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

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posted on 2023-06-09, 08:01 authored by S L Rouse, W J Hawthorne, S Lambert, M L Morgan, Stephen Hare, S Matthews
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, [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 Communications

ISSN

2053-230X

Publisher

International Union of Crystallography

Issue

12

Volume

72

Page range

892-896

Department affiliated with

  • Biochemistry Publications

Full text available

  • Yes

Peer reviewed?

  • Yes

Legacy Posted Date

2017-09-21

First Open Access (FOA) Date

2017-09-21

First Compliant Deposit (FCD) Date

2017-09-21

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