Browsing by Subject "Protein-Protein Interaktion"

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Interaktion des Portalring-Proteins gp20 des Bakteriophagen T4 mit Wirtsproteinen von Escherichia coli
(2012) Quinten, Tobias; Kuhn, Andreas
Bacteriophage T4 is composed of the three structural subunits i. e. capsid, tail and tail fibres. Because of its contractible tail T4 is a member of the Myoviridae. A characteristic feature of its morphogenesis is the membrane-associated assembly of the head structure during a wild-type infection of E. coli. The portal protein, gp20, and the phage-chaperone gp40 are used to form a membrane-bound complex. Most likely, also proteins from the phage-host E. coli are involved in this process. This complex is the starting point for the assembly of the head-related core and scaffold structure. The mature head detaches from the membrane and is filled with DNA thereafter. In the context of this doctoral thesis the role and function of the portal protein gp20 and its interactions with cellular proteins was analyzed. A His-tag was fused to gp20 and it was purified by nickel-affinity chromatography. Also, interactions with the cellular chaperones DnaK, GroEL, Tig and YidC and the phage-chaperone gp40 were detected after formaldehyde crosslinking. Further studies of the cellular localization showed that gp20 and the fusion protein gp20-GFP are membrane-bound. The importance of YidC and DnaK for this membrane-association was demonstrated by fluorescence microscopy. Phage propagation was not affected by YidC depletion, whereas the loss of DnaK led to a reduced propagation. Prohead-structures, that are an intermediate stage of the capsid assembly, were isolated in YidC-free E. coli-cells membrane-unbound when infected with a phage mutant. Previous studies had led to the isolation of different amber mutants. Mutant amber20E481 was used in this thesis to analyze the assembly process in more detail. Here, under non-permissive conditions a 14 amino acid shortened protein, gp20s, is synthesized. Despite the fact that the capsid assembly is blocked in the non-suppressor strain, the localization and expression of the truncated protein was comparable to the wild type gp20. Overexpressed and His-tagged gp20s was found to crosslink with YidC, GroEL and gp40. Structural studies with a transmission electron microscope showed, that mature proheads were found and these were not filled with phage DNA. Most probably, a malfunction of gp20s during DNA packaging accounts for this.
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.