Journal
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Volume 398, Issue 3, Pages 366-371Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.bbrc.2010.06.072
Keywords
Iron transport; Peptidase_M75; SRCD; Mass spectrometry; Crystallisation; EfeUOB; Psyr_3370
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Funding
- FELIX Trust
- Biotechnology and Biological Sciences Research Council (BBSRC)
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The EfeM protein is a component of the putative EfeUOBM iron-transporter of Pseudomonas syringae path-ovar syringae and is thought to act as a periplasmic, ferrous-iron binding protein. It contains a signal peptide of 34 amino acid residues and a C-terminal 'Peptidase_M75' domain of 251 residues. The C-terminal domain contains a highly conserved 'HXXE' motif thought to act as part of a divalent cation-binding site. In this work, the gene (efeM or 'Psyr_3370') encoding EfeM was cloned and over-expressed in Escherichia coli, and the mature protein was purified from the periplasm. Mass spectrometry confirmed the identity of the protein (M-W 27,772 Da). Circular dichroism spectroscopy of EfeM indicated a mainly a-helical structure, consistent with bioinformatic predictions. Purified EfeM was crystallised by hanging-drop vapor diffusion to give needle-shaped crystals that diffracted to a resolution of 1.6 angstrom. This is the first molecular study of a peptidase M75 domain with a presumed iron transport role. (C) 2010 Elsevier Inc. All rights reserved.
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