19 F Paramagnetic Relaxation-Based NMR forQuaternary Structural Restraints of Ion Channels

Quaternary distance restraints are essential to define the three-dimensional structures of protein assemblies. These distances often fall within a range of 10–18 A, which challenges the high and low measurement limits of conventional nuclear magnetic resonance (NMR) and double electron–electron resonance electron spin resonance spectroscopies. Here, we report the use of 19F paramagnetic relaxation enhancement (PRE) NMR in combination with 19F/paramagnetic labeling to equivalent sites in different subunits of a protein complex in micelles to determine intersubunit distances. The feasibility of this strategy was evaluated on a pentameric ligand-gated ion channel, for which we found excellent agreement of the 19F PRE NMR results with previous structural information. The study suggests that 19F PRE NMR is a viable tool in extracting distance restraints to define quaternary structures.