# Determination of protein transporter function using Raman spectroscopy

## TL;DR

This paper introduces a new method using Raman spectroscopy to study protein transporters without radioactive labels.

## Contribution

The novel use of Raman spectroscopy with alkyne-labeled substrates to detect transporter function is presented.

## Key findings

- Raman spectroscopy successfully detected ATP transport by ThNTT4 in Escherichia coli.
- The transporter showed specificity for purine substrates but not pyrimidine substrates.
- Alkyne-labeled substrates enable non-radioactive detection of transporter activity.

## 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.

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 Escherichia coli, 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.