Dokument: Influence of biomolecular environment on fluorophore photophysics
| Titel: | Influence of biomolecular environment on fluorophore photophysics | |||||||
| Weiterer Titel: | Einfluss der biomolekularen Umgebung auf die Photophysik von Fluorophoren | |||||||
| URL für Lesezeichen: | https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=62679 | |||||||
| URN (NBN): | urn:nbn:de:hbz:061-20230519-113750-9 | |||||||
| Kollektion: | Dissertationen | |||||||
| Sprache: | Englisch | |||||||
| Dokumententyp: | Wissenschaftliche Abschlussarbeiten » Dissertation | |||||||
| Medientyp: | Text | |||||||
| Autor: | Prakash, Aiswaria [Autor] | |||||||
| Dateien: |
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| Beitragende: | Prof. Dr. Seidel, Claus A. M. [Gutachter] Prof. Dr. Schmitt, Lutz [Gutachter] | |||||||
| Stichwörter: | Fluorophores, anisotropy, fluorescence lifetime, fluorescence correlation spectroscopy, FRET | |||||||
| Dewey Dezimal-Klassifikation: | 500 Naturwissenschaften und Mathematik » 540 Chemie | |||||||
| Beschreibung: | Fluorescent labels are widely used in biochemistry to study the conformational dynamics and intermolecular interactions in biomolecules. Understanding the basic photophysical mechanisms of these fluorescent labels and their environment dependence can improve the design and accuracy of biophysical techniques. In the presented work, the influence of biomolecular environment on the fluorescent properties of covalently-linked organic dye labels are investigated and the potential of fluorescent probes to sense the local and global biomolecular environments demonstrated.
To reveal the distinct photophysical signatures of different RNA micro-environments, a systematic study of the fluorescence properties of commonly used organic fluorophores, Alexa488 and Cy5, in different RNA environments are carried out. The results are corroborated by accessible volume (AV) and molecular dynamics (MD) simulations to provide structural insights on potential interaction modes. In addition to sensing the local environment, the ability of fluorescent labels to shed light on the tertiary and global structures of RNA is demonstrated. The influence of the protein environment on dye properties is explored with Alexa488 and Alexa647 dyes labeled on the bacteriophage T4 Lysozyme. Combining spectroscopic studies and MD simulations, a detailed insight into the origin, extent and modes of dye-protein interactions is achieved. AV calculations are employed to determine the role of positively charged amino acids in dye trapping and in providing a quenching environment. AV also reveals the manner in which protein dynamics affect dye properties. Detection of substrate binding and protein denaturation are reported as illustrative cases of fluorescence sensing. Förster resonance energy transfer (FRET) is a fluorescence-based technique that employs a pair of fluorescent labels to report on the structural dynamics of a biomolecule. The insights obtained from the photophysical studies of dyes are discussed in the context of FRET measurements and guidelines are provided to avoid or minimize detrimental effects of dye-biomolecular interactions. Those interactions which significantly alter the dye distribution are incorporated into the label description for FRET dyes and these modifications proven to significantly improve the accuracy of FRET-derived structural models. Thus, in future fluorescence-based studies, the knowledge acquired from the present study regarding the environmental sensitivity of dyes in different biomolecular environments can be used to improve the label or model design, the experiment plan or the interpretation of observations. | |||||||
| Lizenz: |  Dieses Werk ist lizenziert unter einer Creative Commons Namensnennung 4.0 International Lizenz | |||||||
| Fachbereich / Einrichtung: | Mathematisch- Naturwissenschaftliche Fakultät | |||||||
| Dokument erstellt am: | 19.05.2023 | |||||||
| Dateien geändert am: | 19.05.2023 | |||||||
| Promotionsantrag am: | 07.12.2022 | |||||||
| Datum der Promotion: | 24.02.2023 |
