Proximity of two tryptophan residues in dihydrofolate reductase determined by ¹⁹F NMR

Abstract

NUCLEAR magnetic resonance methods have provided a wealth of information on the conformational and ligand-binding properties of proteins^1–3. In such studies, it is necessary to resolve resonances from the ligand or from individual residues of the protein. To achieve this, it is often necessary to resort to isotopic substitution methods such as selective deuteration^4,5 or ¹³C enrichment^6–8. An alternative approach is to introduce a fluorine atom (¹⁹F) into the system as an NMR probe, either by using fluorine-containing ligands^9–11 or by labelling the protein itself. This can be done either by chemical modification^12–15 or by the biosynthetic incorporation of selected fluorinated amino acids^16–19. We have used this latter approach to prepare Lactobacillus casei dihydrofolate reductase containing either 3-fluoro-tyrosine or 6-fluorotryptophan residues; by measuring the ¹⁹F spectra at 94.1 MHz we have characterised the effects of ligand binding on individual tyrosine and tryptophan residues in the protein¹⁹. We now report through-space ¹⁹F–¹⁹F spin–spin coupling in 6-fluorotryptophan containing dihydrofolate reductase which demonstrates that two of the tryptophan residues are in close proximity in the folded structure of the protein.