19F Paramagnetic Relaxation-Based NMR for Quaternary 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 angstrom, which challenges the high and low measurement limits of conventional nuclear magnetic resonance (NMR) and double r electron-electron resonance electron spin resonance spectroscopies. Here, we report the use of F-19 paramagnetic relaxation enhancement (PRE) NMR in combination with F-19/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 F-19 PRE NMR results with previous structural information. The study suggests that F-19 PRE NMR is a viable tool in extracting distance restraints to define quaternary structures.