Optimal Control in the High Resolution NMR-Spectroscopy: Transfer Elements and their Application for Structure Elucidation

The design of optimal pulses and NMR pulse sequences is a vivid field of NMR spectroscopy. It is indispensable to provide access to high quality spectral data. This thesis is focused on the development of optimal pulses and pulse sequences using optimal control theory (OCT) and their application in the analysis of organic compounds

Cellular uptake of TP10.

<p>(A, B) Internalization of TP10 WT and of two representative ¹⁹F-labeled analogs Ile8→ <b><i>L</i></b><b>-</b>CF₃-Bpg (C), and Ile20→ <b><i>L</i></b><b>-</b>CF₃-Bpg (D) by HeLa cells. The cells were incubated with 10 µM peptide at 37°C for 30 min.</p

Secondary structure of TP10-WT bound to DMPC/DMPG vesicles evaluated from the CD spectrum (P/L = 1∶50, see Figure S1 A/B in File S1 for details).

a<p>NRMSD = normalized root mean square deviation between calculated and experimental CD spectra.</p

Structural characteristics of TP10.

<p>Summary of features related to the bipartite character of the hybrid peptide TP10 (positions labeled with CF₃-Bpg are marked in red).</p

OCD spectra of TP10.

<p>Representative OCD spectra of TP10 labeled with <b><i>D</i></b><b>-</b>CF₃-Bpg in oriented DMPC/DMPG (3∶1) bilayers at P/L = 1∶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>CF₃-Bpg is placed into the C-terminal region (here: position Leu16), the peptide aggregates with a β-sheet conformation typical of amyloid-like fibrils.</p

Membrane-bound structure of TP10, as derived by solid-state ¹⁹F

<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 τ≈55° and an azimuthal rotation angle of ρ≈120°. (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) ¹⁹F-NMR spectra of TP10 labeled with <b><i>L</i></b><b>-</b>CF₃-Bpg at Ile8, recorded at three different peptide-to-lipid molar ratios (P/L = 1∶50, 1∶200, and 1∶400) in oriented DMPC/DMPG (3∶1) bilayers. The hydrated membrane samples were aligned with their normal parallel (0°) and perpendicular (90°) to the static magnetic field B₀ (indicated by an arrow). (B) Solid-state ³¹P-NMR spectra of the same samples as in (A), recorded before and after the corresponding ¹⁹F-NMR experiment, showing a high quality of lipid alignment. (C) Solid-state ¹⁹F-NMR spectra of the nine <b><i>L</i></b><b>-</b>CF₃-Bpg labeled TP10 analogs at P/L = 1∶400, from which the dipolar couplings of the CF₃-groups were obtained for the structure calculation. All experiments were performed at 40°C.</p

CD spectra of the CF₃-Bpg labeled TP10 analogs.

<p>CD spectra are recorded in the presence of unilamellar DMPC/DMPG (3∶1) vesicles at a P/L ratio of 1∶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 CF₃-Bpg in the galanin part are represented by green lines and in the mastoparan part by red lines.</p

<i>D</i>-amino acid “scan” 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>CF₃-Bpg, as monitored by solid-state ¹⁹F-NMR and OCD in oriented DMPC/DMPG (3∶1) at P/L = 1∶50. The boxed spectral regions show the static powder pattern contributions of immobilized molecules with −8 kHz splittings.</p

Fibril formation of TP10.

<p>TEM images of TP10 analogs (A) Leu16→ <b><i>L</i></b><b>-</b>CF₃<i>-</i>Bpg, (B) Leu16→ <b><i>D</i></b><b>-</b>CF₃-Bpg, showing a network of amyloid-like fibrils.</p