| dc.contributor.advisor | Griesinger, Christian Prof. Dr. | |
| dc.contributor.author | Zhang, Xizhou | |
| dc.date.accessioned | 2022-11-04T09:23:00Z | |
| dc.date.available | 2023-02-14T00:50:10Z | |
| dc.date.issued | 2022-11-04 | |
| dc.identifier.uri | http://resolver.sub.uni-goettingen.de/purl?ediss-11858/14318 | |
| dc.identifier.uri | http://dx.doi.org/10.53846/goediss-9526 | |
| dc.language.iso | eng | de |
| dc.rights.uri | http://creativecommons.org/licenses/by/4.0/ | |
| dc.subject.ddc | 570 | de |
| dc.title | Structural and Functional Investigations of Transmembrane Signaling Histidine Kinase Using NMR | de |
| dc.type | doctoralThesis | de |
| dc.contributor.referee | Griesinger, Christian Prof. Dr. | |
| dc.date.examination | 2022-02-15 | de |
| dc.description.abstracteng | Membrane proteins play a central role in biology as the gatekeepers of all cellular compartments. And transmembrane signaling through membrane bound kinases is a particularly important process for cellular adaptation to a changing environment. In prokaryotes, the membrane bound HK, which is part of the ubiquitous two-component system, is the most common type of signal transduction employing a protein phosphorylation mechanism. Due to their lipid-bound native environment, structural studies of membrane bound HKs have remained challenging for a spectrum of techniques. The first five chapters of this thesis will be dedicated to the functional and structural studies of a membrane HK CitA, which is activated for cross-phosphorylation upon citrate binding by hydrolysis of ATP molecules, employing NMR spectroscopy. The new regime of ultra-fast MAS NMR has allowed the application of 1H-detected experiments to more challenging system. We employed this method for the residue specific assignment of the PASpc construct of CitA and gained chemical shift information of the previously unavailable TM helices, along with their linker regions (Chapter 2). This new piece of information confirms the formation of a P-helix at the end of the PASp domain to further residues downstream, as well as reveals the loosening of a conserved KK linker upon citrate binding between the TM2 helix and the PASc domain.
The role of cytosolic PAS domain in HK activation has remain unsolved since the discovery of HKs over 30 years ago. Based on the functional mutants in the PASc domain of CitA, sPRE profile and NMR visibility, an anti-parallel to parallel dimer rearrangement model for CitA activation in its PASc domain was proposed. However, direct structural evidence and functional data confirming the validity of this structural model is still lacking. We investigated the functional impact of this model on the CitA phosphorylation rate by tracking the ATP hydrolysis using 31P NMR (Chapter 3). An attenuation of CitA’s kinase activity when the anti-parallel N288D mutant PASc domain, instead of the parallel WT PASc domain, was attached to the DHp domain is observed by us. This confirms the functional relevance of this dimer transition in the activity of CitA. The same 31P NMR method provides a convenient way to test the functionality of CitA with different chemical tags, which is a pre-requisite for site-specific inter-dimer distance measurement. We found that tagging CitA with a small CF3 group of a cysteine mutation site, N308C, at the end of the PASc domain retains its functionality and responsiveness to citrate binding, while tagging of the same site with MTSL does not. This paves the way for gaining site specific evidence to prove the anti-parallel to parallel transition upon citrate binding using DNP enhanced 19F NMR (Chapter 4). From this, we found a large change in inter-N308C distance between the citrate free (more than 20 Å apart) and the citrate bound state (11 Å).This supports the drastic anti-parallel to parallel dimer transition directly. Although our data are collected using a fragment of CitA lacking the DHp domain, they provide key information regarding the structural rearrangement of PASc that lead to HK activation in the context of the TM helices. Combined with the 1H-detected MAS NMR assignment, the works presented here suggest that the P-helix formation at the end of PASp, the piston-like upward motion of the TM2 helix, the KK domain loosening at the linker between the TM2 helix and the PASc domain, and the anti-parallel to parallel dimer transition are key events in the activation of CitA with citrate binding.
Internuclear distance determination is the foundation for NMR-based structure calculation. However, high-precision distance measurement is a laborious process requiring lengthy data acquisitions due to the large set of multidimensional spectra needed at different mixing times. This prevents application to large or challenging molecular systems. The last chapter of this thesis will present a new approach, transferred-rotational-echo double resonance (TREDOR), a heteronuclear transfer method in which we simultaneously detect both starting and transferred signals in a single spectrum. This co-acquisition is used to compensate for coherence decay, resulting in accurate and precise distance determination by a single parameter fit using a single spectrum recorded at an ideal mixing time. We showcase TREDOR with the microcrystalline SH3 protein using 3D spectra to resolve resonances. By combining the measured N–C and H–C distances, we calculate the structure of SH3, which converges to the correct fold, with a root-mean-square deviation of 2.1 Å compared to a reference X-ray structure. This demonstrates TREDOR as a fast and straightforward method for determining structures via magic-angle spinning NMR. | de |
| dc.contributor.coReferee | Tittmann, Kai Prof. Dr. | |
| dc.contributor.thirdReferee | De Groot, Bert Prof. Dr. | |
| dc.contributor.thirdReferee | Schwappach, Blanche Prof. Dr. | |
| dc.contributor.thirdReferee | Andreas, Loren Dr. | |
| dc.contributor.thirdReferee | Bennati, Marina Prof. Dr. | |
| dc.subject.eng | NMR | de |
| dc.subject.eng | Membrane protein | de |
| dc.subject.eng | Histidine kinase | de |
| dc.subject.eng | Dipolar coupling | de |
| dc.identifier.urn | urn:nbn:de:gbv:7-ediss-14318-3 | |
| dc.affiliation.institute | Biologische Fakultät für Biologie und Psychologie | de |
| dc.subject.gokfull | Biologie (PPN619462639) | de |
| dc.description.embargoed | 2023-02-14 | de |
| dc.identifier.ppn | 1820789713 | |
| dc.notes.confirmationsent | Confirmation sent 2022-11-04T09:45:02 | de |