Browsing by Subject "Insertion"

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In vitro Interaktionsstudie der bakteriellen Insertase YidC von Escherichia coli mit seinem Substrat Pf3 coat Protein durch Fluoreszenzspektroskopie
(2011) Winterfeld, Sophie; Kuhn, Andreas
With fluorescence spectroscopy the insertion of Pf3 coat protein into YidC proteoliposomes could be characterized. Therefore the binding could be determined by fluorescence titration and with help of single-molecule spectroscopy the insertion process of Pf3 coat protein was analyzed with FRET. Thus the distances of the proteins were determined and the time dependent insertion was observed.
The function of E. coli YidC for the membrane insertion of the M13 procoat protein
(2018) Spann, Dirk; Kuhn, Andreas
The YidC/Oxa1/Alb3 family consists of insertase homologues that facilitate the insertion and folding of membrane proteins. YidC is located in the inner membrane of bacterial cells. Oxa1 is found in the inner membrane of mitochondria and Alb3 facilitates the insertion of membrane proteins in the thylakoid membranes of chloroplasts (Wang and Dalbey 2011, Hennon et al. 2015). An archaeal homologue was found in M. jannaschii showing that this insertase family is present in all domains of life (Dalbey and Kuhn 2015). The insertase family shares a structural feature that is conserved among all discovered members. This is a hydrophilic groove that is open towards the cytoplasm and the membrane core with a hydrophobic slide formed by transmembrane domain (TM) 3 and TM5. YidC functions on its own but also cooperates with the Sec translocon to facilitate the insertion of large membrane proteins. One protein that is membrane-inserted by YidC but is Sec-independent is the major coat protein of the M13 bacteriophage. The main objectives of this work are the analysis of the insertion mechanism of M13 procoat, the major capsid protein of the M13 bacteriophage, via the YidC-only pathway and the oligomeric state of the active YidC. The analysis of interactions between YidC and M13 procoat was performed via radioactive disulfide crosslinking mainly using copper phenanthroline as oxidizing agent. M13 procoat contacts YidC extensively in TM3 and TM5. The observed contacts suggest that the M13 procoat substrate “slides” along TM3 and TM5 of the insertase. Additional crosslinking experiments with the hydrophilic groove and the C1 loop of YidC were also performed to test their importance during the insertion process. A contact was found in the C1 loop that indicates a role in the insertion process, which is consistent with the proposed insertion model from Kumazaki et al. (2014a). Parallel to the radioactive disulfide crosslinking, a protocol using DTNB (Bis(3-carboxy-4-nitrophenyl) disulfide, Ellman’s reagent) as the oxidizing reagent and Western blot for detection was established. This method reliably promoted the formation of crosslinking products in vivo between YidC and M13 procoat over several hours and many, but not all, mapped at the same sites as in the radioactive approach. In addition, this protocol was used to purify small amounts of a YidC-substrate complex for biochemical analysis, which could also be applied to other substrates in the future. The oligomeric state of YidC was investigated by an artificial dimer of the insertase (dYidC) that was constructed by connecting two monomers together with a short linker. This dimer can complement YidC-depleted E. coli MK6S cells and facilitates the insertion of M13 procoat in vivo. For further analysis of the dYidC three functionally defective YidC mutants, T362A (Wickles et al. 2014), delta-C1 (Chen et al. 2014) and the 5S YidC mutant, were tested for their complementation and insertion capability. All three mutants were not able to complement under YidC depletion conditions. These mutants were then cloned in either one or both protomers of the dYidC. Complementation and insertion assays with these dYidC constructs show that in general one active protomer suffices to uphold cell viability and to facilitate the insertion of M13 procoat. Binding studies using cysteine mutants of the dYidC and M13 procoat for disulfide crosslinking with DTNB demonstrated that each protomer individually binds one substrate molecule. In summary, these experiments strongly support a monomer as the active state of the insertase for YidC-only substrates. Taken together, this study contributes to the understanding of the insertion of proteins into the inner bacterial cell membrane.