Conformational heterogeneity of transmembrane residues after the Schiff base reprotonation of bacteriorhodopsin: 15N CPMAS NMR of D85N/T170C membranes

bR, N-like and O-like intermediate states of 15N]methionine-labelled wild type and D85N/T170C bacteriorhodopsin were accumulated in native membranes by controlling the pH of the preparations. 15N cross polarization and magic angle sample spinning (CPMAS) NMR spectroscopy allowed resolution of seven out of nine resonances in the bR-state. It was possible to assign some of the observed resonances by using 13C/15N rotational echo double resonance (REDOR) NMR and Mn2+ quenching as well as D2O exchange, which helps to identify conformational changes after the bacteriorhodopsin Schiff base reprotonation. The significant differences in chemical shifts and linewidths detected for some of the resonances in N- and O-like samples indicate changes in conformation, structural heterogeneity or altered molecular dynamics in parts of the protein.