Synthesis of new, rigid and conformation stabilizing spin labels for analysis of the orientation of transmembrane domains
von Iryna Portnova
Datum der mündl. Prüfung:2021-10-06
Erschienen:2021-11-15
Betreuer:Prof. Dr. Ulf Diederichsen
Gutachter:Prof. Dr. Ulf Diederichsen
Gutachter:Prof. Dr. Marina Bennati
Dateien
Name:eDiss_NewRigidSpin Label_Portnova_2021.pdf
Size:6.50Mb
Format:PDF
Zusammenfassung
Englisch
Lipid-peptide interactions are involved in many essential processes of living organisms, such as cell fusion or transport processes and are therefore, in the center of research interest. One efficient technique to analyze such interactions is the electron paramagnetic resonance (EPR) spectroscopy, in particular the double electron electron resonance (DEER) and pulsed electron double resonance (PELDOR) experiments, which allow to determine intramolecular distances within peptides and to investigate the orientation of peptides incorporated into membranes. One requirement to use this analytical technique is the labeling of the peptides with molecules that bear a paramagnetic center such as a nitroxide radical. Thereby, the peptides can be labeled either by direct incorporation of the spin label into the peptide backbone during the peptide synthesis or by side-directed spin labeling (SDSL). To improve the accuracy of distance and orientation-selection measurements by EPR spectroscopy the aim of this work was to design and synthesize a new spin label with enhanced rigidity for direct incorporation into peptides. The design of the new and rigid spin label was based on the approach to stabilize the peptide conformation by direct incorporation of the spin label. In the course of this work, a new spin label that is capable to induce ß-turn conformations by geometrical preorganization was designed and its synthesis tested. In addition to that, one goal was the investigation of more complex protein systems using the the semi-rigid 4-(3,3,5,5-tetra-methyl-2,6-dioxo-4-oxylpiperazin-1-yl)-l-phenylglycine (TOPP) spin label, which requires a reliable and general method for incorporation of this spin label into large peptide systems. One established method to construct large peptide systems is the native chemical ligation (NCL). This method enables the synthesis of large peptide systems from two or more peptide fragments and allows the incorporation of modified peptide fragments. Thus, the NCL was considered as a promising approach to synthesize complex TOPP spin labeled peptides. The introduction of the TOPP spin label in large peptides constructed by NCL would allow the investigation of large peptide systems by EPR spectroscopy using the semi-rigid TOPP spin label. Therefore, one goal of this work was to synthesize a TOPP spin labeled peptide by NCL.
Keywords: Nitroxide Radical; Spin Label; SPPS; Peptide conformation