TY - JOUR
T1 - Investigation of the allosteric coupling mechanism in a glutamate transporter homolog via unnatural amino acid mutagenesis
AU - Riederer, Erika A.
AU - Valiyaveetil, Francis I.
N1 - Publisher Copyright:
© 2019 National Academy of Sciences. All rights reserved.
PY - 2019/8/6
Y1 - 2019/8/6
N2 - Glutamate transporters harness the ionic gradients across cell membranes for the concentrative uptake of glutamate. The sodium-coupled Asp symporter, GltPh is an archaeal homolog of glutamate transporters and has been extensively used to understand the transport mechanism. A critical aspect of the transport cycle in GltPh is the coupled binding of sodium and aspartate. Previous studies have suggested a major role for hairpin-2 (HP2), which functions as the extracellular gate for the aspartate binding site, in the coupled binding of sodium and aspartate to GltPh. In this study, we develop a fluorescence assay for monitoring HP2 movement by incorporating tryptophan and the unnatural amino acid, p-cyanophenylalanine into GltPh. We use the HP2 assays to show that HP2 opening with Na+ follows an induced-fit mechanism. We also determine how residues in the substrate binding site affect the opening and closing of HP2.
AB - Glutamate transporters harness the ionic gradients across cell membranes for the concentrative uptake of glutamate. The sodium-coupled Asp symporter, GltPh is an archaeal homolog of glutamate transporters and has been extensively used to understand the transport mechanism. A critical aspect of the transport cycle in GltPh is the coupled binding of sodium and aspartate. Previous studies have suggested a major role for hairpin-2 (HP2), which functions as the extracellular gate for the aspartate binding site, in the coupled binding of sodium and aspartate to GltPh. In this study, we develop a fluorescence assay for monitoring HP2 movement by incorporating tryptophan and the unnatural amino acid, p-cyanophenylalanine into GltPh. We use the HP2 assays to show that HP2 opening with Na+ follows an induced-fit mechanism. We also determine how residues in the substrate binding site affect the opening and closing of HP2.
KW - Fluorescence
KW - Glutamate transporters
KW - Unnatural amino acids
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U2 - 10.1073/pnas.1907852116
DO - 10.1073/pnas.1907852116
M3 - Article
C2 - 31332002
AN - SCOPUS:85070200676
SN - 0027-8424
VL - 116
SP - 15939
EP - 15946
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 32
ER -