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Applying bimolecular fluorescence complementation to screen and purify aquaporin protein:protein complexes.

Journal article
Authors Jennie Sjöhamn
Petra Båth
Richard Neutze
Kristina Hedfalk
Published in Protein science : a publication of the Protein Society
Volume 25
Issue 12
Pages 2196-2208
ISSN 1469-896X
Publication year 2016
Published at Department of Chemistry and Molecular Biology
Pages 2196-2208
Language en
Links dx.doi.org/10.1002/pro.3046
www.ncbi.nlm.nih.gov/entrez/query.f...
Subject categories Other Chemistry Topics, Biochemistry

Abstract

Protein:protein interactions play key functional roles in the molecular machinery of the cell. A major challenge for structural biology is to gain high-resolution structural insight into how membrane protein function is regulated by protein:protein interactions. To this end we present a method to express, detect, and purify stable membrane protein complexes that are suitable for further structural characterization. Our approach utilizes bimolecular fluorescence complementation (BiFC), whereby each protein of an interaction pair is fused to nonfluorescent fragments of yellow fluorescent protein (YFP) that combine and mature as the complex is formed. YFP thus facilitates the visualization of protein:protein interactions in vivo, stabilizes the assembled complex, and provides a fluorescent marker during purification. This technique is validated by observing the formation of stable homotetramers of human aquaporin 0 (AQP0). The method's broader applicability is demonstrated by visualizing the interactions of AQP0 and human aquaporin 1 (AQP1) with the cytoplasmic regulatory protein calmodulin (CaM). The dependence of the AQP0-CaM complex on the AQP0 C-terminus is also demonstrated since the C-terminal truncated construct provides a negative control. This screening approach may therefore facilitate the production and purification of membrane protein:protein complexes for later structural studies by X-ray crystallography or single particle electron microscopy.

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