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Projekt Druckansicht

Untersuchung der Umlagerung von holo-TFIID mittels Cross-linking/Massenspectrometrie

Antragstellerin Dr. Therese Thuy Dung Dau
Fachliche Zuordnung Strukturbiologie
Förderung Förderung von 2017 bis 2019
Projektkennung Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 396403853
 
Erstellungsjahr 2020

Zusammenfassung der Projektergebnisse

The aim of this study is the analysis of the conformational changes of the transcription factor IID (TFIID) during transcription initiation with cross-linking mass spectrometry (CLMS) and cryo electron microscopy (cryo-EM). TFIID is a multi-subunit complex, which has been shown to rearrange itself during the initiation of transcription. We collaborated with the Berger lab that provided us with several subcomplexes of TFIID as well as holo-TFIID. Some regions like e.g. the N-terminal region of Taf4 were poorly covered due to missing trypsin cleavage sites. We developed a new digestion protocol, using elastase. The low specificity of elastase has previously prevented the usage of it in cross-linking mass spectrometry. Digesting the sample with trypsin before the addition of elastase allowed us to detect cross-links in the N-terminal region of Taf4. This led us to analyse the optimal usage of non-tryptic proteases in linear proteomics. In general, the addition of trypsin in a sequential manner improves the identification of proteins for all non-tryptic proteases. We then proceed to analyse core-TFIID as well as four different conformations of holo-TFIID using CLMS. The second goal of this project was the structure analysis of proteins in living cells. To this end, we optimised the application of photo-Leu in CLMS and analysed lysates from E.coli. Photo-Leu incorporates instead of Leu into the proteins and cross-linking can be easily initiated through UV. We determined that 0.75 mM photo-Leu cells as the optimal concentration for CLMS. Most of the cross-links were identified within proteins, rather than between proteins. So, while this cross-linker can be used to analyse protein complexes like TFIID, photo-Leu is even better suited for single protein analysis. Especially taking into consideration that the distance restraint from photo-Leu is considerably lower than that of BS3. We then proceeded to look at membrane proteins of E.coli. The most abundant protein OmpA in the outer membrane is OmpA that consists of two domains – a beta-barrel channel and a periplasmic domain. We were able to identify a small linear motif in the region that links these two domains that seems to be a major binding site for other interaction partners of OmpA.

Projektbezogene Publikationen (Auswahl)

 
 

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