Abstract
Quaternary distance restraints are essential to define the three-dimensional structures of protein assemblies. These distances often fall within a range of 10-18 Å, 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 (19)F paramagnetic relaxation enhancement (PRE) NMR in combination with (19)F/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 (19)F PRE NMR results with previous structural information. The study suggests that (19)F PRE NMR is a viable tool in extracting distance restraints to define quaternary structures.