Evaluation of (A) kinetic titration series and (B) parallel sensor kinetics with rabbit IgG binding to GB1 in BLI.

<p>The sensorgrams show the interaction of IgG (analyte) with GB1 (ligand). Applied analyte concentrations were: 0.5, 0.25, 0.125, 0.0625 and 0.03125 µM. The fits are indicated by the red lines, whereas the sensorgrams are shown in black (A) and blue (B). The residuals of the fits are plotted below the respective sensorgram. All other experiments are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106882#pone.0106882.s001" target="_blank">File S1</a>.</p

Comparison of the binding constants obtained by fitting with equivalent models.

<p>Ø: mean, SD: standard deviation, <i>k</i>_d: off rate in 1/s, <i>k</i>_a: on rate in 1/Ms, <i>K</i>_D: dissociation constant in M, #1/2: measurement one and two.</p><p>Comparison of the binding constants obtained by fitting with equivalent models.</p

Final amount of immobilized protein on AR2G sensors.

<p>The value of the response is a measure of the amount of protein on the sensors. A) 1.25 nm, B) 0.15 nm, C) 0.25 nm and D) 0.75 nm have been defined as target for ligand immobilization. Ø and SD: Mean response and the corresponding standard deviation after immobilization.</p><p>Final amount of immobilized protein on AR2G sensors.</p

Comaprison of kinetic titration series (A–C) and parallel sensor kinetics (D–F) with scFv IC16 binding to Aβ(1–42) in BLI.

<p>The sensorgrams show the interaction of scFv IC16 (analyte) with C-terminally biotinylated Aß(1–42) (ligand). The amount of ligand was increased from 0.13 nm (A, D), 0.41 nm (B, E) and 1.01 nm (C, F). Applied analyte concentrations were: 2.4, 1.2, 0.6, 0.3 and 0.15 µM. The fits are indicated by the red lines, whereas the sensorgrams are shown in blue. Each kinetic titration series was reproduced five times. The residuals of the fits are plotted below the respective sensorgram.</p

Role of Hydrophobicity and Charge of Amyloid-Beta Oligomer Eliminating d‑Peptides in the Interaction with Amyloid-Beta Monomers

Inhibition of the self-assembly process of amyloid-beta and even more the removal of already existing toxic amyloid-beta assemblies represent promising therapeutic strategies against Alzheimer’s disease. To approach this aim, we selected a d-enantiomeric peptide by phage-display based on the interaction with amyloid-beta monomers. This lead compound was successfully optimized by peptide microarrays with respect to its affinity and specificity to the target resulting in d-peptides with both increased hydrophobicity and net charge. Here, we present a detailed biophysical characterization of the interactions between these optimized d-peptides and amyloid-beta monomers in comparison to the original lead compound in order to obtain a more thorough understanding of the physicochemical determinants of the interactions. Kinetics and apparent stoichiometry of complex formation were studied using surface plasmon resonance. Potential modes of binding to amyloid-beta were identified, and the influences of ionic strength on complex stability, as well as on the inhibitory effect on amyloid-beta aggregation were investigated. The results reveal a very different mode of interaction of the optimized d-peptides based on a combination of electrostatic and hydrophobic interactions as compared to the mostly electrostatically driven interaction of the lead compound. These conclusions were supported by the thermodynamic profiles of the interaction between optimized d-peptides and Aβ monomers, which indicate an increase in binding entropy with respect to the lead compound

Scheme for the preparation of bR loaded nanodiscs.

<p>Empty nanodiscs were generated by incubating the lipid and MSP1 in the presence of a detergent. Detergent was removed by dialysis and nanodiscs were purified by size exclusion chromatography. The empty nanodiscs were added to the cell-free protein synthesis reaction. bR was incorporated co-translationally into nanodiscs and achieves correct folding. bR loaded nanodiscs were separated from empty nanodiscs by Ni-NTA and are further purified by size exclusion chromatography. bR was shown to be properly folded by its absorption maximum at 550 nm.</p

Sequence alignment of peptides displayed on the surface of representative bR binding phage clones (a) and of nanodisc binding phage clones (b).

<p>Sequence alignment of peptides displayed on the surface of representative bR binding phage clones (a) and of nanodisc binding phage clones (b).</p

List of peptides resembling the extramembranous regions of bR.

<p>Peptides were biotinylated to allow capturing on streptavidin coated plates. Ttds ((N-(3-{2-[2-(3-Amino-propoxy)-ethoxy]-ethoxy}-propyl)-succinamic acid) was used as spacer between the biotin and the peptide to reduce steric hindrance. All peptides were C-terminally amidated.</p

Detergent stability of nanodiscs.

<p>Nanodiscs were incubated for 3 h at room temperature in TBS+0.1% Tween 20, the washing buffer used during phage selection, and separated by size exclusion chromatography using a Superdex 200 10/300 GL column. Solid lines show absorbance at 280 nm of untreated nanodiscs, dotted lines of detergent-treated nanodiscs. The majority of the nanodiscs remained stable upon detergent treatment.</p

Crystal structure of bacteriorhodopsin (PDB accession number 1IW6).

<p><b>A</b> shows a side view of bR and <b>B</b> a view from above. Transmembrane regions are shown in blue and extramembranous regions are shown in green and purple. Putative binding sites for the phage clones 43, 68 and 165 are highlighted in purple. Dotted lines indicate distances between residues of the loops AB and EF: black dotted line between the C^α of the residues A38 and V167 (1.25 nm) and red dotted line between the C^α of the residues V34 and M163 (2.18 nm). Lysine residues including their side chains are marked in grey. Structure visualization of bR was done with pymol.</p