Bicelle Reconstitution Of Ion Channel Domains For Nmr Structural Studies

Solution NMR is an excellent tool for the investigation of protein dynamics and protein-ligand interactions. However, NMR data for membrane proteins often suffers from significantly reduced quality due to the large size of the membrane mimetic-protein complexes. In our study, we show methods to successfully prepare protein-bicelle complexes of suitable size and homogeneity for NMR structural studies. We have optimized the sample preparation and protein reconstitution parameters for two bacterial ion channel domains: firstly, the full-length tetrameric potassium channel KcsA and the isolated voltage sensing domain of the sodium channel NaChBac. Both samples gave well resolved NMR spectra and allowed us to assign a significant portion of the resonances for the two proteins, using a combination of standard triple-resonance experiments and specifically labeled samples. These assignments will form the basis for the structural study of toxin interactions with both channels.