Investigation of the allosteric coupling mechanism in a glutamate transporter homolog via unnatural amino acid mutagenesis

Erika A. Riederer, Francis I. Valiyaveetil

Research output: Contribution to journalArticlepeer-review

11 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)15939-15946
Number of pages8
JournalProceedings of the National Academy of Sciences of the United States of America
Volume116
Issue number32
DOIs
StatePublished - Aug 6 2019

Keywords

  • Fluorescence
  • Glutamate transporters
  • Unnatural amino acids

ASJC Scopus subject areas

  • General

Fingerprint

Dive into the research topics of 'Investigation of the allosteric coupling mechanism in a glutamate transporter homolog via unnatural amino acid mutagenesis'. Together they form a unique fingerprint.

Cite this