Schmitz, Thomas: Influence of disulfide-bonds on structural and functional properties of peptides and proteins - Case studies on FXIIIa inhibitor tridegin and µ-conotoxin PIIIA. - Bonn, 2021. - Dissertation, Rheinische Friedrich-Wilhelms-Universität Bonn.
Online-Ausgabe in bonndoc: https://nbn-resolving.org/urn:nbn:de:hbz:5-62445
@phdthesis{handle:20.500.11811/9110,
urn: https://nbn-resolving.org/urn:nbn:de:hbz:5-62445,
author = {{Thomas Schmitz}},
title = {Influence of disulfide-bonds on structural and functional properties of peptides and proteins - Case studies on FXIIIa inhibitor tridegin and µ-conotoxin PIIIA},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2021,
month = may,

note = {Disulfide-bonded peptides have been studied for many decades serving as tools to investigate various biochemical pathways as well as lead structures for drug development for numerous diseases. Among these, the most fatal are cardiovascular diseases, triggered by dysregulated haemostasis. An essential component of the haemostasis is the blood coagulation cascade, involving the formation of a fibrin network, thereby stabilizing the blood clot. In addition to the central enzyme thrombin, the blood coagulation factor FXIII has a decisive role, as it cross-links the fibrin network and hence stabilizes it. FXIII is thus an attractive target for the development of therapeutically relevant anticoagulants. One of the most promising compounds for inhibiting FXIII activity is the 66mer leech-derived peptide tridegin, which is structurally stabilized by three disulfide bonds. Recent studies focused on the impact of these three disulfide bonds on the structure and function of tridegin. Similar investigations have been carried out earlier for the 15 3-disulfide-bonded µ-conotoxin PIIIA (µ-PIIIA) isomers, that displayed different biological activity towards voltage-gated sodium ion channels. Both studies revealed a significant influence of the disulfide bond connectivity on the functional effectiveness of these peptides encouraging further investigations.
The present dissertation focuses on both these topics providing deeper insights into chemosynthetic, bioanalytical, and biological aspects of the differentially disulfide-linked tridegin and µ-PIIIA variants. First, all 2-disulfide-bonded tridegin analogs deficient in the bond between Cys19 and Cys25 of the native lead structures were synthesized as well as chemically and biologically analyzed. These investigations revealed that the aforementioned disulfide bond is not as crucial for the inhibitory potential towards FXIIIa as supposed. In addition, one of the 2-disulfide-bonded tridegin variants was examined structurally via experimental and computer-based analyses. In this context, the structure of a tridegin analog was determined by NMR-based structural analysis for the first time. The drastic reduction of the synthesis complexity, compounded by the marked retention of inhibitory activity of the disulfide-deficient isomers poise them as attractive candidates as lead structures for therapeutic anticoagulant development.
In a second study of this dissertation, the applicability of LC-TIMS-MS for the discrimination of isomers with different disulfide bond connectivities was examined using ten different µ-PIIIA isomers and analogs. Therefore, the LC and TIMS profiles of pure peptide samples as well as mixtures of three 2- and seven 3-disulfide-bonded variants were compared. This analysis demonstrated that LC-TIMS-MS is a valuable supplement to the commonly used methods HPLC and MS to distinguish different 2- and 3-disulfide-bonded peptide isomers.
In summary, this dissertation provides new insights into the structure-activity relationships of tridegin and serves as a basis for the development of new anticoagulants. Furthermore, it uncovers a methodologically underexplored territory, presenting the suitability of LC-TIMS-MS for the differentiation of peptide isomers with distinct disulfide bond connectivities.},

url = {https://hdl.handle.net/20.500.11811/9110}
}

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