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

DOI: 10.1107/S2053230X16017921 DOI Help

Authors: Sarah L. Rouse (Imperial College London) , Wlliam J. Hawthorne (Imperial College London) , Sebastian Lambert (National University of Singapore) , Marc L. Morgan (Imperial College London) , Stephen A. Hare (Imperial College London) , Stephen Matthews (Imperial College London)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Acta Crystallographica Section F Structural Biology Communications , VOL 72 , PAGES 892 - 896

State: Published (Approved)
Published: December 2016

Open Access Open Access

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 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, α = γ = 90, β = 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 β-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.

Journal Keywords: amyloid transporter; bacterial outer membrane; FapF; Pseudomonas; C8E4.

Subject Areas: Biology and Bio-materials


Instruments: I03-Macromolecular Crystallography

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pg5062.pdf