Probing coiled-coil assembly by paramagnetic NMR spectroscopy.

Here a new method to determine the oligomeric state and orientation of coiled-coil peptide motifs is described. Peptides K and E, which are designed to form a parallel heterodimeric complex in aqueous solution, were labeled with the aromatic amino acids tryptophan and tyrosine on the C-terminus respectively as 'fingerprint' residues. One of the peptides was also labeled with the paramagnetic probe MTSL. One dimensional proton NMR spectroscopy was used to study the peptide quaternary structure by monitoring the signal suppression of the aromatic labels due to proximity of the nitroxyl radical. 1D-NMR confirmed that the peptides K and E form a heterodimeric coiled coil with a parallel orientation. In addition, fluorescence emission quenching of the aromatic labels due to electron exchange with a nitroxyl radical confirmed the parallel coiled coil orientation. Thus, paramagnetic nitroxide and aromatic fluorophore labeling of peptides yields valuable information regarding the quaternary structure from 1D-NMR and steady-state fluorescence measurements. This convenient method is useful not only to investigate coiled coil assembly, but can also be applied to any defined supramolecular assembly.