Purification, crystallization and characterization of the Pseudomonas outer membrane protein FapF, a functional amyloid transporter

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Title: Purification, crystallization and characterization of the Pseudomonas outer membrane protein FapF, a functional amyloid transporter
Author(s): Matthews, SJ
Rouse, S
Hawthorne
Lambert
Hare
Morgan, M
Item Type: Journal Article
Abstract: Bacteria 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 FapF 106–430 from the membrane fraction, reproducible crystals were obtained using the sitting-drop method of vapour diffusion. Diffraction data have been processed to 2.5 A ̊ resolution. These crystals belonged to the monoclinic space group C 121, with unit-cell parameters a = 143.4, b = 124.6, c = 80.4 A ̊ , = = 90, = 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 -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.
Publication Date: 1-Dec-2016
Date of Acceptance: 8-Nov-2016
URI: http://hdl.handle.net/10044/1/42701
DOI: https://dx.doi.org/10.1107/S2053230X16017921
ISSN: 2053-230X
Publisher: International Union of Crystallography
Start Page: 892
End Page: 896
Journal / Book Title: Acta Crystallographica Section F: Structural Biology Communications
Volume: F72
Sponsor/Funder: Medical Research Council (MRC)
Wellcome Trust
Wellcome Trust
Wellcome Trust
Funder's Grant Number: G1100332
100280/Z/12/Z
WT/104933/z/14/z
202926/Z/16/Z
Copyright Statement: © 2016 Rouse et al. This is an open access article distributed under a CC-BY licence (https://creativecommons.org/licenses/by/2.0/uk/legalcode)
Keywords: C8E4
FapF
Pseudomonas
amyloid transporter
bacterial outer membrane
Publication Status: Published
Appears in Collections:Faculty of Natural Sciences



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