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 s ite were added to the N-terminus of a protein, providing a specific lanthan ide ion binding that weakly orients the protein in solution, A comparison o f spectra of the protein with and without the EF-hand residues demonstrates that the structure of the native protein is not perturbed by this modifica tion, 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 ma gnetic field, since residual dipolar couplings can be measured. Since the s igns and magnitudes of the couplings varied with the type of lanthanide, th is demonstrated the ability to obtain multiple orientations of the protein in solution. The sample is a membrane protein in lipid micelles that disrup ted the commonly employed bicelle and filamentous phage solutions; therefor e, 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 lanthanid e ions perturb the protein resonances in ways that provide unique orientati onal and distance constraints. (C) 2000 Academic Press.