Dokument: Development and characterization of low affinity glutamate fluorescent indicators
| Titel: | Development and characterization of low affinity glutamate fluorescent indicators | |||||||
| URL für Lesezeichen: | https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=63094 | |||||||
| URN (NBN): | urn:nbn:de:hbz:061-20230717-081524-3 | |||||||
| Kollektion: | Dissertationen | |||||||
| Sprache: | Englisch | |||||||
| Dokumententyp: | Wissenschaftliche Abschlussarbeiten » Dissertation | |||||||
| Medientyp: | Text | |||||||
| Autor: | Ryndia, Kateryna [Autor] | |||||||
| Dateien: |
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| Beitragende: | Prof. Dr. Fahlke, Christoph [Gutachter] Prof. Dr. Bott, Michael [Gutachter] | |||||||
| Dewey Dezimal-Klassifikation: | 500 Naturwissenschaften und Mathematik » 570 Biowissenschaften; Biologie | |||||||
| Beschreibung: | L-Glutamate (Glu) is the major excitatory neurotransmitter in the vertebrate central nervous system. It is crucial for maintaining normal brain function and contributes to the pathophysiology of various neuropsychiatric diseases. Glutamate releases from presynaptic nerve terminals via exocytosis of synaptic vesicles. Vesicular glutamate transporters (VGLUTs) accumulate glutamate inside synaptic vesicles harnessing the electrochemical proton gradient generated by primary active H+-ATPases. The strength of glutamatergic synapses critically depends on glutamate concentrations inside synaptic vesicles. Whereas various genetically encoded nanosensors were developed to detect local concentration changes in the synaptic and perisynaptic space, sensors that report on glutamate accumulation inside synaptic vesicles are still missing. Since synaptic vesicles exhibit an acidic lumen and since glutamate accumulation is associated with changes of cation and anion concentrations, such a sensor needs to have a pH-, cation- and anion-unaffected affinity to glutamate. Moreover, low affinity is required to report on high mM concentrations in synaptic vesicles.
To engineer and characterize a glutamate sensor capable of following glutamate accumulation in synaptic vesicles, I combined molecular dynamics (MD) simulations with protein biochemistry and fluorescence spectroscopy. Through atomistic MD simulations of the bacterial periplasmic aspartate/glutamate binding protein ybeJ, I studied ligand binding to the sensory domain and its interaction with Na+ / K+ ions. Computational results suggested that glutamate binding is salt-independent. Using the fast-switching alchemical transformations method, I predicted mutants that significantly decrease the affinity to glutamate (T91V and T92V). In order to test the predictions of these simulations, low affinity variants of the novel glutamate fluorescent sensor Fl-GluBP (Fl-GluBP-T91V and Fl-GluBP-T92V) were tested by steady-state fluorescence spectroscopy. Such experiments revealed that the Fl-GluBP-T92V variant has a Kd for glutamate of ~ 120 mM and is virtually pH-, Na+- and K+- independent. Glutamate binding kinetics studies and fluorescence lifetime measurements for this variant showed a small fluorescence dynamic range that makes it unsuited for absolute glutamate concentration measurements inside synaptic vesicles. Despite this, the Fl-GluBP-T92V sensor currently represents the best candidate for following the time course of glutamate accumulation inside synaptic vesicles that may lead to new insights and better understanding of vesicular glutamate accumulation and refilling dynamics. The use of Fl-GluBP-T91V variant (Kd of ~ 49 mM) in synaptic vesicles is impaired by its pH-dependent affinity to glutamate. However, its wide fluorescence dynamic range, glutamate affinity at pH 7.5 and salt stability, qualifies it for the direct quantification of absolute glutamate levels in neuronal cytoplasm. For this, the selectivity between aspartate and glutamate must be improved. | |||||||
| Lizenz: |  Dieses Werk ist lizenziert unter einer Creative Commons Namensnennung 4.0 International Lizenz | |||||||
| Fachbereich / Einrichtung: | Mathematisch- Naturwissenschaftliche Fakultät | |||||||
| Dokument erstellt am: | 17.07.2023 | |||||||
| Dateien geändert am: | 17.07.2023 | |||||||
| Promotionsantrag am: | 14.02.2023 | |||||||
| Datum der Promotion: | 05.06.2023 |
