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.
Keywords: ATP; Microsporidia; Nucleotide transporter; Raman spectroscopy; alkyne; uptake assay.