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

Artikel i vetenskaplig tidskrift
Författare Jennie Sjöhamn
Petra Båth
Richard Neutze
Kristina Hedfalk
Publicerad i Protein science : a publication of the Protein Society
Volym 25
Nummer/häfte 12
Sidor 2196-2208
ISSN 1469-896X
Publiceringsår 2016
Publicerad vid Institutionen för kemi och molekylärbiologi
Sidor 2196-2208
Språk en
Länkar dx.doi.org/10.1002/pro.3046
www.ncbi.nlm.nih.gov/entrez/query.f...
Ämneskategorier Annan kemi, Biokemi

Sammanfattning

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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