Protein fold determined by paramagnetic magic-angle spinning solid-state NMR spectroscopy

Abstract

Biomacromolecules that are challenging for the usual structural techniques can be studied with atomic resolution by solid-state nuclear magnetic resonance. However, the paucity of >5 Å distance restraints, traditionally derived from measurements of magnetic dipole-dipole couplings between protein nuclei, is a major bottleneck that hampers such structure elucidation efforts. Here we describe a general approach that enables the rapid determination of global protein fold in the solid phase via measurements of nuclear paramagnetic relaxation enhancements (PREs) in several analogs of the protein of interest containing covalently-attached paramagnetic tags, without the use of conventional internuclear distance restraints. The method is demonstrated using six cysteine-EDTA-Cu2+ mutants of the 56-residue B1 immunoglobulin-binding domain of protein G, for which ~230 longitudinal backbone 15N PREs corresponding to ~10-20 Å distances were obtained. The mean protein fold determined in this manner agrees with the X-ray structure with a backbone atom root-mean-square deviation of 1.8 Å.