Investigation of Supramolecular Assemblies Based on De Novo Coiled Coil Peptidic Scaffolds
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Investigation of supramolecular assemblies based on de novo coiled coil peptidic scaffolds Inaugural-Dissertation To obtain the academic degree Doctor rerum naturalium (Dr. rer. nat) Submitted to the Department of Biology, Chemistry and Pharmacy of Freie Universität Berlin by Ana Rita de Lima Fernandes First reviewer: Prof. Dr. Beate Koksch (Freie Universität Berlin) Second reviewer: Prof. Dr. Kevin Pagel (Freie Universität Berlin) Disputation: 17.09.2019 iii Declaration This PhD thesis was carried out from April 2015 to April 2019 under supervision of Prof. Dr. Beate Koksch at the Institute of Chemistry and Biochemistry in the Department of Biology, Chemistry and Pharmacy of Freie Universität Berlin. I declare that this PhD thesis was prepared autonomously. Third party content, quotes or images are referred to the original work. Additional contributions are: ▪ Electron microscopy studies were acquired by PD Dr. Christoph Böttcher and Dorian Mikolajczak at the BioSupraMol core facility of Freie Universität Berlin (Germany). ▪ UV-vis absorbance and fluorescence studies were performed in collaboration with Dr. Hans von Berlepsch. ▪ Infrared nano-spectroscopy were performed by Katerina Kanevche and Emanuel Pfitzner from the Department of Physics, Freie Universität Berlin, Arnimallee 14, 14195 Berlin. ▪ Computational modelling was performed by Natalia Ernst and PD Dr. Marcus Weber from the Zuse Institute Berlin (ZIB), Takustrasse 7, 14195 Berlin. ▪ 19F MRI was performed by Dr. Sonia Waiczies and Dr. Min-Chi Ku from the Berlin Ultrahigh Field Facility, Max Delbrück Center for Molecular Medicine, Berlin, Germany. v Publications Vukelić, S., Moschner, J., Huhmann, S., Fernandes, R., Berger, A.A., and Koksch, B., Synthesis of side-chain fluorinated amino acids and their effects on the properties of peptides and proteins, in Modern Synthesis Processes and Reactivity of Fluorinated Compounds: Progress in Fluorine Science, 1st Ed., 2016. Posters Rita Fernandes, Natalia Ernst, Hans v. Berlepsch, Dorian J. Mikolajczak, Marcus Weber, Christoph Böttcher, Beate Koksch, ‘pH triggers the assembly of a helical peptide into distinct microscale ordered structures’, 8th Peptide Engineering Meeting, 8th-10th November 2018, Berlin. Wiley Poster Prize. Presentations Fluorination of self-assembling peptides, FLUORINE21 5th Scientific Training Workshop, Gothemburg, Sweden, June 2017. Protein Engineering Through Fluorinated Amino Acids, FLUORINE21 3rd Scientific Training Workshop, Münster, Germany, November 2016. Fluorine in Peptide and Protein Engineering, XV Iberian Peptide Meeting, Porto, Portugal, February 2016. Protein Engineering Through Fluorinated Amino Acids, GRK 1582 Kollegseminar, Berlin, Germany, January 2016. Fluorine Modification of Ubiquitin, FLUORINE21 2nd Scientific Training Workshop, Dublin, Ireland, September 2015. vi Acknowledgements I would like to thank my supervisor Prof. Dr. Beate Koksch for giving me the opportunity to do my research work in her group. I am thankful for her support and for the freedom to develop a new research area in her group. Many thanks to my collaboration partners Dr. Marcus Weber, Natalia Ernst, Katerina Kanevche, Emanuel Pfitzner and Dr. Hans von Berlepsch for the valuable work and fruitful discussions. A special thanks to Dr. Christoph Böttcher for his guidance and great contribution for the development of the projects described in this thesis. I would like to thank current and past members of the Koksch group, for all the help and good atmosphere throughout these years. A special thanks to Elsa, Jason and Stella for the friendship since day one. Thank you, Dorian and Jakob, for all your help in the lab and for all the good moments outside of it. Thank you, Dr. Allison Berger, for proof-reading the thesis and for the valuable comments. Thank you for my family and friends in Portugal, who have believed in me and supported me throughout these years away from home. A big thanks to my friends in Germany and to my friends from the Fluor21 network. Most of all, I am thankful for Tony. Thank you for the constant care, love and laughs. vii Abstract The ability of peptides to self-assemble into defined supramolecular structures has been of interest to scientists for decades. The expansion of our understanding of the relationships between amino acid sequence and assembly has been accompanied by a growing number of supramolecular structures. There are still, however, some challenges that must be overcome for these materials to be suitable for real-world applications. In particular, the development of simple scaffolds that can self-assemble into stable and ordered structures in a controllable and predictable manner must continue to be pursued. In this context, the results presented in this thesis reports on the development of new, simple and robust coiled-coil peptidic scaffolds that can assemble into different micro- and nanoscale structures. The highlight of this work is the development of a simple 29-mer coiled-coil Cy5 dye conjugate that undergoes stimuli-triggered aggregation resulting in the formation of microscale membranes and rods under acidic and neutral pH conditions, respectively. Spectroscopic and electron microscopy analyses revealed that the presence of Cy5 at the N- terminus was crucial for the formation of high aspect ratio assemblies, whereas peptide sequence governed responsiveness towards pH and structural morphology. Subsequently, a series of newly designed sequences were synthesized and tested to further understand the underlying driving forces for assembly, and to manipulate peptide stability and self-assembly. The first redesigned series aimed to study the impact of substitution of Cy5 with analogous but simpler aromatic systems. It was found that only a compromise between hydrophobicity, charge, and size of the aromatic moieties leads to high order assemblies. In the second redesigned series, the substitution of residues within the hydrophobic core with a fluorinated amino acid or a natural analogue were performed in an attempt to improve peptide stability. The findings showed that the nature and the packing of the side-chains in the helical core were critical for peptide stability and for pH-sensitive aggregation. The final redesign aimed to study the influence of residues placed in solvent exposed positions of the coiled- coil motif on peptide conformation and stability. The results demonstrated that the nature of substitution does not affect the secondary conformation. However, peptides possessing an overall positive net charge exhibit higher thermal stability. viii The peptides presented here enable the development of pH-sensitive supramolecular structures, which have potential applications in biomedicine or material sciences. ix Zusammenfassung Die Fähigkeit von Peptiden, sich in definierte supramolekulare Strukturen zu organisieren erregt die Aufmerksamkeit von Wissenschaftlern bereits seit Jahrzehnten. Die Erweiterung unseres Verständnisses der Beziehungen zwischen Aminosäuresequenz und räumlichen Aufbau wurde von einer wachsenden Anzahl von supramolekularen Strukturen begleitet. Es gibt jedoch noch einige Herausforderungen, die zu bewältigen sind, damit diese Materialien für reale Anwendungen verwendbar sind. Insbesondere die Entwicklung einfacher Gerüste, die sich zu stabilen und geordneten Strukturen in einer kontrollierbaren und vorhersehbaren Weise selbst anordnen können, muss weiterverfolgt werden. In diesem Zusammenhang berichteten die in dieser Arbeit vorgestellten Ergebnisse über die Entwicklung neuer, einfacher und robuster Coiled-Coil- Peptidgerüste, die zu unterschiedlichen Strukturen im Mikro- und Nanomaßstab zusammengesetzt werden können. Das Highlight dieser Arbeit ist die Entwicklung eines einfachen 29-mer-Coiled-Coil-Cy5-Farbstoff Konjugats, das eine reizausgelöste Aggregation erfährt, die im sauren pH Bereich zu einer Bildung von Membranen, und im neutralen zu einer Bildung von Stäben im Mikromaßstab führt. Spektroskopische und elektronenmikroskopische Analysen zeigten, dass das Vorhandensein von Cy5 am N-Terminus entscheidend für die Bildung von Anordnungen mit hohem Seitenverhältnis ist, während die Peptidsequenz die Empfindlichkeit gegenüber pH-Wert und Strukturmorphologie steuert. Darauffolgend wurde eine Serie von neu gestalteten Sequenzen synthetisiert und untersucht, um die zugrunde liegenden treibenden Kräfte für die Anordnung zu verstehen, und die Peptidstabilität und Selbstanordnung zu manipulieren. Die erste überarbeitete Serie zielte darauf ab, die Auswirkungen der Substitution von Cy5 mit analogen, aber einfacheren aromatischen Systemen zu untersuchen. Es wurde festgestellt, dass nur ein Kompromiss zwischen Hydrophobie, Ladung und Größe der aromatischen Einheiten zu Strukturen höherer Ordnung führen. In der Bestrebung die Peptidstabilität zu verbessern, wurden in der zweiten überarbeiteten Serie die Reste innerhalb des hydrophoben Kerns mit einer fluorierten Aminosäure oder einem natürlichen Analog substituiert. Die Ergebnisse zeigten, dass die Beschaffenheit und die Packung x der Seitenketten im hydrophoben helikalen Kern entscheidend für die Peptidstabilität und für pH-sensitive Aggregation sind. Die letzte Neugestaltung zielte darauf ab, den Einfluss lösungsmittelexponierter Reste des Coiled-Coil Motifs auf die Peptidkonformation und -Stabilität zu untersuchen. Die Ergebnisse zeigten, dass die Art des Substituenten die sekundäre Konformation nicht beeinflusst. Peptide, die eine positive Gesamtladung aufweisen, zeigten jedoch eine höhere thermische