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The interactions of outer membrane proteins with the periplasmic chaperone Skp of E.coli and with LPS

The interactions of outer membrane proteins with the periplasmic chaperone Skp of E.coli and with LPS

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QU, Jian, 2007. The interactions of outer membrane proteins with the periplasmic chaperone Skp of E.coli and with LPS

@phdthesis{Qu2007inter-6654, title={The interactions of outer membrane proteins with the periplasmic chaperone Skp of E.coli and with LPS}, year={2007}, author={Qu, Jian}, address={Konstanz}, school={Universität Konstanz} }

Qu, Jian eng application/pdf 2011-03-24T17:28:05Z 2011-03-24T17:28:05Z Die Wechselwirkungen von Außenmembranproteinen mit der periplasmatischen Chaperone Skp aus E. coli und Lipopolysaccharid 2007 The interactions of outer membrane proteins with the periplasmic chaperone Skp of E.coli and with LPS deposit-license This thesis describes several important advancements in the study of outer membrane proteins (OMPs) interacting with Skp and LPS. In the first study, we characterized the complexes that Skp forms with OMPs and how LPS modulates the surface exposure of Skp-bound OmpA. We used tryptophan fluorescence spectroscopy to study the interactions of wild-type Skp, which is devoid of tryptophan, with several OMPs, namely OmpA, OmpG, and YaeT from E. coli, NalP from Neisseria meningitides, FomA from Fusobacterium nucleatum, and hVDAC1 from human mitochondrial OMs. The fluorescence spectroscopic analysis revealed: (1) The Skp trimer binds the bacterial OMPs independent of the bacterium, but not the mitochondrial hVDAC1. (2) The Skp trimer forms stable complexes with YaeT, OmpG, OmpA and NalP at a 1:1 stoichiometry with nanomolar affinity. (3) Skp binding to OMPs is pH-dependent and OMPs did not display binding with Skp at extreme pH values of ~4 and of ~11. Light scattering experiments indicate that Skp forms a stable trimer from pH 3 to pH 11, indicating an essential electrostatic component in binding of OMPs to Skp. (4) The free energy of binding of Skp to OMP was reduced by ~4 kJ/mol at high salt concentration. This reduction indicates that electrostatic interactions of oppositely charged amino acid residues in OMPs and in Skp contribute to binding. The pH and the salt dependencies of OmpA binding to Skp indicated that complexes are kept together by both hydrophobic and electrostatic forces. (5) Skp efficiently shielded the tryptophans of bound OmpA and the addition of LPS weakened the shielding effect. The LPS binding experiment indicated that LPS modulates the conformation of the OmpA Skp3 complex, partially exposing the fluorescent tryptophans to a more polar environment.<br />In a second study, the interactions of 13 single tryptophan mutants of OmpA with the chaperone Skp and with LPS were studied in detail by fluorescence spectroscopy. This study led to the following interesting results: (1) For the first time, the topology of the aqueous folding intermediate of OmpA was described on the level of single amino acid residues. The loops and turns of OmpA are more surface-oriented than β-strands. The periplasmic domain is an autonomous folding unit. The location of this fluorescence maximum is not altered upon Skp binding. (2) Skp binds the entire transmembrane domain of OmpA by multivalent interaction. (3) At low LPS/OmpA ratios of 5 mol/mol, the fluorescence spectra of the single tryptophan mutants of OmpA showed only small changes, with Stokes-shifts of about 1.5 and 2 nm for tryptophan in loops 1 and 3. All other mutants did not exhibit any significant changes in the fluorescence spectra. (4) LPS binding to complexes of Skp and OmpA led to a conformational change: the periplasmic β-turns of OmpA are in the most hydrophobic environment, while the loops are in polar environment. The interaction of both, Skp and LPS, gives OmpA a preferred direction, explaining that both Skp and LPS are necessary to facilitate membrane insertion and folding of OmpA into lipid bilayers. (5) OmpA did not develop native structure, neither in binary complexes with Skp nor in ternary complexes with both, Skp and LPS. Membrane insertion is a requirement for folding of OmpA.<br />In the third chapter, I describe work performed in collaboration with another research group for which I provided the membrane proteins. In this project, polarised attenuated total internal reflection Fourier-transform infrared spectroscopy (ATR-FTIR) was used to determine the orientation of the β-barrels in phosphatidylcholine host matrices of different lipid chainlengths. The linear dichroism of the amide I band from OmpA and FhuA in hydrated membranes generally increased with increasing chain length from diC12:0PC to diC17:0PC phosphatidylcholine, in both the fluid and gel phases. Measurements of the dichroisms of the amide I and amide II bands from dry samples were used to deduce the strand tilt (β = 46° for OmpA, and β = 44.5° for FhuA). These values were then used to deduce the order parameters, , of the β-barrels from the amide I dichroic ratios of the hydrated membranes. The orientational ordering of the β-barrels, and their assembly in the membrane, are discussed in terms of hydrophobic matching with the lipid chains. Qu, Jian

Dateiabrufe seit 01.10.2014 (Informationen über die Zugriffsstatistik)

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