The binding of tetraphenylphosphonium (TPP+) to EmrE, a membrane-bound, 110 residue Escherichia coli multidrug transport protein, has been observed by 31P cross-polarisation–magic-angle spinning nuclear magnetic resonance spectroscopy (CP–MAS NMR). EmrE has been reconstituted into dimyristoyl phosphatidylcholine bilayers. CP–MAS could selectively distinguish binding of TPP+ to EmrE in the fluid membrane. A population of bound ligand appears shifted 4 ppm to lower frequency compared to free ligand in solution, which suggests a rather direct and specific type of interaction of the ligand with the protein. This is also supported by the observed restricted motion of the bound ligand. The observation of another weakly bound substrate population arises from ligand binding to negatively charged residues in the protein loop regions.
