The obvious drawback of the MeTROSY approach is that it is not applicable to 14 out of 20
amino acids. While typically Ganetespib cell line only methyl groups in Ile, Leu, Val are observed , specific isotope labeling strategies have also been developed for Met, Ala (reviewed in ) and Thr . The limited sequence coverage of MeTROSY can be alleviated to some extent by site-specific introduction of 13CH3 groups at desired positions, for example by site-directed mutagenesis, if the structure allows for it. Such MeTROSY-based methionine scanning of solvent exposed residues has recently been proposed to map binding interfaces . Alternatively, a single methyl probe may be introduced by di-sulfide bond formation with a 13CH3–S group from methylmethanethiosulfonate resulting in the methione-mimic S-methylthiocysteine . Both backbone amide-based TROSY and MeTROSY experiments have proven to allow studies of protein structure, dynamics and interaction in systems as large as 1 MDa (Table 1). In addition, other approaches such 13C direct detection
 and  or stereo-selective amino acid labeling  and  can help to study large molecular systems. Yet, despite these advances, low molecular tumbling rates inherently limit the applicability of solution-state NMR. In contrast, the resonance line width in magic-angle spinning (MAS) solid-state NMR (ssNMR) is independent of the protein molecular weight. Recently, Reif find more and co-workers Dimethyl sulfoxide as well as Bertini et al. have shown that also soluble protein complexes can be investigated by ssNMR in an approach referred to as FROSTY
 or sedNMR (sedimented NMR) . Strong centrifugal forces during MAS lead to reversible protein sedimentation at the inner wall of the MAS rotor for protein complexes above 100 kDa, effectively creating a solid. Complexes can also be sedimented into the rotor by conventional ultracentrifugation using a dedicated filling-device  and . Sedimented ssNMR is thus a promising method to overcome the size barrier in solution NMR. Various types of NMR experiments can provide low-resolution structural information even for large systems. Assuming that the stoichiometry and composition of the macromolecular complex under study are known, these can provide useful insights into binding sites, distances between specific pairs or groups of atoms, and relative orientation of subunits. The most frequently used data and their information content are summarized in Table 2. The workhorse of NMR for interaction studies is chemical shift perturbations (CSP) mapping, a simple comparison of peak positions in spectra before and after adding a (unlabeled) binding partner. Ligand binding induces changes in the chemical environment of the observed protein, which can conveniently be monitored by NMR (Fig. 1).