9 research outputs found
CD spectra of the CF<sub>3</sub>-Bpg labeled TP10 analogs.
<p>CD spectra are recorded in the presence of unilamellar DMPC/DMPG (3â¶1) vesicles at a P/L ratio of 1â¶200. (A) <b><i>L</i></b><b>-</b>epimers and (B) <b><i>D</i></b><b>-</b>epimers are compared with the WT peptide (black line). Analogs with CF<sub>3</sub>-Bpg in the galanin part are represented by green lines and in the mastoparan part by red lines.</p
Structural characteristics of TP10.
<p>Summary of features related to the bipartite character of the hybrid peptide TP10 (positions labeled with CF<sub>3</sub>-Bpg are marked in red).</p
Membrane-bound structure of TP10, as derived by solid-state <sup>19</sup>F
<p>-<b>NMR</b>. (A) The N-terminal region is intrinsically unstructured (green) and connected to the C-terminal α-helix (red). The amphiphilic helix is embedded in the lipid membrane with a tilt angle of Ïâ55° and an azimuthal rotation angle of Ïâ120°. (B) The helical wheel projection of the C-terminal mastoparan part illustrates how the charged Lys residues (grey) point towards the aqueous phase, while the hydrophobic residues (yellow) face the interior of the membrane. The yellow box represents the bilayer (not drawn to scale, and without implying any particular insertion depth of the peptide within the bilayer).</p
<i>D</i>-amino acid âscanâ to identify aggregation-prone regions in TP10.
<p>Aggregation of TP10 depends on the position of substitution with the sterically restrictive <b><i>D</i></b><b>-</b>CF<sub>3</sub>-Bpg, as monitored by solid-state <sup>19</sup>F-NMR and OCD in oriented DMPC/DMPG (3â¶1) at P/Lâ=â1â¶50. The boxed spectral regions show the static powder pattern contributions of immobilized molecules with â8 kHz splittings.</p
Solid-state NMR spectra of TP10:
<p>(A) <sup>19</sup>F-NMR spectra of TP10 labeled with <b><i>L</i></b><b>-</b>CF<sub>3</sub>-Bpg at Ile8, recorded at three different peptide-to-lipid molar ratios (P/Lâ=â1â¶50, 1â¶200, and 1â¶400) in oriented DMPC/DMPG (3â¶1) bilayers. The hydrated membrane samples were aligned with their normal parallel (0°) and perpendicular (90°) to the static magnetic field B<sub>0</sub> (indicated by an arrow). (B) Solid-state <sup>31</sup>P-NMR spectra of the same samples as in (A), recorded before and after the corresponding <sup>19</sup>F-NMR experiment, showing a high quality of lipid alignment. (C) Solid-state <sup>19</sup>F-NMR spectra of the nine <b><i>L</i></b><b>-</b>CF<sub>3</sub>-Bpg labeled TP10 analogs at P/Lâ=â1â¶400, from which the dipolar couplings of the CF<sub>3</sub>-groups were obtained for the structure calculation. All experiments were performed at 40°C.</p
Fibril formation of TP10.
<p>TEM images of TP10 analogs (A) Leu16â <b><i>L</i></b><b>-</b>CF<sub>3</sub><i>-</i>Bpg, (B) Leu16â <b><i>D</i></b><b>-</b>CF<sub>3</sub>-Bpg, showing a network of amyloid-like fibrils.</p
OCD spectra of TP10.
<p>Representative OCD spectra of TP10 labeled with <b><i>D</i></b><b>-</b>CF<sub>3</sub>-Bpg in oriented DMPC/DMPG (3â¶1) bilayers at P/Lâ=â1â¶50, measured after 1, 5, and 8 days of ageing. (A) Peptide analogs with a substitution in the N-terminal region (here: position Leu4) have a predominantly α-helical structure, just like the WT peptide. (B) When <b><i>D</i></b><b>-</b>CF<sub>3</sub>-Bpg is placed into the C-terminal region (here: position Leu16), the peptide aggregates with a ÎČ-sheet conformation typical of amyloid-like fibrils.</p
Cellular uptake of TP10.
<p>(A, B) Internalization of TP10 WT and of two representative <sup>19</sup>F-labeled analogs Ile8â <b><i>L</i></b><b>-</b>CF<sub>3</sub>-Bpg (C), and Ile20â <b><i>L</i></b><b>-</b>CF<sub>3</sub>-Bpg (D) by HeLa cells. The cells were incubated with 10 ”M peptide at 37°C for 30 min.</p
Secondary structure of TP10-WT bound to DMPC/DMPG vesicles evaluated from the CD spectrum (P/Lâ=â1â¶50, see Figure S1 A/B in File S1 for details).
a<p>NRMSDâ=ânormalized root mean square deviation between calculated and experimental CD spectra.</p