文摘
In a wide variety of proteins, insolubility presents a challenge to structural biology, as X-raycrystallography and liquid-state NMR are unsuitable. Indeed, no general approach is available as of todayfor studying the three-dimensional structures of membrane proteins and protein fibrils. We here demonstrate,at the example of the microcrystalline model protein Crh, how high-resolution 3D structures can be derivedfrom magic-angle spinning solid-state NMR distance restraints for fully labeled protein samples. First, weshow that proton-mediated rare-spin correlation spectra, as well as carbon-13 spin diffusion experiments,provide enough short, medium, and long-range structural restraints to obtain high-resolution structures ofthis 2 × 10.4 kDa dimeric protein. Nevertheless, the large number of 13C/15N spins present in this protein,combined with solid-state NMR line widths of about 0.5-1 ppm, induces substantial ambiguities in resonanceassignments, preventing 3D structure determination by using distance restraints uniquely assigned on thebasis of their chemical shifts. In the second part, we thus demonstrate that an automated iterative assignmentalgorithm implemented in a dedicated solid-state NMR version of the program ARIA permits to resolve themajority of ambiguities and to calculate a de novo 3D structure from highly ambiguous solid-state NMRdata, using a unique fully labeled protein sample. We present, using distance restraints obtained throughthe iterative assignment process, as well as dihedral angle restraints predicted from chemical shifts, the3D structure of the fully labeled Crh dimer refined at a root-mean-square deviation of 1.33 Å.