Structural analysis of large protein complexes using solvent paramagnetic relaxation enhancements.

Understanding the function of biomolecular complexes requires their structural analysis at atomic resolution. To solve high-resolution structures by ab initio calculations typically data from NMR spectroscopy or X-ray crystallography are employed. In the latter approach, intrinsic flexibility and dynamics may prevent crystallization or introduce artificial conformations linked to crystal packing. Solution NMR spectroscopy does not suffer from such limitations, but is demanding because the adverse relaxation properties of large complexes may lead to extensive signal broadening and severe spectral overlap. Consequently, only sparse restraints can be obtained from such complexes by NMR experiments. Provided that the structures of the individual components of the complex (i.e. proteins, DNA/RNA) are available and that no large-scale conformational changes occur upon complex formation, experimental and computational approaches can be used to obtain the quaternary arrangement of complexes. The assembly of protein complexes by (semi-)rigid-body/ torsion-angle dynamics protocols is widely used and can yield accurate and precise structural models. However, for highmolecular-weight complexes, conventional approaches become highly ambiguous and often cannot distinguish between several possible arrangements of subunits in the complex. Different types of NMR data can provide powerful complementary information for restraining the interface and orientation of the complex and thereby resolve these ambiguities. One technique that has gained popularity in recent years is the site-specific incorporation of paramagnetic spin labels or lanthanide binding tags. These labels are covalently attached to single cysteine residues and provide a rich source of distance (paramagnetic relaxation enhancements) and orientation information (pseudocontact shifts, PCS; residual dipolar couplings, RDCs). A potential drawback of this approach is the requirement of a single accessible cysteine residue for cross-linking with the paramagnetic tag. This requires removal of native cysteines by site-directed mutagenesis which can be difficult for large proteins (that may contain many cysteines). Here, we present an efficient, generally applicable, and robust strategy for improving the precision and accuracy of (semi-)rigid-body/torsion-angle dynamics protocols based on paramagnetic relaxation enhancements (PREs) derived from the soluble paramagnetic agent Gd(DTPA-BMA) (DTPA: diethylenetriamine pentaacetic acid, BMA: bismethylamide). This chemically inert compound can simply be titrated to the sample, does not require any covalent modifications, and can be easily removed by dialysis. Dipolar interactions with the unpaired electron(s) of the chelated lanthanide ion (Gd) lead to a concentrationdependent increase of nuclear relaxation rates (PRE), which result, for example, in line broadening for NMR signals of nuclear spins. 6] The PRE can be translated into direct distance information that reflects the solvent accessibility or, in more quantitative terms, the (minimal) distance to the closest point of the molecular surface (Figure 1).