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Volume 171, Issue 2

Determination of protein transporter function using Raman spectroscopy Open Access

Graphical Abstract

Graphical Abstract

A summary of the workflow for identifying the function of a protein transporter using Raman spectroscopy. A putative transporter gene (ThNTT4) is cloned into a suitable expression vector [ 1 ] and transformed into a heterologous expression system [ 2 ]. Protein expression is induced [ 3 ] and confirmed [ 4 ], followed by an uptake assay using alkyne-labelled substrates such as N6pATP [ 5 ]. Following washing and lysis of the heterologous host cell [ 6 ], the lysate is spotted onto a steel slide, dried down and analysed with the Raman microscope.

Abstract

Transporter proteins are essential across the tree of life as they provide a cell with a means of exchanging vital metabolites with the external milieu. Characterizing the function of transporters is challenging and traditionally uses methods involving radiolabelled substrates, which requires prolonged exposure times and specialist equipment. Here, we provide an alternative method to the classical uptake assay using Raman spectroscopy to detect the uptake of alkyne-labelled substrates and determine transporter function. As a proof of principle, we demonstrate the method using a candidate nucleotide transporter (ThNTT4) expressed in , which is shown to transport alkyne-labelled ATP molecules (N6pATP), which was readily detected using Raman spectroscopy. We show that ATP transport can be detected in a time-dependent manner using alkyne labels and demonstrate the substrate specificity of the transporter for purine but not pyrimidine substrates. This work establishes that Raman spectroscopy is an excellent alternative to using radioactive substrates in analysing, not only pathogen transporters, but potentially any transporter in which its substrate can be alkyne tagged.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): alkyne , ATP , Microsporidia , Nucleotide transporter , Raman spectroscopy and uptake assay
Funding
This study was supported by the:
  • Teesside University
    • Principal Award Recipient: PaulDean
© 2025 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution.
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2025-02-10
2026-02-10

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/content/journal/micro/10.1099/mic.0.001526
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Determination of protein transporter function using Raman spectroscopy
Microbiology 171, 001526 (2025); https://doi.org/10.1099/mic.0.001526
/content/journal/micro/10.1099/mic.0.001526
/content/journal/micro/10.1099/mic.0.001526
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