| dc.contributor.advisor | Stülke, Jörg Prof. Dr. | |
| dc.contributor.author | Bremenkamp, Rica | |
| dc.date.accessioned | 2023-01-12T17:00:15Z | |
| dc.date.available | 2023-01-20T00:50:09Z | |
| dc.date.issued | 2023-01-12 | |
| dc.identifier.uri | http://resolver.sub.uni-goettingen.de/purl?ediss-11858/14456 | |
| dc.identifier.uri | http://dx.doi.org/10.53846/goediss-9661 | |
| dc.format.extent | 146 | de |
| dc.language.iso | eng | de |
| dc.rights.uri | http://creativecommons.org/licenses/by/4.0/ | |
| dc.subject.ddc | 570 | de |
| dc.title | Validation of novel protein-protein interactions in Bacillus subtilis | de |
| dc.type | doctoralThesis | de |
| dc.contributor.referee | Stülke, Jörg Prof. Dr. | |
| dc.date.examination | 2022-12-08 | de |
| dc.description.abstracteng | Protein-protein interactions are the basis for many biological processes in the cell. Understanding
these interactions is crucial for the general analysis of how the cell functions and the potential
identification of novel therapeutical targets. Over the years, many different techniques have been
developed to study these protein interactions, among them high throughput approaches such as
crosslinking mass spectrometry and co-fractionation mass spectrometry. The combination of these
approaches with the artificial intelligence-based protein conformation prediction tool AlphaFold
allowed the identification of many novel protein complexes in B. subtilis, including complexes that
involved proteins of unknown function. The aim of this work was to validate these novel protein
interactions and to prove the functionality of this workflow. An additional objective was assigning
functions to some of the so far understudied proteins that were identified in these complexes. The
complexes between the pyruvate dehydrogenase and YneR, the iron sensor Fur and the essential
protein of unknown function YlaN as well as the interaction of the paralogous protein YabR and YugI
with the ribosome were studied intensively. YneR was identified as the first known inhibitor of the
pyruvate dehydrogenase activity in B. subtilis, and therefore renamed to PdhI. This was achieved
through growth experiments. Electrophoretic mobility shift assays showed that YlaN inhibits Fur-DNA
binding activity. AlphaFold Multimer prediction revealed a potentially strong change in the
conformation of the Fur dimer upon binding of YlaN. Furthermore, we showed that ylaN can be
deleted, if fur had been deleted before. Together, these findings demonstrate that the essentiality of
YlaN is due to the regulation of the DNA binding activity of Fur. The paralogous proteins YabR and YugI
bind to the 30S subunit of the ribosome. This was validated via Western blot analysis of purified
ribosomes and bacterial two-hybrid assays. Deletion of yabR affected growth on minimal medium and
YugI seems to play a role in tetracycline susceptibility. It was suspected that both proteins perform the
same essential function in the cell and can replace each other. This hypothesis could be refuted by the
creation of a double deletion strain. To summarize, this work illustrates that the combination of the
complementary techniques crosslinking mass spectrometry and co-fractionation mass spectrometry
allows the accurate prediction of protein complexes in B. subtilis without the need for genetic
manipulation of the organism. The validation of this concept led to the identification of the function
of multiple proteins and the discovery of so far unknown regulatory mechanisms. | de |
| dc.contributor.coReferee | Ficner, Ralf Prof. Dr. | |
| dc.contributor.thirdReferee | Feußner, Ivo Prof. Dr. | |
| dc.contributor.thirdReferee | Daniel, Rolf Prof. Dr. | |
| dc.contributor.thirdReferee | Pöggeler, Stefanie Prof. Dr. | |
| dc.contributor.thirdReferee | Heimel, Kai Prof. Dr. | |
| dc.subject.eng | Bacillus subtilis | de |
| dc.subject.eng | crosslinking | de |
| dc.subject.eng | protein-protein interactions | de |
| dc.subject.eng | pyruvate dehydrogenase | de |
| dc.subject.eng | iron metabolism | de |
| dc.identifier.urn | urn:nbn:de:gbv:7-ediss-14456-9 | |
| dc.affiliation.institute | Biologische Fakultät für Biologie und Psychologie | de |
| dc.subject.gokfull | Biologie (PPN619462639) | de |
| dc.description.embargoed | 2023-01-20 | de |
| dc.identifier.ppn | 1831019264 | |
| dc.identifier.orcid | 0000-0002-0057-3410 | de |
| dc.notes.confirmationsent | Confirmation sent 2023-01-13T06:15:01 | de |