Application of split-green fluorescent protein for topology mapping membrane proteins in Escherichia coli.
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Toddo S, Söderström B, Palombo I, von Heijne G, Nørholm MH, Daley DO
Protein Sci. 21 (10) 1571-1576 [2012-10-00; online 2012-08-21]
A topology map of a membrane protein defines the location of transmembrane helices and the orientation of soluble domains relative to the membrane. In the absence of a high-resolution structure, a topology map is an essential guide for studying structure-function relationships. Although these maps can be predicted directly from amino acid sequence, the predictions are more accurate if combined with experimental data, which are usually obtained by fusing a reporter protein to the C-terminus of the protein. However, as reporter proteins are large, they cannot be used to report on the cytoplasmic/periplasmic location of the N-terminus of a protein. Here, we show that the bimolecular split-green fluorescent protein complementation system can overcome this limitation and can be used to determine the location of both the N- and C-termini of inner membrane proteins in Escherichia coli.
PubMed 22825803
DOI 10.1002/pro.2131
Crossref 10.1002/pro.2131