Frog oocytes to unveil the structure and supramolecular organization of human transport proteins

Bergeron, Marc J.; Boggavarapu, Rajendra; Meury, Marcel; Ucurum, Zöhre; Caron, Luc; Isenring, Paul; Hediger, Matthias A.; Fotiadis, Dimitrios (2011). Frog oocytes to unveil the structure and supramolecular organization of human transport proteins. PLoS ONE, 6(7), e21901. Lawrence, Kans.: Public Library of Science 10.1371/journal.pone.0021901

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Structural analyses of heterologously expressed mammalian membrane proteins remain a great challenge given that microgram to milligram amounts of correctly folded and highly purified proteins are required. Here, we present a novel method for the expression and affinity purification of recombinant mammalian and in particular human transport proteins in Xenopus laevis frog oocytes. The method was validated for four human and one murine transporter. Negative stain transmission electron microscopy (TEM) and single particle analysis (SPA) of two of these transporters, i.e., the potassium-chloride cotransporter 4 (KCC4) and the aquaporin-1 (AQP1) water channel, revealed the expected quaternary structures within homogeneous preparations, and thus correct protein folding and assembly. This is the first time a cation-chloride cotransporter (SLC12) family member is isolated, and its shape, dimensions, low-resolution structure and oligomeric state determined by TEM, i.e., by a direct method. Finally, we were able to grow 2D crystals of human AQP1. The ability of AQP1 to crystallize was a strong indicator for the structural integrity of the purified recombinant protein. This approach will open the way for the structure determination of many human membrane transporters taking full advantage of the Xenopus laevis oocyte expression system that generally yields robust functional expression.

Item Type:

Journal Article (Original Article)

Division/Institute:

04 Faculty of Medicine > Pre-clinic Human Medicine > Institute of Biochemistry and Molecular Medicine

UniBE Contributor:

Boggavarapu, Venkata Ravi Naga Rajendr; Meury, Marcel; Ucurum Fotiadis, Zöhre; Hediger, Matthias and Fotiadis, Dimitrios José

ISSN:

1932-6203

Publisher:

Public Library of Science

Language:

English

Submitter:

Factscience Import

Date Deposited:

04 Oct 2013 14:23

Last Modified:

14 Dec 2014 20:42

Publisher DOI:

10.1371/journal.pone.0021901

PubMed ID:

21760919

Web of Science ID:

000292655400025

BORIS DOI:

10.7892/boris.8145

URI:

https://boris.unibe.ch/id/eprint/8145 (FactScience: 213632)

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