Fluorine-19 labeling of the tryptophan residues in the G protein-coupled receptor NK1R using the 5-fluoroindole precursor in Pichia pastoris expression

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Fluorine-19 labeling of the tryptophan residues in the G protein-coupled receptor NK1R using the 5-fluoroindole precursor in Pichia pastoris expression

Abstract

In NMR spectroscopy of biomolecular systems, the use of fluorine-19 probes benefits from a clean background and high sensitivity. Therefore, 19F-labeling procedures are of wide-spread interest. Here, we use 5-fluoroindole as a precursor for cost-effective residue-specific introduction of 5-fluorotryptophan (5F-Trp) into G protein-coupled receptors (GPCRs) expressed in Pichia pastoris. The method was successfully implemented with the neurokinin 1 receptor (NK1R). The 19F-NMR spectra of 5F-Trp-labeled NK1R showed one well-separated high field-shifted resonance, which was assigned by mutational studies to the â??toggle switch tryptophanâ??. Residue-selective labeling thus enables site-specific investigations of this functionally important residue. The method described here is inexpensive, requires minimal genetic manipulation and can be expected to be applicable for yeast expression of GPCRs at large.



Source: Journal of Biomolecular NMR