Abstract:
:Blue native electrophoresis (BNE) has become the most popular method for the global analysis of membrane protein complexes. Although it has been shown to be very useful for that purpose, it can produce the dissociation of complexes with weak interactions and, due to the use of Coomassie Brilliant Blue, does not allow the subsequent application of fluorimetric and/or enzymatic techniques. Recently, we have successfully used the high resolution clear native electrophoresis (hrCNE) for the analysis of Neisseria meningitidis outer membrane porin complexes. The aim of this study was to determine the composition of the complexome of the Escherichia coli envelope by using hrCNE and to compare our results with those previously obtained using BNE. The bidimensional electrophoresis approaches used, hrCN/hrCNE and hrCN/SDS-PAGE, coupled to mass spectrometry allowed a detailed analysis of the complexome of E. coli membranes. For the first time, the three subunits of the formate dehydrogenase FDH-O were identified forming a single complex and hrCNE also allowed the identification of both the HflK and HflC proteins as components of the HflA complex. This technique also allowed us to suggest a relationship between OmpF and DLDH and, although OmpA is considered to be monomeric in vivo, we found this protein structured as homodimers. Thus hrCNE provides a good tool for future analyses of bacterial membrane proteins and complexes and is an important alternative to the commonly used BNE.
journal_name
J Microbiol Methodsjournal_title
Journal of microbiological methodsauthors
Diéguez-Casal E,Freixeiro P,Costoya L,Criado MT,Ferreirós C,Sánchez Sdoi
10.1016/j.mimet.2014.05.003subject
Has Abstractpub_date
2014-07-01 00:00:00pages
45-54eissn
0167-7012issn
1872-8359pii
S0167-7012(14)00124-9journal_volume
102pub_type
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