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Structural insights into Omp85-mediated protein translocation and insertion in the bacterial outer membrane

Gruss, Fabian. Structural insights into Omp85-mediated protein translocation and insertion in the bacterial outer membrane. 2015, PhD Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_11514

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Abstract

Proteins of the Omp85 superfamily reside in the outer membranes of Gram-negative bacteria, mitochondria and chloroplasts and are responsible for the insertion of outer membrane β-barrel proteins into or the translocation of soluble proteins across the membrane. They contain a C-terminal membrane-embedded 16-stranded β-barrel and soluble substrate-interacting POTRA domains, which in Gram-negative bacteria locate to the periplasm. The underlying translocation and insertion mechanisms are poorly understood and atomic structures of Omp85 insertases have been missing.
This thesis provides the structural basis for the insertion mechanism of substrates by the Escherichia coli Omp85 insertase TamA. The crystal structure of TamA reveals minimal interactions between the first and the last β-strand of the barrel with a lipid-occupied lateral gate, suggesting substrate β-barrel assembly via hybrid barrel formation and lateral release. Exemplified by the crystallization of TamA, a general crystal seeding protocol for optimization of membrane protein crystals grown from bicelle solution is described. Furthermore, interactions of TamA with its associated periplasmic complex partner TamB are investigated by experimental approaches and bioinformatics, revealing potential interaction sites between these two proteins. The organization of Omp85 insertases is then compared to Omp85 translocases, represented by FhaC from Bordetella pertussis, and a mechanism for substrate selection by FhaC is deduced from a newly determined crystal structure of an FhaC double mutant defective in substrate recognition.
Whereas protein import into chloroplasts is mediated by a member of the Omp85 superfamily, in mitochondria this task is fulfilled by Tom40, a 19-stranded β-barrel outer membrane protein that lacks POTRA domains. As a basis for experimental in vitro approaches to gaining insights into the Tom40 translocation mechanism, a protocol for recombinant Tom40 over-expression, refolding and sample preparation is provided. NMR spectroscopy of isotope-labeled protein evidences the presence of folded Tom40 in our samples.
Advisors:Maier, Timm
Committee Members:Spiess, Martin
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Structural Biology & Biophysics > Structural Biology (Maier)
Item Type:Thesis
Thesis no:11514
Bibsysno:Link to catalogue
Number of Pages:143 S.
Language:English
Identification Number:
Last Modified:30 Jun 2016 10:59
Deposited On:07 Dec 2015 14:10

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