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Application of split‐green fluorescent protein for topology mapping membrane proteins in Escherichia coli
Author(s) -
Toddo Stephen,
Söderström Bill,
Palombo Isolde,
von Heijne Gunnar,
Nørholm Morten H. H.,
Daley Daniel O.
Publication year - 2012
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.2131
Subject(s) - periplasmic space , green fluorescent protein , transmembrane protein , topology (electrical circuits) , escherichia coli , membrane protein , membrane topology , complementation , transmembrane domain , vesicle associated membrane protein 8 , inner membrane , peptide sequence , protein structure , chemistry , biophysics , biology , biochemistry , membrane , gene , receptor , mathematics , combinatorics , phenotype
Abstract 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 .