Probing surface accessibility of proteins using paramagnetic relaxation in solid-state NMR spectroscopy

Paramagnetic Relaxation Enhancement (PRE) can be used to accelerate NMR data acquisition by reducing the longitudinal proton relaxation time T ₁ in the solid state. We show that the presence of paramagnetic compounds in the bulk solvent induces a site-specific relaxation in addition to local dynamics, which is dependent on the surface accessibility of the respective amide proton in the protein. Differentiation between paramagnetic relaxation and dynamics was achieved by a comparison of ¹H T ₁ times obtained from microcrystalline protein samples prepared with different concentrations of the Cu^II(edta) chelate. We find that relaxation can in addition be mediated by hydroxyl groups, which transfer relaxation by their ability to exchange with the quickly relaxing bulk solvent. Furthermore, relaxation seems to be transferred by water molecules which diffuse into the protein structure and yield an efficient difference PRE in flexible regions of the protein. The experiments are demonstrated using a perdeuterated sample of the α-spectrin SH3 domain, which was microcrystallized from a buffer containing 90% D₂O. Deuteration is a prerequisite to avoid spin diffusion which would otherwise compromise site specific resolution.