Journal
PROTEIN EXPRESSION AND PURIFICATION
Volume 114, Issue -, Pages 30-36Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2015.05.014
Keywords
FeoB; Unnatural amino acid; Spin label; EPR; Membrane protein
Categories
Funding
- German Research Foundation (DFG) [SFB 813]
Ask authors/readers for more resources
The ferrous iron transporter FeoB is an important factor in the iron metabolism of various bacteria. As a membrane bound GTPase it also represents an interesting evolutionary link between prokaryotic and eukaryotic membrane signalling pathways. To date, structural information for FeoB is limited to the cytosolic GTPase domain and structural features such as the oligomeric state of the transporter in the membrane, and thereby the nature of the transport pore are a matter of constant debate. Recently, EPR distance measurements have become an important tool to investigate such questions in frozen solution. As a prerequisite for these experiments, we designed protocols to express and purify both the cytosolic domain of FeoB (NFeoB) and full-length FeoB from Escherichia coli BL21 in purity, quantity and quality needed for EPR studies. Since FeoB from E. coli contains 12 native cysteines, we incorporated the unnatural amino acid para-acetylphenylalanine (pAcF) into the protein. We spin labelled the mutant protein using the HO4120 spin label and performed preliminary EPR experiments using cw-X-band EPR spectroscopy. Our results provide new insights concerning the oligomeric state of full-length FeoB. (C) 2015 Elsevier Inc. All rights reserved.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available