Int J Biol Macromol. 2006 Aug 15;39(1-3):3-9. Epub 2006 Jan 6.

Purification of the YadA membrane anchor for secondary structure analysis and crystallization.

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Max Planck Institute for Biochemistry, Department Membrane Biochemistry, Am Klopferspitz 18a, 82152 Martinsried, Germany.

Abstract

Non-fimbrial adhesins, such as Yersinia YadA, Moraxella UspA1 and A2, Haemophilus Hia and Hsf, or Bartonella BadA, represent an important class of molecules by which pathogenic proteobacteria adhere to their hosts. They form trimeric surface structures with a head-rod-anchor architecture. Whereas their head and rod domains may be of heterologous origin, their anchor domains are homologous and display the properties of autotransporters. Conflicting topology models exist for these membrane anchors. Here, we describe the expression and purification of the membrane anchor of YadA from Yersinia enterocolitica for structural biology experiments. We expressed YadA-M in the Escherichia coli outer membrane. After solubilization and purification, it is a trimer of extreme stability. Using protein FTIR and secondary structure analysis, we show that the anchor is a beta-barrel, but contains a helical part at its N-terminus. We have crystallized the protein under various conditions and present X-ray data to 3.8 A resolution.

PMID:: 16405993; [PubMed - indexed for MEDLINE]

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