The recent observation of pseudocontact shifts (pcs) in 13 C highresolution solid-state NMR of paramagnetic proteins opens the way to their application as structural restraints. Here, by investigating a microcrystalline sample of cobalt(II)-substituted matrix metalloproteinase 12 [CoMMP-12 (159 AA, 17.5 kDa)], it is shown that a combined strategy of protein labeling and dilution of the paramagnetic species (i.e., 13 C-, 15 N-labeled CoMMP-12 diluted in unlabeled ZnMMP-12, and 13 C-, 15 N-labeled ZnMMP-12 diluted in unlabeled CoMMP-12) allows one to easily separate the pcs contributions originated from the protein internal metal (intramolecular pcs) from those due to the metals in neighboring proteins in the crystal lattice (intermolecular pcs) and that both can be used for structural purposes. It is demonstrated that intramolecular pcs are significant structural restraints helpful in increasing both precision and accuracy of the structure, which is a need in solid-state structural biology nowadays. Furthermore, intermolecular pcs provide unique information on positions and orientations of neighboring protein molecules in the solid phase.S olid-state NMR (SSNMR) on biomolecules is a rapidly growing technique, with an increased interest based on its ability to determine protein structures in the solid phase (1, 2) and to permit the study of noncrystalline biomolecular systems such as membrane proteins (3) and fibrils (4, 5).Limitations in biomolecular structural determination through SSNMR are due to the difficulties in obtaining a large number of restraints to be used for structural purposes (4, 6-9). Most of the structural information is obtained through distance restraints, analogous to nuclear Overhauser effects (NOE) in solution NMR, which in the solid state are obtained through experiments such as proton-driven spin diffusion (PDSD) (1, 6, 10), and CHHC (11), whereas specifically designed sequences can be applied on short peptides (4,(12)(13)(14). The ability to obtain a large number of distance restraints is hampered by the reduced resolution of SSNMR spectra, which increases the amount of ambiguities in the assigned restraints (15), whereas relayed transfer (10, 16) and the effects of the dipolar truncation (17) affect the accuracy of these restraints. These problems have been tackled by working on samples prepared with selective labeling schemes (1, 6) and, more recently, on uniformly labeled proteins with the help of software able to provide automated PDSD/ CHHC assignment and on dealing with a large number of ambiguous restraints (10,15,18). However, even when additional dihedral angle restraints from backbone chemical shiftsthrough Chemical Shift Index (CSI) (19) or TALOS (20) programs-are used, the size of the affordable proteins has been, up to now, limited to small systems (Ͻ100 aa) (10,15,18). In this work, we show how SSNMR paramagnetic restraints such as pseudocontact shifts (pcs) can be used as additional sources of restraints for protein structural determination, even providing information abou...

The use of pseudocontact shifts arising from paramagnetic metal ions in a microcrystalline protein sample is proposed as a strategy to obtain unambiguous signal assignments in solid-state NMR spectra enabling distance extraction for protein structure calculation. With this strategy, 777 unambiguous (281 sequential, 217 medium-range, and 279 long-range) distance restraints could be obtained from PDSD, DARR, CHHC, and the recently introduced PAR and PAIN-CP solid-state experiments for the cobalt(II)-substituted catalytic domain of matrix metalloproteinase 12 (159 amino acids, 17.6 kDa). The obtained structure is a high resolution one, with backbone rmsd of 1.0 ± 0.2 Å, and is in good agreement with the X-ray structure (rmsd to X-ray 1.3 Å). The proposed strategy, which may be generalized for nonmetallo-proteins with the use of paramagnetic tags, represents a significant step ahead in protein structure determination using solid-state NMR.

The idea of NMR crystallography was conceived as soon as NMR spectroscopy was invented. Over the years, several efforts have been devoted to the development of NMR tools to complement X-ray diffraction results. Many of the NMR-related observables are short-ranged in nature, but the paramagnetic ones are not. Therefore, paramagnetism-based restraints are in principle suitable to assess spatial relationships among molecules. We will here review some of the underlying concepts and see how they apply to the problem of obtaining structural information on molecules in the lattice, and on the lattice itself. We will provide positive examples and discuss the negative issues that still haunt paramagnetism-based NMR crystallography.