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Purification, crystallization and characterization of the Pseudomonas outer membrane protein FapF, a functional amyloid transporter
journal contribution
posted on 2023-06-09, 08:01 authored by S L Rouse, W J Hawthorne, S Lambert, M L Morgan, Stephen Hare, S MatthewsBacteria often produce extracellular amyloid fibres via a multi-component secretion system. Aggregation-prone, unstructured subunits cross the periplasm and are secreted through the outer membrane, after which they self-assemble. Here, significant progress is presented towards solving the high-resolution crystal structure of the novel amyloid transporter FapF from Pseudomonas, which facilitates the secretion of the amyloid-forming polypeptide FapC across the bacterial outer membrane. This represents the first step towards obtaining structural insight into the products of the Pseudomonas fap operon. Initial attempts at crystallizing full-length and N-terminally truncated constructs by refolding techniques were not successful; however, after preparing FapF106-430 from the membrane fraction, reproducible crystals were obtained using the sitting-drop method of vapour diffusion. Diffraction data have been processed to 2.5 Å resolution. These crystals belonged to the monoclinic space group C121, with unit-cell parameters a = 143.4, b = 124.6, c = 80.4 Å, [alpha] = [gamma] = 90, [beta] = 96.32° and three monomers in the asymmetric unit. It was found that the switch to complete detergent exchange into C8E4 was crucial for forming well diffracting crystals, and it is suggested that this combined with limited proteolysis is a potentially useful protocol for membrane [beta]-barrel protein crystallography. The three-dimensional structure of FapF will provide invaluable information on the mechanistic differences of biogenesis between the curli and Fap functional amyloid systems.
History
Publication status
- Published
File Version
- Published version
Journal
Acta Crystallographica Section F: Structural Biology CommunicationsISSN
2053-230XPublisher
International Union of CrystallographyExternal DOI
Issue
12Volume
72Page range
892-896Department affiliated with
- Biochemistry Publications
Full text available
- Yes
Peer reviewed?
- Yes
Legacy Posted Date
2017-09-21First Open Access (FOA) Date
2017-09-21First Compliant Deposit (FCD) Date
2017-09-21Usage metrics
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