1,720,965 research outputs found
Structure of Nanobody Nb23
Get PDFBackground: Nanobodies, or VHHs, are derived from heavy chain-only antibodies (hcAbs) found in camelids. They overcome some of the inherent limitations of monoclonal antibodies (mAbs) and derivatives thereof, due to their smaller molecular size and higher stability, and thus present an alternative to mAbs for therapeutic use. Two nanobodies, Nb23 and Nb24, have been shown to similarly inhibit the self-aggregation of very amyloidogenic variants of β2-microglobulin. Here, the structure of Nb23 was modeled with the Chemical-Shift (CS)-Rosetta server using chemical shift assignments from nuclear magnetic resonance (NMR) spectroscopy experiments, and used as prior knowledge in PONDEROSA restrained modeling based on experimentally assessed internuclear distances. Further validation was comparatively obtained with the results of molecular dynamics trajectories calculated from the resulting best energy-minimized Nb23 conformers. Methods: 2D and 3D NMR spectroscopy experiments were carried out to determine the assignment of the backbone and side chain hydrogen, nitrogen and carbon resonances to extract chemical shifts and interproton separations for restrained modeling. Results: The solution structure of isolated Nb23 nanobody was determined. Conclusions: The structural analysis indicated that isolated Nb23 has a dynamic CDR3 loop distributed over different orientations with respect to Nb24, which could determine differences in target antigen affinity or complex lability
Molecular electrostatics and pKa shifts calculations with the Generalized Born model. A tutorial through examples with Bluues2
Get PDFBiomolecular electrostatics is of key importance for biological function and recognition. The continuum electrostatic model based on the Poisson-Boltzmann (PB) equation has been widely used to study biomolecular electrostatics. The solution of the PB equation gives the electrostatic potential over the space enclosing the molecule(s) of interest, typically obtained at points of a suitable grid. The Generalized Born (GB) model has been used to provide a useful approximation to the solution obtained by solving numerically the PB equation. The main advantage of the GB approach is to express the electrostatic energy of the system as the sum of pairwise interactions. The latter depend on geometric parameters (the GB radii) which depend on all atoms, and their radii, of the system. In this work we present a tutorial through examples for a more efficient and general version of the program Bluues which is able to compute: 1) the generalized Born radius of each atom; 2) the electrostatic potential at the surface of the molecule mapped to solvent accessible atoms; 3) the solvent accessible surface in a PDB formatted file; 4) the electrostatic potential in the volume surrounding the molecule; 5) the electrostatic free energy and different contributions to it; 6) the pH-dependent properties of proteins (total charge and pH-dependent free energy of folding) in the pH range −4 to 18; 7) the pKa shifts due to molecular structure of all ionizable groups. Program summary: Program Title: bluues2 CPC Library link to program files: https://doi.org/10.17632/bx98gcrzbg.1 Developer's repository link: https://github.com/federico-fogolari/bluues2 Code Ocean capsule: https://codeocean.com/capsule/9338710 Licensing provisions: GPLv3 Programming language: C Nature of problem: Biomolecular electrostatics is of key importance for biological function and recognition. Computation of electrostatic effects and their dissection in atomic contributions is a general problem in molecular modeling. Solution method: The GBR6 version of the Generalized Born (GB) model is implemented through surface integrals and all common molecular electrostatic analyses are performed by a single command line program. Additional comments including restrictions and unusual features: The program requires, as input, files in pqr format, which may be generated by independent software (e.g. babel, pdb2pqr). For the optional generation of the solvent excluded surface, the software msms must be installed and present in the path
Insights into a Protein-Nanoparticle System by Paramagnetic Perturbation NMR Spectroscopy
Get PDFBACKGROUND: The interaction between proteins and nanoparticles is a very relevant subject because of the potential applications in medicine and material science in general. Further interest derives from the amyloidogenic character of the considered protein, β2-microglobulin (β2m), which may be regarded as a paradigmatic system for possible therapeutic strategies. Previous evidence showed in fact that gold nanoparticles (AuNPs) are able to inhibit β2m fibril formation in vitro. METHODS: NMR (Nuclear Magnetic Resonance) and ESR (Electron Spin Resonance) spectroscopy are employed to characterize the paramagnetic perturbation of the extrinsic nitroxide probe Tempol on β2m in the absence and presence of AuNPs to determine the surface accessibility properties and the occurrence of chemical or conformational exchange, based on measurements conducted under magnetization equilibrium and non-equilibrium conditions. RESULTS: The nitroxide perturbation analysis successfully identifies the protein regions where protein-protein or protein-AuNPs interactions hinder accessibility or/and establish exchange contacts. These information give interesting clues to recognize the fibrillation interface of β2m and hypothesize a mechanism for AuNPs fibrillogenesis inhibition. CONCLUSIONS: The presented approach can be advantageously applied to the characterization of the interface in protein-protein and protein-nanoparticles interactions
Exploring exchange processes in proteins by paramagnetic perturbation of NMR spectra
No full textThe effect of extrinsic paramagnetic probes on NMR relaxation rates for surface mapping of proteins and other biopolymers is a widely investigated and powerful NMR technique. Here we describe a new application of those probes. It relies on the setting of the relaxation delay to generate magnetization equilibrium and off-equilibrium conditions, in order to tailor the extent of steady state signal recovery with and without the water-soluble nitroxide Tempol. With this approach it is possible to identify signals whose relaxation is affected by exchange processes and, from the relative assignments, to map the protein residues involved in association or conformational interconversion processes on a micro-to-millisecond time scale. This finding is confirmed by the comparison with the results obtained from relaxation dispersion measurements. This simple and convenient method allows preliminary inspection to highlight regions where structural or chemical exchange events are operative, in order to focus on quantitative subsequent determinations by transverse relaxation dispersion experiments or analogous NMR relaxation studies, and/or to gain insights into the predictions of calculations
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Exopolysaccharide produced by the potential probiotic Lactococcus garvieae C47: Structural characteristics, rheological properties, bioactivities and impact on fermented camel milk
No full textFermented camel milk possesses a weak (liquid-like) gel structure. We aimed to 1) investigate the characteristics, bioactivities and rheological properties of the exopolysaccharide (EPS) produced by Lactococcus garvieae-C47 (exopolysaccharide-C47 product), a potential probiotic bacterium, on milk extracted from camels and 2) examine the rheological properties of the fermented camel milk produced by L. garvieae-C47. Exopolysaccharide-C47 product (molecular weight: 7.3 × 106 Da) was composed of the following monosaccharides: glucose (82.51%), arabinose (5.32%) and xylose (12.17%). The antioxidant, antitumor and α-amylase inhibitory activities of exopolysaccharide-C47 product reached up to 67.52, 59.35 and 91.0%, respectively. The apparent viscosity of exopolysaccharide-C47 product decreased with the increase in shear rate and declined by increasing the temperature up to 50 °C. The rheological properties of exopolysaccharide-C47 product are influenced by the salt type and pH value. The exopolysaccharide product produced by L. garvieae C47 possesses valuable health benefits and has the ability to improve the weak structure of fermented camel milk
Assessing nanobody interaction with SARS-CoV-2 Nsp9
Get PDFThe interaction between SARS-CoV-2 non-structural protein Nsp9 and the nanobody 2NSP90 was investigated by NMR spectroscopy using the paramagnetic perturbation methodology PENELOP (Paramagnetic Equilibrium vs Nonequilibrium magnetization Enhancement or LOss Perturbation). The Nsp9 monomer is an essential component of the replication and transcription complex (RTC) that reproduces the viral gRNA for subsequent propagation. Therefore preventing Nsp9 recruitment in RTC would represent an efficient antiviral strategy that could be applied to different coronaviruses, given the Nsp9 relative invariance. The NMR results were consistent with a previous characterization suggesting a 4:4 Nsp9-to-nanobody stoichiometry with the occurrence of two epitope pairs on each of the Nsp9 units that establish the inter-dimer contacts of Nsp9 tetramer. The oligomerization state of Nsp9 was also analyzed by molecular dynamics simulations and both dimers and tetramers resulted plausible. A different distribution of the mapped epitopes on the tetramer surface with respect to the former 4:4 complex could also be possible, as well as different stoichiometries of the Nsp9-nanobody assemblies such as the 2:2 stoichiometry suggested by the recent crystal structure of the Nsp9 complex with 2NSP23 (PDB ID: 8dqu), a nanobody exhibiting essentially the same affinity as 2NSP90. The experimental NMR evidence, however, ruled out the occurrence in liquid state of the relevant Nsp9 conformational change observed in the same crystal structure
The corona of protein-gold nanoparticle systems: The role of ionic strength
No full textThe nature of the nanoparticle-protein corona is emerging as a key aspect in determining the impact of nanomaterials on proteins and in general on the biological response. We previously demonstrated that citrate-stabilized gold nanoparticles (Cit-AuNPs) interact with β2-microglobulin (β2m) preserving the protein native structure. Moreover, Cit-AuNPs are able to hinder in vitro fibrillogenesis of a β2m pathologic variant, namely D76N, by reducing the oligomeric association of the protein in solution. Here, we clarify the characteristics of the interaction between β2m and Cit-AuNPs by means of different techniques, i.e. surface enhanced Raman spectroscopy, NMR and quartz crystal microbalance with dissipation monitoring. All the results obtained clearly show that by simply changing the ionic strength of the medium it is possible to switch from a labile and transient nature of the protein-NP adduct featuring the so-called soft corona, to a more "hard"interaction with a layer of proteins having a longer residence time on the NP surface. This confirms that the interaction between β2m and Cit-AuNPs is dominated by electrostatic forces which can be tuned by modifying the ionic strength. This journal i
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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