Browsing by Subject "YidC"

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Biogenese und Virusassembly des filamentösen Coliphagen M13
(2012) Ploß, Martin; Kuhn, Andreas
Taxonomically, the bacteriophage M13 is assigned to the single-stranded DNA phage and belongs to the family of Inoviridae. For propagation the Gram-negative bacteria Escherichia coli with F-pili is required. The host cell is not lysed by the phage. New findings about the M13 phage biogenesis are presented here within four essential areas of the M13 phage cycle concerning the sections infection, assembly, and phage secretion. Phage adsorption experiments in which the host bacterium E. coli K38 was infected by M13 phage showed that the phage adsorption to the cells takes place within the first 5 minutes and because of a limitation of F-pili per cell a maximum of 7 phages per cell were found to be adsorbed. The insertion of the phage coat protein gp9 into the cytoplasmic membrane of the host cell was verified by the periplasmic location of antigenic epitopes introduced into the N-terminal domain of gp9. The membrane insertion of gp9 was found to depend on the host protein YidC. Plasmid-encoded gp9 exhibiting antigenic epitopes at the N-terminal domain did not interfere with the assembly of new progeny phage. Therefore, the development of a phage display system with gp9 by introducing short peptide sequences (17 ? 36 amino acids) is feasible. After overexpression of gp1/11 assembly complexes in E. coli and size exclusion chromatography, respectively, the complex was characterized and a molecular weight of ~ 300 kDa was assigned. Examinations of the purified gp1/11 assembly complexes by transmission electron microscopy (TEM) revealed ring-like structures with ~ 7 ? 8 nm in inner diameter and ~ 11 ? 12 nm in outer diameter. The investigation of M13 wild-type infection showed that the secretion of new progeny phage starts after a short lag period (eclipse). An infected E. coli cell secreted upto 925 progeny in a time period of 115 minutes which corresponds to an average of 7 secreted phages per minute. The generation time of the infected E. coli K38 cells rose from 24 minutes to 48 minutes. Experiments were carried out with genetically manipulated phages which were hindered to synthesize the major coat protein gp8 in the host cell by a nonsense mutation in the phage genome. Therefore, phage replication was only observed in host cells bearing plasmid encoding gene 8. Since the quantity of the protein was limited the lag period (eclipse) was extended to 12 minutes and the efficiency of phage secretion was decreased to about 2 phages per minute. The M13 phage secretion from infected E. coli cells was visualized by atomic force microscopy (AFM). The identity of the phage was verified by labeling with protein-A conjugated gold and transmission electron microscopy (TEM). The secretion of M13 progeny was first observed at the cell poles of E. coli and then spreaded within 4 minutes along the cell surface. After 16 minutes the secretion was observed over the entire cell surface.
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.
Membraneinbau von MscL und MscL-Mutanten aus Escherichia coli
(2012) Neugebauer, Stella; Kuhn, Andreas
About one third of all synthesized proteins in a cell are membrane proteins. To accomplish their function, it is important to ensure, that they safely reach their destination, insert efficiently into the membrane, where they fold into their correct tertiary structure. Previous studies have shown that various molecules are responsible for the targeting and insertion of membrane proteins in Escherichia coli that operate as individual modules. The mechanosensitive channel MscL is a pentameric complex in the cytoplasmic membrane of E. coli. By its action as a safety valve, MscL allows the adaption to hypoosmotic conditions of bacteria living under varying circumstances. The two transmembrane segments of the MscL monomer are connected by a periplasmic loop of 29 amino acid residues. In previous studies, the membrane insertion of MscL was analyzed in vivo in depletion strains and was monitored by modification of a single cysteine residue in the periplasmic domain of the MscL protein (Facey et al., 2007). The targeting of MscL to the inner membrane occurs in a cotranslational manner via the signal recognition particle (SRP). At the membrane, the MscL protein inserts independently of the membrane potential and the Sec-components SecAYEG, but requires YidC for the insertion process. The present thesis is about the molecular mechanisms regarding the decision whether the nascent polypeptide chain of MscL is recognized and bound by YidC or by the Sec-translocase. The periplasmic localized loop of MscL was altered by introducing negatively or positively charged residues as well as uncharged side chains and the effects on the translocation were investigated. Translocation of the periplasmic domain of MscL was detected using AMS-derivatization (4-acetamido-4´-maleimidylstilbene-2, 2´-disulfonic acid) of a single cysteine residue. The extension of the loop region by one, two or three negatively charged residues (aspartic acid residues) made the insertion of MscL dependent on the membrane potential and the Sec translocon. The requirement of SecYE was gradually affected by increasing the number of charged residues. Efficient translocation of the periplasmic loop with three additional uncharged (asparagines) residues also required the Sec-complex. The insertion of these MscL mutants was independent on the SecA component, but all the investigated mutants still showed a strict dependence on YidC. The ability of the altered MscL proteins to form functional pentameric channels was verified by growth tests and native gel electrophoresis. The presence of three additional positively charged arginine residues in the periplasmic domain inhibited MscL insertion into the lipid bilayer as well as the mutant with five additional negatively charged aspartic acid residues. As a logical consequence, the expression of these two MscL proteins could not protect the cells from osmolysis within growth tests. The direct involvement of the membrane insertase YidC with MscL and the MscL mutants was corroborated with in vivo crosslinking. YidC interacts with both transmembrane regions of MscL. Earlier studies have shown that YidC makes contact with the Pf3 coat protein in the center of the membrane. Here, the same interaction sites of YidC were identified contacting MscL during its insertion. Besides considering the significance of YidC for efficient membrane insertion, the present work has demonstrated that YidC is also essential for oligomerization of MscL into a functional channel.
Membraninsertion des Phagenproteins M13 procoat in Lipidvesikel mit rekonstituiertem Escherichia coli YidC
(2011) Stiegler, Natalie; Kuhn, Andreas
Translocation of proteins across or into the cytoplasmic membrane of Escherichia coli is accomplished by several mechanisms. The cellular secretion machinery, the translocase SecYEG, mediates the transport of unfolded proteins into the periplasm with the help of the ATPase SecA or passes the membrane proteins for bilayer integration to the insertase YidC. Membrane insertion is catalysed by YidC, whereby the native conformation of the proteins in the lipid bilayer is achieved. The translocation of a few membrane proteins occurs Sec-independently solely with the help of the insertase YidC. One of these Sec-independent proteins is the major capsid protein of the bacteriophage M13. This protein is inserted as preprotein, termed M13 procoat, with the orientation Nin-Cin into the inner membrane and a central loop domain located in the periplasm. This process is catalysed by the electrochemical membrane potential and YidC. M13 procoat is then processed by the leader peptidase to its mature form, M13 coat (orientation Nout-Cin). In the present thesis an analysis of the different transport systems of the inner membrane is performed using the example of the M13 procoat protein and its mutants. One mutant is the procoat H5EE which has 2 additional acidic residues introduced between residues +2 and +3. The insertion of this mutant requires the Sec translocase and strictly depends on the electrochemical potential. Membrane insertion of M13 procoat and derived proteins into the cytoplasmic membrane was followed in an in vitro reconstitution and translocation system. Therefore, all components of the Sec translocase (SecYEG and SecA), the insertase YidC and the different procoat proteins were purified and tested with the in vitro translocation system. Reconstitution of YidC into phospholipid vesicles depended on the lipid composition for its orientation. The cytoplasmic-out orientation corresponds to the active topology in E. coli where both termini are located in the cytoplasm. Certain lipid compositions caused the inversed orientation, which affected the catalytic activity of the reconstituted insertase. The procoat mutants H5 und H5EE were membrane inserted only in the presence of reconstituted YidC. Both proteins inserted efficiently into the vesicles with the periplasmic loop in the interior of the vesicles like the mutant PClep of procoat H5 with the C-terminal extension of the leader peptidase. Spontaneous insertion of H5 und H5EE into liposomes occurred only into leaky vesicles of the E. coli lipids. The membrane integrity was improved by the addition of an adequate amount of diacylglycerol (DAG) to the phospholipids. The leaky phospholipids were sealed by the addition of 3-4% DAG. The proteins H5 und H5EE showed a dependency of the membrane potential. Insertion occured more efficiently into YidC proteoliposomes when a stable membrane potential was generated. Proteoliposomes with reconstituted SecYEG translocase were also tested for protein insertion. Remarkedly, the protein M13 procoat H5EE efficiently inserted into SecYEG proteoliposomes, where the wildtype-like protein H5 did not.
Molekulare Dynamik der YidC-Membraninsertase aus Escherichia coli
(2011) Imhof, Nora; Kuhn, Andreas
The membrane insertase YidC of the Gram-negative bacterium E. coli enables the insertion of proteins into the cytoplasmic membrane. YidC itself is localized in the cytoplasmic membrane and spans the membrane six times with its N- and C-termini localized in the cytoplasm. These six transmembrane segments are connected by three periplasmic loops (P1, P2 and P3) and two cytoplasmic loops (C1 and C2). It is known that the binding of the YidC-dependent protein Pf3 coat induces conformational changes in the tertiary structure of YidC. This molecular dynamic of YidC was examined in detail with steady-state and time-resolved fluorescence spectroscopy. Therefore, three tryptophan mutants of YidC with one tryptophan residue each, at position 354 in the first periplasmic domain P1, at position 454 in the second periplasmic region and at position 508 near the third periplasmic region, respectively, were used. Additionally, a double tryptophan mutant was used which contained two tryptophan residues at position 332/334 of the domain P1. These tryptophan residues were used as intrinsic fluorophores. First, it was shown that the tryptophan mutants of YidC complemented the growth defect of the E. coli YidC-depletion strain JS7131. Additionally, the mutants were able to insert the strictly YidC-dependent PClep protein into the cytoplasmic membrane of the depletion strain. Thus, the functionality of the tryptophan mutants of YidC was ensured. Purified tryptophan mutants of YidC were reconstituted into liposomes and titrated with Pf3W0 coat, a tryptophan free mutant of Pf3 coat protein allowing spectroscopic studies of each periplasmic region (P1, P2 and P3) before and after binding of Pf3W0 coat protein. Analysis of the emission spectra and the fluorescence lifetimes of detergent solubilized as well as of the reconstituted YidC tryptophan mutants before binding of Pf3W0 coat revealed that the tryptophan residue of each single tryptophan mutant (YidCW354, YidCW454 and YidCW508) was localized at the membrane/water interface. These results are consistent with the proposed membrane topology of YidC. The tryptophan residues of the double tryptophan mutant of YidC (YidC2W) showed fluorescence properties consistent with their localization in a partially exposed alpha-helical segment of the P1 domain. Analysis of the emission spectra and the fluorescence lifetimes provided additional evidence that binding of Pf3W0 coat induced conformational changes of all periplasmic regions (P1, P2 and P3) within YidC. Measurements of fluorescence anisotropy showed that the conformational changes affected motions within all three periplasmic regions of the YidC tryptophan mutants, whereas the periplasmic domain P1 with the tryptophan residues W332/W334 and the third periplasmic domain P3 with the tryptophan residue W508 were affected most significantly.
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.
Tth-IM60, eine Membraninsertase aus Thermus thermophilus
(2011) Meyer, Susanne H.; Kuhn, Andreas
The evolutionarily conserved YidC/Oxa1/Alb3 family of proteins catalyzes the insertion of integral membrane proteins in bacteria, mitochondria, and chloroplasts. In this work Tth-IM60 from Thermus thermophilus was identified as a member of this family. The function and structure of the protein was analysed in detail. Complementation studies in a Escherichia coli YidC-depletion strain show a functional replacement of the essential YidC by Tth-IM60 in vivo. A heterologous expression of the his-tagged Tth-IM60 protein was achieved in the E. coli strain C43 and pMS as a plasmid vector. It was shown that Tth-IM60 protein is located in the inner membrane probably in a dimeric state. After purification the protein tends to oligomerize in a higher, but very stable oligomeric state. The Tth-IM60 oligomeric protein was stable for 50 days at least. By size-exclusion chromatography the zwitterionic detergent LDAO and a buffer containing 20 mM TrisHCl pH 8,5, 500 mM NaCl and 10 % glycerol were identified as the best conditions for purification and stability. With this buffer, Tth-IM60 was purified as a dimer via its C-terminal histag by two Ni-IMACs (immobilized metal affinity chromatography) and a size-exclusion chromatography. A final protein concentration up to 10 mg/ml was feasible. The purified Tth-IM60 protein was used for functional and structural studies. A weak binding of Tth-IM60 to the first periplasmic loop of SecF as shown by pulldown assays, suggests a Sec-dependent function of Tth-IM60. In addition, the essential Sec-independent function of Tth-IM60 was demonstrated by the translocation of the YidC substrate Pf3 coat into Tth-IM60 proteoliposomes. Moreover, the results of the single molecule spectroscopy measurements implicate that the translocation of Pf3 coat proteins occurs with a fast kinetics within 5 minutes. The secondary structure of the Tth-IM60 protein was analysed by circular dichroism spectroscopy: 49 ? 55 % alpha-helical, 13 - 14 % beta-sheet, 14 ? 17 % beta-turns and 18 - 21% unordered structures were calculated. Tth-IM60 comprises more alpha-helical structures, but less beta-sheets than YidC, probably because of the first periplasmic loop of Tth-IM60 being shorter. The melting point of Tth-IM60 was determined to 68 °C, which is 10 degrees higher than the melting point of YidC. Furthermore, the Tth-IM60 protein was crystallized and X-ray analysis was performed. However, due to the low resolution the structure of the Tth-IM60 protein could not be determined so far. The second part of this thesis concerns the ?translocating chain-associated membrane? (TRAM) protein. The TRAM protein is involved in the insertion of integral membrane proteins of the endoplasmic reticulum (ER) together with the Sec61-complex. In contrast to the Sec-components, no homologous YidC protein exists in the ER-membrane. Therefore, it was postulated that TRAM and YidC could have functional similarities. For functional studies the TRAM protein from X. laevis was expressed in E. coli as a fusion protein with a N-terminal MBP (maltose-binding protein) and a C-terminal histag. TRAM was purified via two different affinity matrices: Ni sepharose and an amylose resin. It was possible to reconstitute the fusion protein into liposomes. However a translocation of the YidC-substrate Pf3 coat into these proteoliposomes was not detectable. In addition, complementation studies with a YidC-depletion strain did not show that the essential YidC function can be replaced by TRAM in vivo.