novel host compounds for molecular encapsulations.
Abstract: Cyclodextrin glycosyltransferases (CGTases), members of glycoside hydrolase family 13, catalyze the conversion of amylose to cyclodextrins (CDs), circular alpha-(1,4)-linked glucopyranose oligosaccharides of different ring sizes. The CD containing 12 alpha-D-glucopyranose residues was preferentially synthesized by molecular imprinting of CGTase from Paenibacillus sp. A11 with cyclomaltododecaose (CD(12)) as the template molecule. The imprinted CGTase was stabilized by cross-linking of the derivatized protein. A high proportion of CD(12) and larger CDs was obtained with the imprinted enzyme in an aqueous medium. The molecular imprinted CGTase showed an increased catalytic efficiency of the CD(12)-forming cyclization reaction, while decreased k(cat)/K(m) values of the reverse ring-opening reaction were observed. The maximum yield of CD(12) was obtained when the imprinted CGTase was reacted with amylose at 40 degrees C for 30 min. Molecular imprinting proved to be an effective means toward increase in the yield of large-ring CDs of a specific size in the biocatalytic production of these interesting novel host compounds for molecular encapsulations.
TL;DR: Observation of serine protease like activity suggest presence of an atypical catalytic framework in a few pathological IgM Abs, which is independent of IgM‐RF titer value.
Abstract: The IgM antibodies from rheumatoid arthritis (RA) patients' sera were screened for peptide hydrolyzing activity. Recovery of structurally intact IgM antibodies (Abs), in a single step, was achieved using a weak anion-exchange methacrylate monolith disk. The IgM Abs from patients' sera hydrolyzed the Pro-Phe-Arg-4-methyl-coumaryl-7-amide (PFR-MCA) substrate appreciably compared to the healthy donors. The apparent Km values of IgM Abs from patients' sera were between 0.4 and 0.7 mM. Furthermore, IgM Abs displayed 5 to 10-folds greater proteolysis activity than IgG Abs, recovered from the same pathological serum. The proteolysis activity, as a function, was found to be independent of IgM-RF titer value. Affinity labeling approach targeted at the catalytic site histidine was studied, using a specific irreversible inhibitor, N-α-tosyl-L-lysine chloromethyl ketone (TLCK). Despite modification of catalytic His, observation of serine protease like activity suggest presence of an atypical catalytic framework in a few pathological IgM Abs. Copyright © 2010 John Wiley & Sons, Ltd.
TL;DR: The thermodynamic analysis of the binding of the 7C8 antibody to the chloramphenicol phosphonate antigens—one containing a trifluoroacetyl group (CP‐F) and the other containing an acetyl group(CP‐H)—by using isothermal titration calorimetry (ITC) showed that this binding to CP‐H is accompanied with less favorable enthalpy and more favorable entropy changes.
Abstract: We analyzed the binding of the 7C8 antibody to the chloramphenicol phosphonate antigens—one containing a trifluoroacetyl group (CP-F) and the other containing an acetyl group (CP-H)—by using isothermal titration calorimetry (ITC). The thermodynamic difference due to the substitution of F by H was evaluated using free energy calculations based on molecular dynamics (MD) simulations. We have previously shown that another antibody, namely, 6D9, binds more weakly to CP-H than to CP-F, mainly due to the different hydration free energies of the dissociated state and not due to the unfavorable hydrophobic interactions with the antibody in the bound state. Unlike in the binding of the trifluoroacetyl group with 6D9, in its binding with 7C8, it is exposed to the solvent, as seen in the crystal structure of the complex of 7C8 with CP-F. The thermodynamic analysis performed in this study showed that the binding affinity of 7C8 for CP-H is similar to that for CP-F, but this binding to CP-H is accompanied with less favorable enthalpy and more favorable entropy changes. The free energy calculations indicated that, upon the substitution of F by H, enthalpy and entropy changes in the associated and dissociated states were decreased, but the magnitude of enthalpy and entropy changes in the dissociated state was larger than that in the associated state. The differences in binding free energy, enthalpy, and entropy changes determined by the free energy calculations for the substitution of F by H are in good agreement with the experimental results. Copyright © 2009 John Wiley & Sons, Ltd.
TL;DR: Miniaturized protein–protein interaction assays were designed to analyze Rho GTPase activation based on its interaction with RHo GDI or p21‐activated kinase (PAK).
Abstract: Bead-based interaction assays are excellently suited to study protein-protein interactions, as they require only minimal amounts of sample material. Miniaturized protein-protein interaction assays were designed to analyze Rho GTPase activation based on its interaction with Rho GDI or p21-activated kinase (PAK).Rho GDI plays a key role in the regulation of a variety of cellular functions through its interaction with Rho GTPases. Rho GDI is frequently overexpressed in many human cancers. Therefore, there is a growing and as yet unfulfilled demand for screening assays to identify biologically active compounds that may inhibit the Rho GTPase-Rho GDI interaction. Bead-based interaction assays provide an interesting alternative that facilitate such assays to be performed faster with only small amounts of material compared to routinely used co-immunoprecipitation followed by Western Blot analysis.Bead-based protein interaction assays for overexpressed HA-tagged Rho GTPases were established to study the GTPγS-dependent interaction of five different Rho GTPases with the regulatory protein Rho GDIα and the downstream effector PAK1. In addition, it was demonstrated that the ability of Rho GTPases to interact with Rho GDI in this experimental system was markedly, but differentially sensitive to post-translational modification of their carboxyl terminus. Importantly, this modification also notably affected the ability of Rac1 and Rac2, but not of Cdc42, to interact with PAK1.
TL;DR: A method to select peptide nucleic acid (PNA) encoded molecules with specific functional properties from combinatorially generated libraries and harnesses the sensitivity of DNA detection methods and the multiplexed and miniaturized nature of molecule screening to efficiently select and identify target‐binding molecules from large nucleic Acid encoded chemical libraries.
Abstract: The lack of efficient identification and isolation methods for specific molecular binders has fundamentally limited drug discovery. Here, we have developed a method to select peptide nucleic acid (PNA) encoded molecules with specific functional properties from combinatorially generated libraries. This method consists of three essential stages: (1) creation of a Lab-on-Bead™ library, a one-bead, one-sequence library that, in turn, displays a library of candidate molecules, (2) fluorescence microscopy-aided identification of single target-bound beads and the extraction – wet or dry – of these beads and their attached candidate molecules by a micropipette manipulator, and (3) identification of the target-binding candidate molecules via amplification and sequencing. This novel integration of techniques harnesses the sensitivity of DNA detection methods and the multiplexed and miniaturized nature of molecule screening to efficiently select and identify target-binding molecules from large nucleic acid encoded chemical libraries. Beyond its potential to accelerate assays currently used for the discovery of new drug candidates, its simple bead-based design allows for easy screening over a variety of prepared surfaces that can extend this technique's application to the discovery of diagnostic reagents and disease markers.
TL;DR: The use of labels has limitations, such as changes to the binding kinetics due to the alterations in structure and function that occur with label addition, difficulty in detecting biochemical activities and the need for additional steps in assay development.
Abstract: Detecting the molecular basis of protein–protein recognition is an essential element in understanding protein function because their ability to form specific complexes with other proteins underlies most cellular processes. The use of labels has limitations, such as changes to the binding kinetics due to the alterations in structure and function that occur with label addition, difficulty in detecting biochemical activities and the need for additional steps in assay development. These issues have driven the development of label-free formats for identifying the full range of biochemical activities.
Although optical-based systems dominate the label-free biosensor market, electrochemical, piezoelectric and acoustic devices represent similar but significantly less expensive alternatives. Acoustic biosensors have been employed in the label-free detection of an incredibly broad range of analytes, from interfacial chemistries and lipid membranes, to small molecules and whole cells.
Resonant acoustic profiling (RAP) technology offers label-free, real-time analysis of biomolecular interactions and offers an efficient way to optimize the development and production process of recombinant proteins. RAP measures only the physical binding events and is insensitive to refractive index and colour changes. This enables direct measurement in undiluted crude and complex samples, such as cell culture media or periplasmic extracts, without intensive assay calibration. This advantage simplifies experimental design and eliminates expensive time-consuming purification of often limited material, while delivering high content information. In this respect RAP technology reduces costs and increases the throughput and the density of information to optimize and control the processes more effectively. Copyright © 2010 John Wiley & Sons, Ltd.