Quantifying Ligand-to-Protein Distances in Complex Environments Using Intermolecular 19F PRE NMR Spectroscopy.

The determination of structural features is crucial to understand the interplay between structure and function of biomolecules and biomolecular complexes. In this context, nuclear magnetic resonance (NMR) spectroscopy provides experimental approaches, one of which is paramagnetic relaxation enhancement (PRE). Thus, placing a paramagnetic center and fluorine at strategic sites within (bio)molecules forming a complex enables the determination of distances through a straightforward, one-dimensionally guided NMR spectroscopic setup. Moreover, the almost absence of fluorine in biomolecules found in nature allows performing experimental work using cell-like or in cell conditions. Here, we made use of a single-cysteine mutant of Bacillus subtilis cold shock protein B (BsCspB) equipped with a paramagnetic spin label in complex with a fluorine-labeled variant of singly stranded DNA ligand dT4 to acquire intermolecular, 19F-based PREs. The distance between BsCspB and fluorine in dT4 has then been probed using three different experimental settings: in vitro, molecular crowding, and cell lysate conditions. Our data suggests that the intermolecular distance between the paramagnetically spin-labeled protein and the fluorine-labeled ligand does not change significantly using the three different conditions. This matches results regarding the conservation of binding affinities determined for this biomolecular complex using the three different conditions.