PSEUDYANA for NMR structure calculation of paramagnetic metalloproteins using torsion angle molecular dynamics (original) (raw)
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Paramagnetic shifts in solid-state NMR of proteins to elicit structural information
Proceedings of the National Academy of Sciences, 2008
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. matrix metalloproteinase ͉ pseudocontact shift ͉ microcrystal ͉ cobalt(II) S olid-state NMR (SSNMR) on biomolecules is a rapidly
PARAssign—paramagnetic NMR assignments of protein nuclei on the basis of pseudocontact shifts
Journal of Biomolecular NMR, 2013
The use of paramagnetic NMR data for the refinement of structures of proteins and protein complexes is widespread. However, the power of paramagnetism for protein assignment has not yet been fully exploited. PARAssign is software that uses pseudocontact shift data derived from several paramagnetic centers attached to the protein to obtain amide and methyl assignments. The ability of PARAssign to perform assignment when the positions of the paramagnetic centers are known and unknown is demonstrated. PARAssign has been tested using synthetic data for methyl assignment of a 47 kDa protein, and using both synthetic and experimental data for amide assignment of a 14 kDa protein. The complex fitting space involved in such an assignment procedure necessitates that good starting conditions are found, both regarding placement and strength of paramagnetic centers. These starting conditions are obtained through automated tensor placement and user-defined tensor parameters. The results presented herein demonstrate that PARAssign is able to successfully perform resonance assignment in large systems with a high degree of reliability. This software provides a method for obtaining the assignments of large systems, which may previously have been unassignable, by using 2D NMR spectral data and a known protein structure.
Journal of the American Chemical Society, 2012
A natural bond orbital (NBO) analysis of unpaired electron spin density in metalloproteins is presented, which allows a fast and robust calculation of paramagnetic NMR parameters. Approximately 90% of the unpaired electron spin density occupies metal−ligand NBOs, allowing the majority of the density to be modeled by only a few NBOs that reflect the chemical bonding environment. We show that the paramagnetic relaxation rate of protons can be calculated accurately using only the metal−ligand NBOs and that these rates are in good agreement with corresponding rates measured experimentally. This holds, in particular, for protons of ligand residues where the point-dipole approximation breaks down. To describe the paramagnetic relaxation of heavy nuclei, also the electron spin density in the local orbitals must be taken into account. Geometric distance restraints for 15 N can be derived from the paramagnetic relaxation enhancement and the Fermi contact shift when local NBOs are included in the analysis. Thus, the NBO approach allows us to include experimental paramagnetic NMR parameters of 15 N nuclei as restraints in a structure optimization protocol. We performed a molecular dynamics simulation and structure determination of oxidized rubredoxin using the experimentally obtained paramagnetic NMR parameters of 15 N. The corresponding structures obtained are in good agreement with the crystal structure of rubredoxin. Thus, the NBO approach allows an accurate description of the geometric structure and the dynamics of metalloproteins, when NMR parameters are available of nuclei in the immediate vicinity of the metal-site.
Perspectives in paramagnetic NMR of metalloproteins
Dalton Trans., 2008
NMR experiments and tools for the characterization of the structure and dynamics of paramagnetic proteins are presented here. The focus is on the importance of 13 C direct-detection NMR for the assignment of paramagnetic systems in solution, on the information contained in paramagnetic effects observed both in solution and in the solid state, and on novel paramagnetism-based tools for the investigation of conformational heterogeneity in protein-protein complexes or in multi-domain proteins.
Long-range paramagnetic NMR data can provide a closer look on metal coordination in metalloproteins
Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry, 2018
Paramagnetic NMR data can be profitably incorporated in structural refinement protocols of metalloproteins or metal-substituted proteins, mostly as distance or angle restraints. However, they could in principle provide much more information, because the magnetic susceptibility of a paramagnetic metal ion is largely determined by its coordination sphere. This information can in turn be used to evaluate changes occurring in the coordination sphere of the metal when ligands (e.g.: inhibitors) are bound to the protein. This gives an experimental handle on the molecular structure in the vicinity of the metal which falls in the so-called blind sphere. The magnetic susceptibility anisotropy tensors of cobalt(II) and nickel(II) ions bound to human carbonic anhydrase II in free and inhibited forms have been determined. The change of the magnetic susceptibility anisotropy is directly linked to the binding mode of different ligands in the active site of the enzyme. Indication about the metal c...
Efficient χ-tensor determination and NH assignment of paramagnetic proteins
Journal of Biomolecular NMR, 2006
Anisotropic magnetic susceptibility tensors v of paramagnetic metal ions are manifested in pseudocontact shifts, residual dipolar couplings, and other paramagnetic observables that present valuable long-range information for structure determinations of protein-ligand complexes. A program was developed for automatic determination of the v-tensor anisotropy parameters and amide resonance assignments in proteins labeled with paramagnetic metal ions. The program requires knowledge of the three-dimensional structure of the protein, the backbone resonance assignments of the diamagnetic protein, and a pair of 2D 15 N-HSQC or 3D HNCO spectra recorded with and without paramagnetic metal ion. It allows the determination of reliable v-tensor anisotropy parameters from 2D spectra of uniformly 15 N-labeled proteins of fairly high molecular weight. Examples are shown for the 185-residue N-terminal domain of the subunit e from E. coli DNA polymerase III in complex with the subunit h and La 3+ in its diamagnetic and Dy 3+ , Tb 3+ , and Er 3+ in its paramagnetic form.
Pseudo-Contact NMR Shifts over the Paramagnetic Metalloprotein CoMMP-12 from First Principles
Angewandte Chemie International Edition, 2016
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.
Journal of The American Chemical Society, 2007
An innovative analytical/computational approach is presented to provide maximum allowed probabilities (MAPs) of conformations in protein domains not rigidly connected. The approach is applied to calmodulin and to its adduct with R-synuclein. Calmodulin is a protein constituted by two rigid domains, each of them composed by two calcium-binding EF-hand motifs, which in solution are largely free to move with respect to one another. We used the N60D mutant of calmodulin, which had been engineered to selectively bind a paramagnetic lanthanide ion to only one of its four calcium binding sites, specifically in the second EF-hand motif of the N-terminal domain. In this way, pseudocontact shifts (pcs's) and selforientation residual dipolar couplings (rdc's) measured on the C-terminal domain provide information on its relative mobility with respect to the domain hosting the paramagnetic center. Available NMR data for terbium(III) and thulium(III) calmodulin were supplemented with additional data for dysprosium(III), analogous data were generated for the R-synuclein adduct, and the conformations with the largest MAPs were obtained for both systems. The MAP analysis for calmodulin provides further information on the variety of conformations experienced by the system. Such variety is somewhat reduced in the calmodulin-R-synuclein adduct, which however still retains high flexibility. The flexibility of the calmodulin-R-synuclein adduct is an unexpected result of this research.