Lanthanide ions bind specifically to an added EF-hand and orient a membrane protein in micelles for solution NMR spectroscopy

Twelve amino acid residues corresponding to an EF-hand'' calcium-binding site were added to the N-terminus of a protein, providing a specific lanthanide ion binding that weakly orients the protein in solution, A comparison of spectra of the protein with and without the EF-hand residues demonstrates that the structure of the native protein is not perturbed by this modification, since there are minimal chemical shift changes. With a lanthanide but not calcium bound to the EF-hand, the protein is weakly oriented by the magnetic field, since residual dipolar couplings can be measured. Since the signs and magnitudes of the couplings varied with the type of lanthanide, this demonstrated the ability to obtain multiple orientations of the protein in solution. The sample is a membrane protein in lipid micelles that disrupted the commonly employed bicelle and filamentous phage solutions; therefore, the addition of a specific metal binding site in the form of an EF-hand may provide a general approach to orienting proteins where the addition of external agents is problematic. An additional benefit is that the lanthanide ions perturb the protein resonances in ways that provide unique orientational and distance constraints. (C) 2000 Academic Press.