Search

The recently solved crystal structure of YidC protein suggests that it mediates membrane protein insertion by means of an intramembrane cavity rather than a transmembrane (TM) pore. This concept of protein translocation prompted us to characterize the native, membrane-integrated state of YidC with respect to the hydropathic nature of its TM region. Here, we show that the cavity-forming region of the stage III sporulation protein J (SpoIIIJ), a YidC homolog, is indeed open to the aqueous milieu of the Bacillus subtilis cells and that the overall hydrophilicity of the cavity, along with the presence of an Arg residue on several alternative sites of the cavity surface, is functionally important. We propose that YidC functions as a proteinaceous amphiphile that interacts with newly synthesized membrane proteins and reduces energetic costs of their membrane traversal.

YidC, a member of the YidC/Oxa1/Alb3 family, inserts proteins into the membrane and facilitates membrane-protein folding in bacteria. YidC plays key roles in both Sec-mediated integration and Sec-independent insertion of membrane proteins. Here, {\it Bacillus halodurans} YidC2, which has five transmembrane helices conserved among the other family members, was identified as a target protein for structure determination by a fluorescent size-exclusion chromatography analysis. The protein was overexpressed, purified and crystallized in the lipidic cubic phase. The crystals diffracted X-rays to 2.4{\AA} resolution and belonged to space group {\it P}2${\sb 1}$, with unit-cell parameters {\it a} = 43.9, {\it b} = 60.6, {\it c} = 58.9{\AA}, {$\beta$} = 100.3{$^\circ$}. The experimental phases were determined by the multiwavelength anomalous diffraction method using a mercury-derivatized crystal.

The YidC family of proteins are membrane insertases that catalyze the translocation of the periplasmic domain of membrane proteins via a hydrophilic groove located within the inner leaflet of the membrane. All homologs have a strictly conserved, positively charged residue in the center of this groove. In Bacillus subtilis, the positively charged residue has been proposed to be essential for interacting with negatively charged residues of the substrate, supporting a hypothesis that YidC catalyzes insertion via an early-step electrostatic attraction mechanism. Here, we provide data suggesting that the positively charged residue is important not for its charge but for increasing the hydrophilicity of the groove. We found that the positively charged residue is dispensable for Escherichia coli YidC function when an adjacent residue at position 517 was hydrophilic or aromatic, but was essential when the adjacent residue was apolar. Additionally, solvent accessibility studies support the idea that the conserved positively charged residue functions to keep the top and middle of the groove sufficiently hydrated. Moreover, we demonstrate that both the E. coli and Streptococcus mutans YidC homologs are functional when the strictly conserved arginine is replaced with a negatively charged residue, provided proper stabilization from neighboring residues. These combined results show that the positively charged residue functions to maintain a hydrophilic microenvironment in the groove necessary for the insertase activity, rather than to form electrostatic interactions with the substrates.

The YidC family of proteins are membrane insertases that catalyze the translocation of the periplasmic domain of membrane proteins via a hydrophilic groove located within the inner leaflet of the membrane. All homologs have a strictly conserved, positively charged residue in the center of this groove. In Bacillus subtilis, the positively charged residue has been proposed to be essential for interacting with negatively charged residues of the substrate, supporting a hypothesis that YidC catalyzes insertion via an early-step electrostatic attraction mechanism. Here, we provide data suggesting that the positively charged residue is important not for its charge but for increasing the hydrophilicity of the groove. We found that the positively charged residue is dispensable for Escherichia coli YidC function when an adjacent residue at position 517 was hydrophilic or aromatic, but was essential when the adjacent residue was apolar. Additionally, solvent accessibility studies support the idea that the conserved positively charged residue functions to keep the top and middle of the groove sufficiently hydrated. Moreover, we demonstrate that both the E. coli and Streptococcus mutans YidC homologs are functional when the strictly conserved arginine is replaced with a negatively charged residue, provided proper stabilization from neighboring residues. These combined results show that the positively charged residue functions to maintain a hydrophilic microenvironment in the groove necessary for the insertase activity, rather than to form electrostatic interactions with the substrates.

Recently, we engineered a tunable rhamnose promoter-based setup for the production of recombinant proteins in E. coli. This setup enabled us to show that being able to precisely set the production rate of a secretory recombinant protein is critical to enhance protein production yields in the periplasm. It is assumed that precisely setting the production rate of a secretory recombinant protein is required to harmonize its production rate with the protein translocation capacity of the cell. Here, using proteome analysis we show that enhancing periplasmic production of human Growth Hormone (hGH) using the tunable rhamnose promoter-based setup is accompanied by increased accumulation levels of at least three key players in protein translocation; the peripheral motor of the Sec-translocon (SecA), leader peptidase (LepB), and the cytoplasmic membrane protein integrase/chaperone (YidC). Thus, enhancing periplasmic hGH production leads to increased Sec-translocon capacity, increased capacity to cleave signal peptides from secretory proteins and an increased capacity of an alternative membrane protein biogenesis pathway, which frees up Sec-translocon capacity for protein secretion. When cells with enhanced periplasmic hGH production yields were harvested and subsequently cultured in the absence of inducer, SecA, LepB, and YidC levels went down again. This indicates that when using the tunable rhamnose-promoter system to enhance the production of a protein in the periplasm, E. coli can adapt its protein translocation machinery for enhanced recombinant protein production in the periplasm.

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) disul, Die YidC/Oxa1/Alb3 Proteinfamilie besteht aus Insertase-Homologen, die die Insertion und Faltung von Membranproteinen vermittelt. YidC ist dabei in der inneren Zellmembran von Bakterien lokalisiert. Oxa1 befindet sich in der inneren Membran von Mitochondrien und Alb3 vermittelt die Insertion von Membranproteinen in der Thylakoidmembran von Chloroplasten (Wang und Dalbey 2011, Hennon et al. 2015). Ein weiteres Homolog wurde im Archaeum M. jannaschii gefunden wodurch gezeigt werden konnte, dass diese Insertasefamilie in allen Domänen des Lebens konserviert ist (Dalbey und Kuhn 2015). Alle Homologe besitzen eine hydrophile Kavität als gemeinsames Strukturmerkmal. Diese Kavität ist zum Zytoplasma und zum hydrophoben Membrankern geöffnet und besitzt eine hydrophobe „Rutsche“ die von den Transmembrandomänen 3 und 5 gebildet wird. YidC ist in der Lage alleine kleine Proteine zu inserieren kann aber auch mit dem Sec Translokon zusammenarbeiten um die Insertion von großen Membranproteinen zu ermöglichen. Das Haupthüllprotein des M13 Bakteriophagen ist wiederum ein Substrat, das von YidC allein inseriert wird. Ziele dieser Arbeit sind die Untersuchung des Insertionsmechanismus von M13 Procoat, dem Haupthüllprotein des M13 Bakteriophagen, sowie des oligomeren Zustands des aktiven YidCs. Um die Interaktion zwischen YidC und M13 Procoat zu untersuchen wurden Disulfid-Crosslinkingexperimente mit radioaktiver Markierung der Proteine durchgeführt. Dabei kam fast ausschließlich Kupferphenanthrolin als Oxidationsmittel zum Einsatz. Es konnte gezeigt werden, dass M13 Procoat dabei sehr häufig mit den Transmembrandomänen 3 und 5 von YidC in Kontakt kommt. Die beobachteten Kontaktprofile deuten dabei auf ein „Gleiten“ von M13 Procoat entlang der hydrophoben „Rutsche“ in YidC hin. Zusätzlich durchgeführte Crosslinkingexperimente wurden in der hydrophilen Kavität und mit der C1 Schlaufe von YidC durchgeführt. Ein gefundener Kontakt auf der C1 Schlaufe zeigt, dass diese eine Rolle im Inser

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) disul, Die YidC/Oxa1/Alb3 Proteinfamilie besteht aus Insertase-Homologen, die die Insertion und Faltung von Membranproteinen vermittelt. YidC ist dabei in der inneren Zellmembran von Bakterien lokalisiert. Oxa1 befindet sich in der inneren Membran von Mitochondrien und Alb3 vermittelt die Insertion von Membranproteinen in der Thylakoidmembran von Chloroplasten (Wang und Dalbey 2011, Hennon et al. 2015). Ein weiteres Homolog wurde im Archaeum M. jannaschii gefunden wodurch gezeigt werden konnte, dass diese Insertasefamilie in allen Domänen des Lebens konserviert ist (Dalbey und Kuhn 2015). Alle Homologe besitzen eine hydrophile Kavität als gemeinsames Strukturmerkmal. Diese Kavität ist zum Zytoplasma und zum hydrophoben Membrankern geöffnet und besitzt eine hydrophobe „Rutsche“ die von den Transmembrandomänen 3 und 5 gebildet wird. YidC ist in der Lage alleine kleine Proteine zu inserieren kann aber auch mit dem Sec Translokon zusammenarbeiten um die Insertion von großen Membranproteinen zu ermöglichen. Das Haupthüllprotein des M13 Bakteriophagen ist wiederum ein Substrat, das von YidC allein inseriert wird. Ziele dieser Arbeit sind die Untersuchung des Insertionsmechanismus von M13 Procoat, dem Haupthüllprotein des M13 Bakteriophagen, sowie des oligomeren Zustands des aktiven YidCs. Um die Interaktion zwischen YidC und M13 Procoat zu untersuchen wurden Disulfid-Crosslinkingexperimente mit radioaktiver Markierung der Proteine durchgeführt. Dabei kam fast ausschließlich Kupferphenanthrolin als Oxidationsmittel zum Einsatz. Es konnte gezeigt werden, dass M13 Procoat dabei sehr häufig mit den Transmembrandomänen 3 und 5 von YidC in Kontakt kommt. Die beobachteten Kontaktprofile deuten dabei auf ein „Gleiten“ von M13 Procoat entlang der hydrophoben „Rutsche“ in YidC hin. Zusätzlich durchgeführte Crosslinkingexperimente wurden in der hydrophilen Kavität und mit der C1 Schlaufe von YidC durchgeführt. Ein gefundener Kontakt auf der C1 Schlaufe zeigt, dass diese eine Rolle im Inser

The YidC/Oxa1/Alb3 family of membrane proteins function to insert proteins into membranes in bacteria, mitochondria, and chloroplasts. Recent X-ray structures of YidC from Bacillus halodurans and Escherichia coli revealed a hydrophilic groove that is accessible from the lipid bilayer and the cytoplasm. Here, we explore the water accessibility within the conserved core region of the E. coli YidC using in vivo cysteine alkylation scanning and molecular dynamics (MD) simulations of YidC in POPE/POPG membranes. As expected from the structure, YidC possesses an aqueous membrane cavity localized to the membrane inner leaflet. Both the scanning data and the MD simulations show that the lipid-exposed transmembrane helices 3, 4, and 5 are short, leading to membrane thinning around YidC. Close examination of the MD data reveals previously unrecognized structural features that are likely important for protein stability and function.

application/pdf, The recently solved crystal structure of YidC protein suggests that it mediates membrane protein insertion by means of an intramembrane cavity rather than a transmembrane (TM) pore. This concept of protein translocation prompted us to characterize the native, membrane-integrated state of YidC with respect to the hydropathic nature of its TM region. Here, we show that the cavity-forming region of the stage III sporulation protein J (SpoIIIJ), a YidC homolog, is indeed open to the aqueous milieu of the Bacillus subtilis cells and that the overall hydrophilicity of the cavity, along with the presence of an Arg residue on several alternative sites of the cavity surface, is functionally important. We propose that YidC functions as a proteinaceous amphiphile that interacts with newly synthesized membrane proteins and reduces energetic costs of their membrane traversal.

The recently solved crystal structure of YidC protein suggests that it mediates membrane protein insertion by means of an intramembrane cavity rather than a transmembrane (TM) pore. This concept of protein translocation prompted us to characterize the native, membrane-integrated state of YidC with respect to the hydropathic nature of its TM region. Here, we show that the cavity-forming region of the stage III sporulation protein J (SpoIIIJ), a YidC homolog, is indeed open to the aqueous milieu of the Bacillus subtilis cells and that the overall hydrophilicity of the cavity, along with the presence of an Arg residue on several alternative sites of the cavity surface, is functionally important. We propose that YidC functions as a proteinaceous amphiphile that interacts with newly synthesized membrane proteins and reduces energetic costs of their membrane traversal., journal article

application/pdf, YidC, a member of the YidC/Oxa1/Alb3 family, inserts proteins into the membrane and facilitates membrane-protein folding in bacteria. YidC plays key roles in both Sec-mediated integration and Sec-independent insertion of membrane proteins. Here, {\it Bacillus halodurans} YidC2, which has five transmembrane helices conserved among the other family members, was identified as a target protein for structure determination by a fluorescent size-exclusion chromatography analysis. The protein was overexpressed, purified and crystallized in the lipidic cubic phase. The crystals diffracted X-rays to 2.4{\AA} resolution and belonged to space group {\it P}2${\sb 1}$, with unit-cell parameters {\it a} = 43.9, {\it b} = 60.6, {\it c} = 58.9{\AA}, {$\beta$} = 100.3{$^\circ$}. The experimental phases were determined by the multiwavelength anomalous diffraction method using a mercury-derivatized crystal.

YidC, a member of the YidC/Oxa1/Alb3 family, inserts proteins into the membrane and facilitates membrane-protein folding in bacteria. YidC plays key roles in both Sec-mediated integration and Sec-independent insertion of membrane proteins. Here, {\it Bacillus halodurans} YidC2, which has five transmembrane helices conserved among the other family members, was identified as a target protein for structure determination by a fluorescent size-exclusion chromatography analysis. The protein was overexpressed, purified and crystallized in the lipidic cubic phase. The crystals diffracted X-rays to 2.4{\AA} resolution and belonged to space group {\it P}2${\sb 1}$, with unit-cell parameters {\it a} = 43.9, {\it b} = 60.6, {\it c} = 58.9{\AA}, {$\beta$} = 100.3{$^\circ$}. The experimental phases were determined by the multiwavelength anomalous diffraction method using a mercury-derivatized crystal., journal article

To understand how YidC and SecYEG function together in membrane protein topogenesis, insertion and folding of the lactose permease of Escherichia coli (LacY), a 12-transmembrane helix protein LacY that catalyzes symport of a galactoside and an H(+), was studied. Although both the SecYEG machinery and signal recognition particle are required for insertion of LacY into the membrane, YidC is not required for translocation of the six periplasmic loops in LacY. Rather, YidC acts as a chaperone, facilitating LacY folding. Upon YidC depletion, the conformation of LacY is perturbed, as judged by monoclonal antibody binding studies and by in vivo cross-linking between introduced Cys pairs. Disulfide cross-linking also demonstrates that YidC interacts with multiple transmembrane segments of LacY during membrane biogenesis. Moreover, YidC is strictly required for insertion of M13 procoat protein fused into the middle cytoplasmic loop of LacY. In contrast, the loops preceding and following the inserted procoat domain are dependent on SecYEG for insertion. These studies demonstrate close cooperation between the two complexes in membrane biogenesis and that YidC functions primarily as a foldase for LacY.

To understand how YidC and SecYEG function together in membrane protein topogenesis, insertion and folding of the lactose permease of Escherichia coli (LacY), a 12-transmembrane helix protein LacY that catalyzes symport of a galactoside and an H(+), was studied. Although both the SecYEG machinery and signal recognition particle are required for insertion of LacY into the membrane, YidC is not required for translocation of the six periplasmic loops in LacY. Rather, YidC acts as a chaperone, facilitating LacY folding. Upon YidC depletion, the conformation of LacY is perturbed, as judged by monoclonal antibody binding studies and by in vivo cross-linking between introduced Cys pairs. Disulfide cross-linking also demonstrates that YidC interacts with multiple transmembrane segments of LacY during membrane biogenesis. Moreover, YidC is strictly required for insertion of M13 procoat protein fused into the middle cytoplasmic loop of LacY. In contrast, the loops preceding and following the inserted procoat domain are dependent on SecYEG for insertion. These studies demonstrate close cooperation between the two complexes in membrane biogenesis and that YidC functions primarily as a foldase for LacY.

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 loc

Die Translokation von Proteinen über und in die Cytoplasmamembran von Escherichia coli kann über verschiedene Mechanismen bewerkstelligt werden. Die zelluläre Sekretionsmaschinerie, die Translokase SecYEG, transportiert mithilfe der ATPase SecA große ungefaltete Proteine ins Periplasma oder leitet Membranproteine an die Insertase YidC weiter. Die Membraninsertion wird von YidC katalysiert, wodurch die native Konformation der Proteine in der Lipid-doppelschicht erreicht wird. Die Translokation einiger weniger Membranproteine verläuft Sec-unabhängig nur mithilfe der Insertase YidC. Eines dieser Sec-unabhängigen Proteine stellt das Haupthüllprotein des Bakteriophagens M13 dar. Dieses Protein wird als Präprotein, genannt M13 procoat, mit der Orientierung Nin-Cin in die innere Membran inseriert und besitzt eine zentrale Loop-Domäne im Periplasma. Dieser Vorgang wird vom elektrochemischen Membran-potential und YidC katalysiert. Anschließend wird das M13 procoat durch die Leaderpeptidase zu seiner maturen Form, M13 coat (Orientierung Nout-Cin), prozessiert. Die vorliegende Arbeit befasst sich anhand des Modellproteins M13 procoat und M13 procoat-Mutanten mit der Analyse der verschiedenen Transportsysteme der inneren Membran. Eine Mutante ist das Procoat H5EE, welches zwei zusätzliche saure Aminosäurereste zwischen Position +2 und +3 besitzt. Die Insertion dieser Mutante benötigt die Sec-Translokase und ist strikt vom Membranpotential abhängig. Die Membraninsertion von M13 procoat und abgeleiteten Proteinen in die cytoplasmatische Membran wurde in einem in vitro-Rekonstitutions- und Translokationssystem untersucht. Hierfür wurden die einzelnen Komponenten der Sec-Translokase (SecYEG und SecA), die Insertase YidC, sowie die verschiedenen Procoatproteine gereinigt und in dem in vitro-Translokationssystem eingesetzt. Die Rekonstitution von YidC in Phospholipidvesikel erfolgte abhängig von der Lipid-zusammensetzung der Membran. Die cytoplasmic-out-Orientierung entspricht der ak, 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. Ce

Mit Hilfe der Fluoreszenzspektroskopie konnte die Insertion von Pf3 coat Protein in YidC Proteoliposomen näher charakterisiert werden. Somit wurde die Bindung mittels Fluoreszenztitration untersucht und mit Hilfe der Einzelmolekülspektroskopie konnte der Insertionsvorgang vom Pf3 coat Protein mit FRET analysiert werden. Auf diese Weise wurden die Abstände der Proteine bestimmt und der zeitliche Verlauf der Insertion beobachtet., 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.

In the bacterium Escherichia coli all proteins are synthesized in the cytoplasm. However, at least 25% of them then need to be exported to another cellular compartment where they exert their function. E. coli has several different targeting pathways to ensure the correct localization of its proteins. When a nascent chain emerges at the ribosomal tunnel exit the signal recognition particle (SRP) can bind to it. The ribosomal nascent chain -SRP- complex is then targeted either to the Sec-translocon or the YidC insertase at the cytoplasmic membrane. Integral cytoplasmic membrane proteins are inserted into the lipid bilayer whilst periplasmic and outer membrane proteins are translocated across the membrane. In order to be fully functional, proteins need to be both correctly localized and folded. In many cases, they also assemble into complexes with other proteins or co-factors. In my thesis I will present an improved protocol for two-dimensional blue native/SDS-PAGE (2D BN/SDS-PAGE) that makes it very suitable to study the biogenesis of integral cytoplasmic membrane proteins and especially the complexes in the cytoplasmic membrane. Using this and other methods I have studied the biogenesis of integral cytoplasmic membrane proteins and other exported proteins. This has been done on a global scale in mutant strains where the SRP-targeting pathway, the Sec-translocon and the integral cytoplasmic membrane chaperone/insertase YidC have been compromised.