Expanding the utility of NMR restraints with paramagnetic compounds: background and practical aspects.

NMR spectroscopy is one of the most important methods for determining protein structures. The scientific community is constantly pushing the limits of NMR spectroscopy by investigating proteins of increasing sizes including membrane proteins, decreasing acquisition times by alternative sampling techniques, and automating signal assignment for high-throughput protein structure determination. Application of NMR spectroscopy to large or membrane proteins is one of the long-standing limitations as slow tumbling of the protein/membrane-mimetic complex results in line-broadening that complicates the acquisition of distance restraints based on the Nuclear Overhauser Effect (NOE) for structure elucidation. Furthermore, the spectral dispersion for alpha-helical membrane proteins is typically smaller than for beta-barrels resulting in peak overlap that complicates signal assignment. Therefore, other types of restraints are needed that complement or replace NOEs for structure elucidation. The present review focuses on a set of structural restraints that can be observed when a paramagnetic center is introduced into the protein. A paramagnetic center in a protein leads to an interaction of the unpaired electron with the nuclear spins of the protein. This results in distance- and sometimes orientation-dependent effects that can be exploited as structural restraints. The three practically most often utilized phenomena are paramagnetic relaxation enhancements (PREs, i.e. contributions to the relaxation rate), Pseudo-Contact Shifts (PCSs, i.e. contributions to the chemical shift), and Residual Dipolar Couplings (RDCs). The introduction of a paramagnetic group into the protein matrix can be achieved by either substitution of the metal ion in metalloproteins (which make up to about 25% of the proteins in living organisms [1]) or by attachment of metal-binding peptides or small molecule tags coordinating a paramagnetic metal ion. Metal ions suitable for the measurement of paramagnetic restraints are those from the transition or lanthanide series where each of the metal ions offers different characteristics. 1.1. Magnetic Susceptibility Anisotropy and resulting effects Fig.1 shows a scheme that puts the paramagnetic restraints in context to relaxation and alignment. Which of these restraints are measureable depends primarily on the presence of magnetic susceptibility anisotropy (MSA), a deviation of the magnetic susceptibility tensor from symmetry. All paramagnetic species exhibit dipolar PREs: in compounds with (nearly) isotropic magnetic susceptibility, such as nitroxide spin-labels (methane-thio-sulfonate (MTSL) for instance), Gd, Mn, Cu, doxyl-stearic acid (DSA) the dipolar interactions of the unpaired electron with the nuclei of the protein result in distance-dependent line-broadening. The efficiency of the line-broadening depends on the magnetic properties of the metal ion. Figure 1 Overall context If the paramagnetic center possesses MSA, as is the case for lanthanide ions (except for Gd, and the diamagnetic species Lu and La), other PRE contributions emerge that add to the dipolar PREs. The largest of these paramagnet-induced relaxation phenomena are the Curie and CSA relaxation that can interfere with each other in so called cross-correlation effects. Additionally, MSA induces hyperfine shifts consisting of two contributions: the contact shift that is only observed at very short distances around the metal ion and the PCS whose orientation- and distance-dependence can be exploited as structural restraints. Furthermore, the presence of MSA will lead to partial alignment of the protein in the magnetic field – this is called internal alignment. While direct dipolar couplings between nuclei average out for isotropic tumbling of the molecule, partial alignment retains this spatial anisotropy and results in RDCs. These RDCs are a factor of ~1000 smaller than full dipolar couplings allowing their convenient determination. Experimentally, RDCs are observed as a perturbation of the J-couplings, if the nuclei are connected by a chemical bond. As RDCs depend on the mutual orientation of the internuclear vectors in the molecular frame they are useful restraints in structure determination. RDCs gained importance in conjunction with protein structure prediction in the last decade, as RDCs can also be measured if the protein is aligned by other means than a paramagnetic center, for instance by using external alignment media such as bicelles [2], poly-acrylamide gels [3], or bacteriophages [4]. These external alignment methods are not the subject of this review.