We apply the Kurland-McGarvey (J. Magn. Reson. 1970, 2, 286) theory for the NMR shielding of paramagnetic molecules, particularly its special case limited to the ground-state multiplet characterized by zero-field splitting (ZFS) interaction of the form S·D·S. The correct formulation for this problem was recently presented by Soncini and Van den Heuvel (J. Chem. Phys. 2013, 138, 054113). With the effective electron spin quantum number S, the theory involves 2S+1 states, of which all but one are low-lying excited states, between which magnetic couplings take place by Zeeman and hyperfine interactions. We investigate these couplings as a function of temperature, focusing on both the high- and low-temperature behaviors. As has been seen in work by others, the full treatment of magnetic couplings is crucial for a realistic description of the temperature behavior of NMR shielding up to normal measurement temperatures. At high temperatures, depending on the magnitude of ZFS, the effect of magnetic couplings diminishes, and the Zeeman and hyperfine interactions become effectively averaged in the thermally occupied states of the multiplet. At still higher temperatures, the ZFS may be omitted altogether, and the shielding properties may be evaluated using a doublet-like formula, with all the 2S+1 states becoming effectively degenerate at the limit of vanishing magnetic field. We demonstrate these features using first-principles calculations of Ni(II), Co(II), Cr(II), and Cr(III) complexes, which have ZFS of different sizes and signs. A non-monotonic inverse temperature dependence of the hyperfine shift is predicted for axially symmetric integer-spin systems with a positive D parameter of ZFS. This is due to the magnetic coupling terms that are proportional to kT at low temperatures, canceling the Curie-type 1/kT prefactor of the hyperfine shielding in this case.

We apply the theory of the nuclear magnetic resonance (NMR) chemical shift for paramagnetic systems to demanding cobalt(II) complexes. Paramagnetic NMR (pNMR) chemical shift results by density-functional theory (DFT) can be very far from the experimental values. Therefore, it is of interest to investigate the applicability of electron-correlated ab initio computational methods to achieve useful accuracy. Here, we use ab initio wave function based electronic structure methods to calculate the pNMR chemical shift within the theoretical framework established recently. We applied the N-electron valence-state perturbation theory (NEVPT2) on three Co(II) systems, where the active space of the underlying complete active space self-consistent field (CASSCF) wave function consists of seven electrons in the five metal 3d orbitals. These complexes have the S = 3/2 electronic ground state consisting of two doublets separated by zero-field splitting (ZFS). To calculate the hyperfine coupling tensor A, DFT was used, while the g- and ZFS-tensors were calculated using the ab initio CASSCF and NEVPT2 methods. These results were combined to obtain the total chemical shifts. The shifts obtained from these calculations are in generally good agreement with the experimental results, in some cases suggesting a reassignment of the signals. The accuracy of this mixed ab initio/DFT approach is very promising for further applications to demanding pNMR problems involving transition metals.

In the light of occurrence of bacterial strains with multiple resistances against most antibiotics, antimicrobial peptides that interact with the outer layer of Gram-negative bacteria, such as polymyxin (PMX), have recently received increased attention. Here we present a study of the interactions of PMX-B, -E, and -M with lipopolysaccharide (LPS) from a deep rough mutant strain of Escherichia coli. A method for efficient purification of biosynthetically produced LPS using reversed-phase high-performance liquid chromatography in combination with ternary solvent mixtures was developed. LPS was incorporated into a membrane model, dodecylphosphocholine micelles, and its interaction with polymyxins was studied by heteronuclear NMR spectroscopy. Data from chemical shift mapping using isotopelabeled LPS or labeled polymyxin, as well as from isotope-filtered nuclear Overhauser effect spectroscopy experiments, reveal the mode of interaction of LPS with polymyxins. Using molecular dynamics calculations the complex of LPS with PMX-B in the presence of dodecylphosphocholine micelles was modeled using restraints derived from chemical shift mapping data and intermolecular nuclear Overhauser effects. In the modeled complex the macrocycle of PMX is centered around the phosphate group at GlcN-B, and additional contacts from polar side chains are formed to GlcN-A and Kdo-C, whereas hydrophobic side chains penetrate the acyl-chain region.

Caspases are proteases with an active-site cysteine and aspartate specificity in their substrates. They are involved in apoptotic cell death and inflammation, and dysfunction of these enzymes is directly linked to a variety of diseases. Caspase-8 initiates an apoptotic pathway triggered by external stimuli. It was previously characterized in its active inhibitor bound state by crystallography. Here we present the solution structure of the monomeric unprocessed catalytic domain of the caspase-8 zymogen, procaspase-8, showing for the first time the position of the linker and flexibility of the active site forming loops. Biophysical studies of carefully designed mutants allowed disentangling dimerization and processing, and we could demonstrate lack of activity of monomeric uncleaved procaspase-8 and of a processed but dimerization-incompetent mutant. The data provide experimental support in so-far unprecedented detail, and reveal why caspase-8 (and most likely other initiator caspases) needs the dimerization platform during activation.

Long-range pseudo-contact NMR shifts (PCSs) provide important restraints for the structure refinement of proteins when a paramagnetic metal center is present, either naturally or introduced artificially. Here we show that ab initio quantum-chemical methods and a modern version of the Kurland-McGarvey approach for paramagnetic NMR (pNMR) shifts in the presence of zero-field splitting (ZFS) together provide accurate predictions of all PCSs in a metalloprotein (high-spin cobalt-substituted MMP-12 as a test case). Computations of 314 13 C PCSs via g-and ZFS-tensors based on multi-reference methods provide a reliable bridge between EPRparameter-and susceptibility-based pNMR formalisms. Due to the high sensitivity of PCSs to even small structural differences, local structures based either on X-ray diffraction or on various DFT optimizations could be evaluated critically by comparing computed and experimental PCSs. Many DFT functionals provide insufficiently accurate structures. We also found the available 1RMZ PDB X-ray structure to exhibit deficiencies related to binding of a hydroxamate inhibitor. This has led to a newly refined PDB structure for MMP-12 (5LAB) that provides a more accurate coordination arrangement and PCSs.The anisotropic magnetic susceptibility [1] of paramagnetic metal ions induces the so-called pseudo-contact shifts (PCSs) in NMR spectra, which can be observed for nuclei between 5 Å and 40 Å from the metal center. [2] PCSs provide precious structural information on the biomolecules on which they are measured, both in solution and in the solid state. [3] PCS-based structural restraints have also become important for protein NMR crystallography. [4] Their importance is further enhanced by recent developments in fast magic-angle spinning (MAS) combined with high-field instruments. [4d, 5] While PCSs can thus provide crucial information on the structure of a metalloprotein as a whole, NMR is typically blind to nuclei near the paramagnetic metal center due to fast paramagnetic relaxation. The computation of pNMR shifts by first-principles quantum-chemical (QC) methods, on the other hand, has recently progressed appreciably, in particular by inclusion of the non-contact terms in small to medium-sized molecules, with no fundamental limitations close to the metal center. [6] There has so far been no attempt to access the longrange PCSs in larger biological systems by first-principles calculations, as the molecular sizes needed for an explicit treatment of the hyperfine coupling (HFC) anisotropies appeared prohibitive.Here we show that introduction of the point-dipole approximation (PDA), appropriate for the long-range spin-dipolar HFCs, into modern quantum-chemical pNMR shift machinery can be used to compute long-range PCSs based on accurate multireference ab initio calculations of g-and zero-field splitting (ZFS) D-tensors. Using such a combined approach, we have computed the entire set of 314 previously measured 13 C long-range PCSs [4a] (and further shifts from nuclei closer to the meta...