title: Purification, crystallization and characterization of the Pseudomonas outer membrane protein FapF, a functional amyloid transporter.
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 FapF106-430from 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.
authors.affiliation: Life Sciences, Imperial College London, South Kensington, London SW7 2AZ, England.
authors.forename: Sarah L
authors.initials: SL
authors.lastname: Rouse
authors.affiliation: Life Sciences, Imperial College London, South Kensington, London SW7 2AZ, England.
authors.forename: Wlliam J
authors.initials: WJ
authors.lastname: Hawthorne
authors.affiliation: Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543, Singapore.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Lambert
authors.affiliation: Life Sciences, Imperial College London, South Kensington, London SW7 2AZ, England.
authors.forename: Marc L
authors.initials: ML
authors.lastname: Morgan
authors.affiliation: Life Sciences, Imperial College London, South Kensington, London SW7 2AZ, England.
authors.forename: Stephen A
authors.initials: SA
authors.lastname: Hare
authors.affiliation: Life Sciences, Imperial College London, South Kensington, London SW7 2AZ, England.
authors.forename: Stephen
authors.initials: S
authors.lastname: Matthews
mesh_terms: D000595:Amino Acid Sequence; D000682:Amyloid / Q000737:chemistry* / Q000378:metabolism; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019897:Periplasm / Q000737:chemistry / Q000378:metabolism; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D011549:Pseudomonas / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014961:X-Ray Diffraction
chemical_list: D000682:Amyloid; D001425:Bacterial Outer Membrane Proteins; D026901:Membrane Transport Proteins; D011994:Recombinant Proteins
keywords: C8E4; FapF; Pseudomonas; amyloid transporter; bacterial outer membrane