11 research outputs found
Incorporation of <i>cis</i>- and <i>trans</i>-4,5-Difluoromethanoprolines into Polypeptides
Substituted prolines exert diverse effects on the backbone conformation of proteins. Novel difluoro-analogues were obtained by adding difluorocarbene to N-Boc-4,5-dehydroproline methyl ester, which gave the <i>trans</i>-adduct as the sole product with 71% yield. Upon cleavage of the N-protection group the free amino acid decomposed rapidly. Its incorporation into the proline-rich cell-penetrating âsweet arrow peptideâ was thus accomplished using a dipeptide strategy. Two building blocks, containing either <i>cis</i>- or <i>trans</i>-4,5-difluoromethanoproline, were obtained by difluorocyclopropanation of the aminoacyl derivatives of 4,5-dehydroproline. The resulting dipeptides were stable under standard conditions of Fmoc solid phase peptide synthesis and, thus, suitable to study conformational effects
Curvature Engineering: Positive Membrane Curvature Induced by Epsin NâTerminal Peptide Boosts Internalization of Octaarginine
Epsin-1
is a representative protein for inducing the positive curvature
necessary for the formation of clathrin-coated pits. Here we demonstrate
that the N-terminus 18-residue peptide of epsin-1 (EpN18) has this
ability <i>per se</i>, as proved by differential scanning
calorimetry (DSC) and solid-state NMR. Moreover, it is shown how this
positive curvature promotion can be exploited for promoting the direct
penetration of a representative cell-penetrating peptide (CPP), octaarginine
(R<sub>8</sub>), through artificial and plasma membranes. This synergistic
effect has been used for the efficient delivery of a proapoptotic
domain peptide (PAD), which induced high level of apoptosis only when
coadministered with R<sub>8</sub> and EpN18, thus emphasizing the
importance of positive curvature induction for achieving the desired
ultimate cargo bioavailability
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
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
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
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
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
<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
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