CIRRI, Erica; BRIER, Sebastien; ASSAL, Reda; CANUL TEC, Juan Carlos; CHAMOT ROOKE, Julia; REYES, Nicolas

doi:10.7554/eLife.40110

CIRRI, Erica
Institut Pasteur [Paris] [IP]

BRIER, Sebastien
Institut Pasteur [Paris] [IP]

ASSAL, Reda

Langue

Article de revue

Ce document a été publié dans

eLife. 2018-10-18, vol. 7

Résumé en anglais

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.< Réduire

Mots clés en anglais

Amino Acid Sequence

Amino Acid Transport System X-AG

Consensus Sequence

Deuterium Exchange Measurement

Excitatory Amino Acid Transporter 1

Humans

Kinetics

Models

Molecular

Mutant Proteins

Neurotransmitter Agents

Protein Stability

Protein Subunits

Protein Unfolding

Temperature

URI

https://oskar-bordeaux.fr/handle/20.500.12278/186021

DOI

http://dx.doi.org/10.7554/eLife.40110

Unités de recherche

MFP (Laboratoire Microbiologie Fondamentale et Pathogénicité) - UMR 5234

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Consensus designs and thermal stability determinants of a human glutamate transporter.

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Ce document a été publié dans

Résumé en anglais

Mots clés en anglais

URI

DOI

Unités de recherche