Solid-state NMR of a large membrane protein by paramagnetic relaxation enhancement

Membrane proteins play an important role in many biological functions. Solid-state NMR spectroscopy is uniquely suited for studying the structure and dynamics of membrane proteins in a membranous environment. The major challenge to obtain high quality solid-state NMR spectra of membrane proteins is sensitivity, due to limited quantities of labeled high-molecular-weight proteins. Here we demonstrate the incorporation of paramagnetic metal (Cu ²⁺) ions, through either ethylenediaminetetraacetic acid (EDTA) or a chelator lipid, into membrane protein samples for rapid data collection under fast magic-angle spinning (MAS) and low power ¹H decoupling. Spectral sensitivity of DsbB (20 kDa), an integral membrane protein, more than doubles in the same experimental time due to ¹H T₁ relaxation enhancement by Cu²⁺ ions, with DsbB native fold and active site intact. This technique can be implemented to acquire multidimensional solid-state NMR spectra for chemical shift assignments and structure elucidation of large membrane proteins with small sample quantities.