%0 Journal Article %T Consensus designs and thermal stability determinants of a human glutamate transporter %+ Mécanismes moléculaires du transport membranaire - Molecular mechanisms of membrane transport %+ Spectrométrie de Masse pour la Biologie – Mass Spectrometry for Biology (UTechS MSBio) %A Cirri, Erica %A Brier, Sébastien %A Assal, Reda %A Canul-Tec, Juan Carlos %A Chamot-Rooke, Julia %A Reyes, Nicolas %Z H2020 Excellent Science (ERC Starting grant 309657)Agence Nationale de la Recherche (CACSICE grant ANR-11-EQPX-008)Centre National de la Recherche Scientifique (UMR 3528) %< avec comité de lecture %J eLife %I eLife Sciences Publication %V 7 %8 2018-10-18 %D 2018 %R 10.7554/eLife.40110 %M 30334738 %K molecular biophysics %K permeation and transport %K protein dynamics %K protein folding %K protein stability %K structural biology %K biochemistry %K chemical biology %K consensus amino acid %K human %Z Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics %Z Chemical Sciences/Analytical chemistryJournal articles %X 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. %G English %2 https://pasteur.hal.science/pasteur-01916344/document %2 https://pasteur.hal.science/pasteur-01916344/file/elife-40110-v2.pdf %L pasteur-01916344 %U https://pasteur.hal.science/pasteur-01916344 %~ PASTEUR %~ CNRS %~ OPENAIRE %~ INC-CNRS %~ TEST-HALCNRS %~ ANR