30 research outputs found

    Inhibition of Ligand Exchange Kinetics via Active-Site Trapping with an Antibody Fragment

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    We describe the first example of an inhibitory antibody fragment (nanobody ca1697) that binds simultaneously to an enzyme (the enzyme dihydrofolate reductase from <i>Escherichia coli</i>) and its bound substrate (folate). Binding of the antibody to the substrate causes a 20-fold reduction in the rate of folate exchange kinetics. This work opens up the prospect of designing new types of antibody-based inhibitors of enzymes and receptors through suitable design of immunogens

    Η() can be used for the estimation of the electrostatic interaction and the hydrogen bonding ability

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    <p><b>Copyright information:</b></p><p>Taken from "Influence of the π–π interaction on the hydrogen bonding capacity of stacked DNA/RNA bases"</p><p>Nucleic Acids Research 2005;33(6):1779-1789.</p><p>Published online 23 Mar 2005</p><p>PMCID:PMC1069514.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p

    () Electrostatic interaction energy (Δ) between cytosine and the substituted benzenes Ph-X (kcal/mol) versus the local hardness η()

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    <p><b>Copyright information:</b></p><p>Taken from "Influence of the π–π interaction on the hydrogen bonding capacity of stacked DNA/RNA bases"</p><p>Nucleic Acids Research 2005;33(6):1779-1789.</p><p>Published online 23 Mar 2005</p><p>PMCID:PMC1069514.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> () Correlation part of the interaction energy (Δ) between cytosine and the substituted benzenes Ph-X (kcal/mol) versus the benzene ring polarizability divided by (see ) (a.u.)

    Correlation part of the interaction energy (Δ) computed for the 10 stacked DNA/RNA base dimers (kcal/mol) versus the product of the polarizabilities of each base over (see ) (a

    No full text
    <p><b>Copyright information:</b></p><p>Taken from "Influence of the π–π interaction on the hydrogen bonding capacity of stacked DNA/RNA bases"</p><p>Nucleic Acids Research 2005;33(6):1779-1789.</p><p>Published online 23 Mar 2005</p><p>PMCID:PMC1069514.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p>u.)

    Probing the N‑Terminal β‑Sheet Conversion in the Crystal Structure of the Human Prion Protein Bound to a Nanobody

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    Prions are fatal neurodegenerative transmissible agents causing several incurable illnesses in humans and animals. Prion diseases are caused by the structural conversion of the cellular prion protein, PrP<sup>C</sup>, into its misfolded oligomeric form, known as prion or PrP<sup>Sc</sup>. The canonical human PrP<sup>C</sup> (HuPrP) fold features an unstructured N-terminal part (residues 23–124) and a well-defined C-terminal globular domain (residues 125–231). Compelling evidence indicates that an evolutionary N-terminal conserved motif AGAAAAGA (residues 113–120) plays an important role in the conversion to PrP<sup>Sc</sup>. The intrinsic flexibility of the N-terminal has hampered efforts to obtain detailed atomic information on the structural features of this palindromic region. In this study, we crystallized the full-length HuPrP in complex with a nanobody (Nb484) that inhibits prion propagation. In the complex, the prion protein is unstructured from residue 23 to 116. The palindromic motif adopts a stable and fully extended configuration to form a three-stranded antiparallel β-sheet with the β1 and β2 strands, demonstrating that the full-length HuPrP<sup>C</sup> can adopt a more elaborate β0-β1-α1-β2-α2-α3 structural organization than the canonical β1-α1-β2-α2-α3 prion-like fold. From this structure, it appears that the palindromic motif mediates β-enrichment in the PrP<sup>C</sup> monomer as one of the early events in the conversion of PrP<sup>C</sup> into PrP<sup>Sc</sup>

    Rev and Nb<sub>190</sub> protein organization and mutation scheme.

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    <p>(<b>A</b>) Schematic representation of the HIV-1 Rev functional domain organization and secondary structures. The N-terminal domain of Rev forms a helix-loop-helix while de C-terminal domain is intrinsically unfolded. Three functional domains are shown: the Nuclear Export Signal (NES), the Nucleolar Localization Signal (NoLS) that also serves as RNA Binding Domain (RBD) and the first and second multimerization domain (Multimer. 1 and Multimer. 2). (<b>B</b>) Mutation scheme of the Nb<sub>190</sub> alanine scan. The three hyper-variable CDR regions are underlined as defined by the IMGT <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060259#pone.0060259-Lefranc1" target="_blank">[50]</a>. Residues that were mutated to alanine are shown in bold.</p

    Mapping of the Rev epitope.

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    <p>(<b>A</b>) Relative affinity of wild-type Nb<sub>190</sub> for the K20A and Y23A Rev mutants by FRAP of Nb<sub>190</sub>-mKO when bound to RevM10-GFP. Values are averages ± SEM (n ≥8). (<b>B</b>) Half-time values for the recovery times after photobleaching in panel A. (<b>C</b>) Relative affinity of Nb<sub>190</sub>T33A for the V16A, H53A and L60A Rev mutants by FRAP of Nb<sub>190</sub>-mKO when bound to RevM10-GFP. Values are averages ± SEM (n ≥6). (<b>D</b>) Half-time values for the recovery times after photobleaching in panel C. (<b>E</b>) Schematic overview of the alanine scan performed on the head multimerization surface of Rev. Residues that were mutated to alanine are shown in bold. Mutated positions that resulted in a decreased affinity for the Nb<sub>190</sub>T33A mutant are shown in bold red. Mutated positions that resulted in a decreased affinity for the wild-type nanobody have a red circle.</p

    Nb<sub>190</sub>-Rev interaction model.

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    <p>(<b>A</b>) Cartoon representation of the Nb<sub>190</sub> homology model. The coloring corresponds to the conservation of the amino acids calculated with the Consurf server, ranging from highly variable residues (red), over intermediate conservation (green), to highly conserved residues (blue). (<b>B</b>) Electrostatic surface of Nb<sub>190</sub> and helix-tun-helix motif of Rev: blue (negative), red (positive) and white (neutral). The interaction between Nb<sub>190</sub> and Rev is a combination of electrostatic interactions between RevK20 and the negative pocket of Nb<sub>190</sub> and further hydrophobic stabilization by the surrounding residues. (<b>C</b>) Cartoon representation of Nb<sub>190</sub> (green) docked onto Rev (N-terminal helix-turn-helix motive) (light blue). Important residues for binding interaction according to the alanine scanning results are colored dark blue for Rev and yellow for Nb<sub>190</sub>.). Aliphatic hydrogens and backbone atoms have been hidden for clarity. (<b>D</b>) Close-up of the Nb<sub>190</sub>-Rev interaction pattern, with residue RevK20 forming an extensive hydrogen bonding network (depicted by blue lines) with neighboring residues T33 and D107 in Nb<sub>190</sub>. In addition, RevY23 makes a π-π interaction with Nb<sub>190</sub>F100. This latter residue is also further stabilized by hydrophobic contacts with RevK20 and RevH53. RevH53 and RevL60 interact with Nb<sub>190</sub>L101, while RevV16 makes contact with Nb<sub>190</sub>Y105. Nb<sub>190</sub>D98 stabilizes the CDR3 loop by hydrogen bonds with Nb<sub>190</sub>N96.</p

    Binding Specificities of Nanobody•Membrane Protein Complexes Obtained from Chemical Cross-Linking and High-Mass MALDI Mass Spectrometry

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    The application of nanobodies as binding partners for structure stabilization in protein X-ray crystallography is taking an increasingly important role in structural biology. However, the addition of nanobodies to the crystallization matrices might complicate the optimization of the crystallization process, which is why analytical techniques to screen and characterize suitable nanobodies are useful. Here, we show how chemical cross-linking combined with high-mass matrix-assisted laser/desorption ionization mass spectrometry can be employed as a fast screening technique to determine binding specificities of intact nanobody•membrane protein complexes. Titration series were performed to rank the binding affinity of the interacting nanobodies. To validate the mass spectrometry data, microscale thermophoresis was used, which showed binding affinities of the stronger binding nanobodies, in the low μM range. In addition, mass spectrometry provides access to the stoichiometry of the complexes formed, which enables the definition of conditions under which homogeneous complex states are present in solution. Conformational changes of the membrane protein were investigated and competitive binding experiments were used to delimit the interaction sites of the nanobodies, which is in agreement with crystal structures obtained. The results show the diversity of specifically binding nanobodies in terms of binding affinity, stoichiometry, and binding site, which illustrates the need for an analytical screening approach

    Modular Integrated Secretory System Engineering in <i>Pichia pastoris</i> To Enhance G‑Protein Coupled Receptor Expression

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    Membrane protein research is still hampered by the generally very low levels at which these proteins are naturally expressed, necessitating heterologous expression. Protein degradation, folding problems, and undesired post-translational modifications often occur, together resulting in low expression levels of heterogeneous protein products that are unsuitable for structural studies. We here demonstrate how the integration of multiple engineering modules in <i>Pichia pastoris</i> can be used to increase both the quality and the quantity of overexpressed integral membrane proteins, with the human CXCR4 G-protein coupled receptor as an example. The combination of reduced proteolysis, enhanced ER folding capacity, GlycoDelete-based <i>N</i>-Glycan trimming, and nanobody-based fold stabilization improved the expression of this GPCR in <i>P. pastoris</i> from a low expression level of a heterogeneously glycosylated, proteolyzed product to substantial quantities (2–3 mg/L shake flask culture) of a nonproteolyzed, homogeneously glycosylated proteoform. We expect that this set of tools will contribute to successful expression of more membrane proteins in a quantity and quality suitable for functional and structural studies
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