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PLoS One. 2013 Jun 25;8(6):e67112. doi: 10.1371/journal.pone.0067112. Print 2013.

Multicolour fluorescence-detection size-exclusion chromatography for structural genomics of membrane multiprotein complexes.

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1
Institute of Biochemistry, Biocenter, Goethe-University Frankfurt, Frankfurt am Main, Germany.

Abstract

Many interesting and important membrane proteins are hetero-oligomeric. However, besides naturally abundant examples, the structures of relatively few such complexes are known. Partly, this is due to difficulties in expression, stoichiometric assembly, and in the evaluation of their stability prior to crystallization trials. Here we describe a new approach, which allows rapid assessment of protein complex quality, assembly and stoichiometry, simplifying the search for conditions conducive to long-term stability and crystallization. Multicolour fluorescence size-exclusion chromatography (MC-FSEC) is used to enable tracking of individual subunits through expression, solubilization and purification steps. We show how the method has been applied to the heterodimeric transporter associated with antigen processing (TAP) and demonstrate how it may be extended in order to analyse membrane multisubunit assemblies.

PMID:
23825631
PMCID:
PMC3692423
DOI:
10.1371/journal.pone.0067112
[Indexed for MEDLINE]
Free PMC Article
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