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Title: Purification, crystallization and preliminary X-ray diffraction analysis of the Escherichia coli common pilus chaperone EcpB

In Escherichia coli, the common pilus (Ecp) belongs to an alternative chaperone–usher pathway that plays a major role in both early biofilm formation and host-cell adhesion. Initial attempts at crystallizing the chaperone EcpB using natively purified protein from the bacterial periplasm were not successful; however, after the isolation of EcpB under denaturing conditions and subsequent refolding, crystals were obtained at pH 8.0 using the sitting-drop method of vapour diffusion. This is the first time that this refolding strategy has been used to purify CU chaperones. Pili are key cell-surface components that allow the attachment of bacteria to both biological and abiotic solid surfaces, whilst also mediating interactions between themselves. In Escherichia coli, the common pilus (Ecp) belongs to an alternative chaperone–usher (CU) pathway that plays a major role in both early biofilm formation and host-cell adhesion. The chaperone EcpB is involved in the biogenesis of the filament, which is composed of EcpA and EcpD. Initial attempts at crystallizing EcpB using natively purified protein from the bacterial periplasm were not successful; however, after the isolation of EcpB under denaturing conditions and subsequent refolding, crystals were obtained at pH 8.0 using the sitting-drop method of vapour diffusion. Diffraction data have been processedmore » to 2.4 Å resolution. These crystals belonged to the trigonal space group P3{sub 1}21 or P3{sub 2}21, with unit-cell parameters a = b = 62.65, c = 121.14 Å and one monomer in the asymmetric unit. Molecular replacement was unsuccessful, but selenomethionine-substituted protein and heavy-atom derivatives are being prepared for phasing. The three-dimensional structure of EcpB will provide invaluable information on the subtle mechanistic differences in biogenesis between the alternative and classical CU pathways. Furthermore, this is the first time that this refolding strategy has been used to purify CU chaperones, and it could be implemented in similar systems where it has not been possible to obtain highly ordered crystals.« less
Authors:
; ;  [1]
  1. Imperial College London, South Kensington, London SW7 2AZ (United Kingdom)
Publication Date:
OSTI Identifier:
22375722
Resource Type:
Journal Article
Resource Relation:
Journal Name: Acta crystallographica. Section F, Structural biology communications; Journal Volume: 71; Journal Issue: Pt 6; Other Information: PMCID: PMC4461329; PMID: 26057794; PUBLISHER-ID: tt5069; PUBLISHER-ID: S2053230X15006354; OAI: oai:pubmedcentral.nih.gov:4461329; Copyright (c) Garnett et al. 2015; This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND SUPERFLUIDITY; ADHESION; ATOMS; CRYSTALLIZATION; CRYSTALS; DIFFUSION; ESCHERICHIA COLI; FILAMENTS; INTERACTIONS; PROTEINS; RESOLUTION; SOLIDS; SPACE GROUPS; SURFACES; X-RAY DIFFRACTION