Protein structural studies by paramagnetic solid-state NMR spectroscopy aided by a compact cyclen-type Cu(II) binding tag

Paramagnetic relaxation enhancements (PREs) are a rich source of structural information in protein solid-state NMR spectroscopy. Here we demonstrate that PRE measurements in natively diamagnetic proteins are facilitated by a thiol-reactive compact, cyclen-based, high-affinity Cu 2+ binding tag, 1-[2-(pyridin-2-yldisulfanyl)ethyl]-1,4,7,10-tetraazacyclododecane (TETAC), that overcomes the key shortcomings associated with the use of larger, more flexible metal-binding tags. Using the TETAC–Cu 2+ K28C mutant of B1 immunoglobulin-binding domain of protein G as a model, we find that amino acid residues located within ~10 Å of the Cu 2+ center experience considerable transverse PREs leading to severely attenuated resonances in 2D 15 N– 13 C correlation spectra. For more distant residues, electron–nucleus distances are accessible via quantitative measurements of longitudinal PREs, and we demonstrate such measurements for 15 N–Cu 2+ distances up to ~20 Å.