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© Institut Pasteur
La protéine EVER 2 (en rouge) est une protéine membranaire localisée dans la membrane du noyau et le cytoplasme au niveau du réticulum endoplasmique. Elle est absente de la membrane cytoplasmique (en vert).
Scientific Fields
Diseases
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Technique

Published in eLife - 18 Oct 2018

Cirri E, Brier S, Assal R, Canul-Tec JC, Chamot-Rooke J, Reyes N

Link to Pubmed [PMID] – 30334738

Elife 2018 10;7

Human excitatory amino acid transporters (EAATs) take up the neurotransmitter glutamate in the brain and are essential to maintain excitatory neurotransmission. Our understanding of the EAATs’ molecular mechanisms has been hampered by the lack of stability of purified protein samples for biophysical analyses. Here, we present approaches based on consensus mutagenesis to obtain thermostable EAAT1 variants that share up to ~95% amino acid identity with the wild type transporters, and remain natively folded and functional. Structural analyses of EAAT1 and the consensus designs using hydrogen-deuterium exchange linked to mass spectrometry show that small and highly cooperative unfolding events at the inter-subunit interface rate-limit their thermal denaturation, while the transport domain unfolds at a later stage in the unfolding pathway. Our findings provide structural insights into the kinetic stability of human glutamate transporters, and introduce general approaches to extend the lifetime of human membrane proteins for biophysical analyses.

https://www.ncbi.nlm.nih.gov/pubmed/30334738