Structure and dynamics studies of the enzymes hCAII and GlpG via NMR spectroscopy
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2024
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Abstract
Lösungs- und Festkörper-NMR-Spektroskopie eignen sich gut, um Moleküle wie Proteine mit atomarer Auflösung zu charakterisieren. Hier wurden insbesondere strukturelle und dynamische Änderungen durch die Interaktion mit Inhibitoren, Strategien zur NMR-Probenoptimierung und verschiedene Methoden zur Zuordnung von NMR-Peaks eruiert. Lösungs- und Festkörper-NMR-Experimente wurden entwickelt, um die Flexibilität von SBR in hCAII-gebundenem Zustand zu messen. Das Enzym hCAII reguliert osmotische Prozesse in humanen Zellen und ist deshalb ein Zielprotein für Arzneimittel. Die entwickelte Methode zeigte, dass sich der Ligand trotz hoher Affinität in der Bindungstasche bewegt. Dies könnte von Interesse für die Wirkstoffentwicklung sein. Des Weiteren wurden sogenannte „time-shared“ 3D Lösungs- und Festkörper-NMR-Experimente für die Zuordnung von Methyl-Gruppen durch die Korrelation von Amid- und Methyl-Protonen in räumlicher Nähe zueinander genutzt.
Intramembranproteasen der Rhomboid-ähnlichen Familie sind an Prozessen beteiligt, die mit Krebs- und neurodegenerativen Erkrankungen[1] assoziiert sind. Die Intramembranprotease GlpG aus E. coli wurde als Vertreter dieser Familie betrachtet. Nachdem die Expression und Aufreinigung von GlpG optimiert worden waren, wurden die NMR-Peaks der Methyl-Gruppen durch Punktmutationen zugeordnet. Diese wurden dazu genutzt lokale Änderungen der chemischen Umgebung und der Flexibilität der Seitenketten im Mikro- bis Millisekunden-Bereich für GlpG ohne und im Komplex mit Inhibitoren mittels Lösungs-NMR-Spektroskopie zu verfolgen. Die Ergebnisse deuten darauf hin, dass die Interaktion mit dem Inhibitor STS1775 Konformationsänderungen bzw. Änderungen in der Dynamik von Methyl-Gruppen induziert, die bis zu 18 Å weit vom aktiven Zentrum entfernt sind.
Solution and solid-state NMR are valuable tools to characterize the chemical environments and dynamics of molecules like proteins with atomic resolution. Here, the effects of protein-ligand interactions on structural features and flexibility, strategies for NMR sample optimization, as well as different tools for peak assignments were elucidated. For an unlabelled ligand in hCAII-bound state, dynamics were detected for a high-affinity complex via dedicated solution and solid-state NMR experiments. The enzyme hCAII is a well-studied drug target involved in, for example, pH regulation in human cells. The approach allowed to connect protein-ligand interactions with ligand flexibility, which might be of interest for medicinal chemistry. Furthermore, time-shared 3D NOESY/RFDR experiments were utilized both in solution and the solid state to assign methyl groups of hCAII. Intra-membrane proteases of the rhomboid-like family are involved in processes associated with devastating conditions like cancer and neurodegeneration[1]. Here, GlpG from E. coli as a well-studied representative of the family was studied. After optimizing several expression and purification conditions, selected methyl groups were assigned via mutation to study chemical shift perturbations and microsecond to millisecond motion via solution NMR spectroscopy, comparing GlpG in apo and inhibitor-bound state. Our data indicate that upon binding of inhibitor STS1775, the conformational ensemble changes despite of up to 18 Å distance from the active site. Consequently, the occupancy of the active site modulates conformations close to but also far away from the substrate binding site.
Solution and solid-state NMR are valuable tools to characterize the chemical environments and dynamics of molecules like proteins with atomic resolution. Here, the effects of protein-ligand interactions on structural features and flexibility, strategies for NMR sample optimization, as well as different tools for peak assignments were elucidated. For an unlabelled ligand in hCAII-bound state, dynamics were detected for a high-affinity complex via dedicated solution and solid-state NMR experiments. The enzyme hCAII is a well-studied drug target involved in, for example, pH regulation in human cells. The approach allowed to connect protein-ligand interactions with ligand flexibility, which might be of interest for medicinal chemistry. Furthermore, time-shared 3D NOESY/RFDR experiments were utilized both in solution and the solid state to assign methyl groups of hCAII. Intra-membrane proteases of the rhomboid-like family are involved in processes associated with devastating conditions like cancer and neurodegeneration[1]. Here, GlpG from E. coli as a well-studied representative of the family was studied. After optimizing several expression and purification conditions, selected methyl groups were assigned via mutation to study chemical shift perturbations and microsecond to millisecond motion via solution NMR spectroscopy, comparing GlpG in apo and inhibitor-bound state. Our data indicate that upon binding of inhibitor STS1775, the conformational ensemble changes despite of up to 18 Å distance from the active site. Consequently, the occupancy of the active site modulates conformations close to but also far away from the substrate binding site.
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NMR Spektroskopie, Protein Biochemie