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

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

Abstract

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 Cu2+ 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 TETACâ??Cu2+ K28C mutant of B1 immunoglobulin-binding domain of protein G as a model, we find that amino acid residues located within ~10Â*Ã? of the Cu2+ center experience considerable transverse PREs leading to severely attenuated resonances in 2D 15Nâ??13C correlation spectra. For more distant residues, electronâ??nucleus distances are accessible via quantitative measurements of longitudinal PREs, and we demonstrate such measurements for 15Nâ??Cu2+ distances up to ~20Â*Ã?.



Source: Journal of Biomolecular NMR