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Consensus designs and thermal stability determinants of a human glutamate transporter
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Consensus designs and thermal stability determinants of a human glutamate transporter
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Journal Article
Consensus designs and thermal stability determinants of a human glutamate transporter
2018
Overview
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.
Publisher
eLife Science Publications, Ltd,eLife Sciences Publications Ltd,eLife Sciences Publication,eLife Sciences Publications, Ltd
Subject
/ Amino Acid Transport System X-AG - chemistry
/ Amino Acid Transport System X-AG - metabolism
/ Analysis
/ Biochemistry and Chemical Biology
/ Biochemistry, Molecular Biology
/ Design
/ Deuterium Exchange Measurement
/ Excitatory Amino Acid Transporter 1 - chemistry
/ Excitatory Amino Acid Transporter 1 - metabolism
/ Excitatory amino acid transporters
/ Humans
/ Hydrogen
/ Kinetics
/ Neurotransmitter Agents - metabolism
/ Peptides
/ Protein Subunits - chemistry
/ Proteins
/ Structural Biology and Molecular Biophysics
