6 research outputs found
Synthesis of Oligo-(alkyne-triplet)peptide Constructs
CopperÂ(I)-catalyzed azide–alkyne
cycloaddition (CuAAC) click
synthesis of an Fmoc-(trispropargyl)Âamino acid building block for
solid phase peptide synthesis (SPPS) of oligo-(trialkyne)Âpeptide constructs
is reported. These can carry potentially indefinite numbers of inherent
alkyne-triplets, which are click derivatized with GlcNAc-azide into
the corresponding glycopeptides
The combinatory microarray screening assay.
<p><b>A</b>) Capture antibody and peptides are printed on a microarray slide before adding scFv in a spot-on-spot print. Subsequent detection of captured biotinylated peptide with streptavidin-conjugated fluorophore (SA-AF647), and bound scFv clone with anti-His and labelled anti-mouse antibodies. <b>B</b>) Analysis of five scFv antibodies. In the capture assay, three clones (a-c) are identified as positive binders to the biotinylated Ku80 peptide <b>7</b>, while d and e are negative. Two scFv clones (b and c) were identified as Ku80 peptide <b>7</b> binders) and one scFv clone (<i>d</i>) was bound to Ku80 peptide <b>6</b>. One scFv clone (<i>e</i>) was negative for all tested peptides. The table summary of the combinatory microarray screening assay shows the four possible outcomes of screening.</p
Phage pool evaluation using phage stock solution from selection with Ku80 peptide 6.
<p><b>A</b>) Titration curve for phages (from third round of selection) binding to Ku80 peptide 6 (â– ) and peptide <b>7</b> (â—Ź) in the phage binding (assay average value of triplicates). Detection was possible down to 1/2000 dilution. <b>B</b>) Titration curve for phages (from third round of selection) binding to Ku80 peptide <b>7</b> (â—Ź) and the negative control, peptide <b>12</b> (â– ) in ELISA (single value). Detection was possible at 1/284 dilution. <b>C</b>) Analysis of phage binding to a representative peptide (black bar) and negative control peptide (red bar) after selection rounds 1, 2 and 3 using the phage-binding assay.</p
Flow cytometry evaluation of clones from the combinatory microarray screening assay.
<p>Tn-positive Jurkat cells (A-B) were stained with anti-Tn scFv G2-H7, GOD3-2C4 (positive control) and a non-binding scFv clone or mouse Ig (negative controls), and analysed with image stream flow cytometry. <b>A</b>) Geomean of APC fluorescence signal from the secondary antibody. <b>B</b>) Three individual representative pictures of stained cells from image stream flow cytometry analysis, showing (Ch01 = bright field, Ch06 = side scatter, Ch11 = APC) cells or clusters of cells with characteristic cell surface staining. The cell line LnCap (<b>C</b>-<b>D</b>) was stained for viability with Sytox® Green (AF488) and with scFv H5 and control antibodies. <b>C</b>) Geomean of APC fluorescence signal from secondary antibody of viable cells (AF488 negative). <b>D</b>) Cell-sized objects were gated with forward and side scatter and analysed for APC and AF488 fluorescence.</p
Results of scFv screening using the combinatory microarray screening assay, in terms of the number of peptide-positive clones and the total number of clones.
<p>Results of scFv screening using the combinatory microarray screening assay, in terms of the number of peptide-positive clones and the total number of clones.</p
Probing the Role of Backbone Hydrogen Bonds in Protein–Peptide Interactions by Amide-to-Ester Mutations
One of the most frequent
protein–protein interaction modules
in mammalian cells is the postsynaptic density 95/discs large/zonula
occludens 1 (PDZ) domain, involved in scaffolding and signaling and
emerging as an important drug target for several diseases. Like many
other protein–protein interactions, those of the PDZ domain
family involve formation of intermolecular hydrogen bonds: C-termini
or internal linear motifs of proteins bind as β-strands to form
an extended antiparallel β-sheet with the PDZ domain. Whereas
extensive work has focused on the importance of the amino acid side
chains of the protein ligand, the role of the backbone hydrogen bonds
in the binding reaction is not known. Using amide-to-ester substitutions
to perturb the backbone hydrogen-bonding pattern, we have systematically
probed putative backbone hydrogen bonds between four different PDZ
domains and peptides corresponding to natural protein ligands. Amide-to-ester
mutations of the three C-terminal amides of the peptide ligand severely
affected the affinity with the PDZ domain, demonstrating that hydrogen
bonds contribute significantly to ligand binding (apparent changes
in binding energy, ΔΔ<i>G</i> = 1.3 to >3.8
kcal mol<sup>–1</sup>). This decrease in affinity was mainly
due to an increase in the dissociation rate constant, but a significant
decrease in the association rate constant was found for some amide-to-ester
mutations suggesting that native hydrogen bonds have begun to form
in the transition state of the binding reaction. This study provides
a general framework for studying the role of backbone hydrogen bonds
in protein–peptide interactions and for the first time specifically
addresses these for PDZ domain–peptide interactions