Browsing by Subject "Pf3 coat"

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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.
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.
Untersuchungen zur autonomen und YidC-vermittelten Membraninsertion von Pf3 coat-Protein mit Hilfe Fluoreszenz-spektroskopischer Einzelmolekülmessungen
(2011) Schönbauer, Anne-Kathrin; Kuhn, Andreas
Pf3 coat is the capsid protein of the bacteriophage Pf3. The phage leaves the host cell by continuous extrusion without damaging the cell. The protein itself consists of 44 amino acid residues and has a rod-like shape. Because of its simple structure, the protein needs only the help of the insertase YidC to insert into the bacterial inner membrane. 3L-Pf3 coat, a protein mutant with three additional leucine residues in the center of the transmembrane region (TMD), has an increased hydrophobicity. It is independent of YidC and inserts into the membrane autonomously (Serek et al., 2004). In this work, a newly developed physical method was used to find out whether the elongation or the increased hydrophobicity accounts for the autonomous insertion of the protein. For this reason, two new protein mutants were constructed. Each mutant has only one of the changed properties of the 3L-Pf3 coat protein: GAT-Pf3 coat has an elongated TMD with three additional residues (glycine, alanin and threonine). The second mutant, 2M-Pf3 coat, shows an increased hydrophobicity due to the substitution of two alanine residues by two methionine residues at the positions 30 and 31. So it had an increased hydrophobicity like 3L-Pf3 coat. The above mentioned proteins, wt-Pf3 coat and its mutants, were modified with a fluorescent label to follow the proteins with optical methods. The Proteins were first modified with a single cysteine and then labeled by a fluorescent marker, Atto520 maleimid. Proteins with a labeled N-terminal tail were called NC-Pf3 coat, whereas CC-Pf3 coat had a labeled C-terminal tail. In addition, the orientation of the protein in the membrane was identified by quenching the fluorescence of the NC- and CC- labeled proteins. A new method employing single molecules was developed using fluorescence correlation spectroscopy. This method allows real time observations of binding and insertion of the protein into semisynthetical liposomes. By using fluorescent quenching the membrane insertion and binding were distinguished. It became clear that both the elongation of the TMD as well as an increased hydrophobicity play a crucial role in the autonomous insertion of the protein into the membrane. Therefore, the interaction between the hydrophobic region of the protein and the hydrophobic core region of the membrane is important for the binding of the protein and its insertion into the membrane.