PREDITOR: a web server for predicting protein torsion angle restraints

Every year between 500 and 1000 peptide and protein structures are determined by NMR and deposited into the Protein Data Bank. However, the process of NMR structure determination continues to be a manually intensive and time-consuming task. One of the most tedious and error-prone aspects of this process involves the determination of torsion angle restraints including phi, psi, omega and chi angles. Most methods require many days of additional experiments, painstaking measurements or complex calculations. Here we wish to describe a web server, called PREDITOR, which greatly accelerates and simplifies this task. PREDITOR accepts sequence and/or chemical shift data as input and generates torsion angle predictions (with predicted errors) for phi, psi, omega and chi-1 angles. PREDITOR combines sequence alignment methods with advanced chemical shift analysis techniques to generate its torsion angle predictions. The method is fast (<40 s per protein) and accurate, with 88% of phi/psi predictions being within 30° of the correct values, 84% of chi-1 predictions being correct and 99.97% of omega angles being correct. PREDITOR is 35 times faster and up to 20% more accurate than any existing method. PREDITOR also provides accurate assessments of the torsion angle errors so that the torsion angle constraints can be readily fed into standard structure refinement programs, such as CNS, XPLOR, AMBER and CYANA. Other unique features to PREDITOR include dihedral angle prediction via PDB structure mapping, automated chemical shift re-referencing (to improve accuracy), prediction of proline cis/trans states and a simple user interface. The PREDITOR website is located at:

FlexOracle: predicting flexible hinges by identification of stable domains

<p>Abstract</p> <p>Background</p> <p>Protein motions play an essential role in catalysis and protein-ligand interactions, but are difficult to observe directly. A substantial fraction of protein motions involve hinge bending. For these proteins, the accurate identification of flexible hinges connecting rigid domains would provide significant insight into motion. Programs such as GNM and FIRST have made global flexibility predictions available at low computational cost, but are not designed specifically for finding hinge points.</p> <p>Results</p> <p>Here we present the novel FlexOracle hinge prediction approach based on the ideas that energetic interactions are stronger <it>within </it>structural domains than <it>between </it>them, and that fragments generated by cleaving the protein at the hinge site are independently stable. We implement this as a tool within the Database of Macromolecular Motions, MolMovDB.org. For a given structure, we generate pairs of fragments based on scanning all possible cleavage points on the protein chain, compute the energy of the fragments compared with the undivided protein, and predict hinges where this quantity is minimal. We present three specific implementations of this approach. In the first, we consider only pairs of fragments generated by cutting at a <it>single </it>location on the protein chain and then use a standard molecular mechanics force field to calculate the enthalpies of the two fragments. In the second, we generate fragments in the same way but instead compute their free energies using a knowledge based force field. In the third, we generate fragment pairs by cutting at <it>two </it>points on the protein chain and then calculate their free energies.</p> <p>Conclusion</p> <p>Quantitative results demonstrate our method's ability to predict known hinges from the Database of Macromolecular Motions.</p

A simple method to predict protein flexibility using secondary chemical shifts

Protein motions play a critical role in many biological processes, such as enzyme catalysis, allosteric regulation, antigen-antibody interactions, and protein-DNA binding. NMR spectroscopy occupies a unique place among methods for investigating protein dynamics due to its ability to provide site-specific information about protein motions over a large range of time scales. However, most NMR methods require a detailed knowledge of the 3D structure and/or the collection of additional experimental data (NOEs, T\u2081, T\u2082, etc.) to accurately measure protein dynamics. Here we present a simple method based on chemical shift data that allows accurate, quantitative, site-specific mapping of protein backbone mobility without the need of a three-dimensional structure or the collection and analysis of NMR relaxation data. Further, we show that this chemical shift method is able to quantitatively predict per-residue RMSD values (from both MD simulations and NMR structural ensembles) as well as model-free backbone order parameters.NRC publication: N

The RCI server: rapid and accurate calculation of

doi:10.1093/nar/gkm32

A Simple Method to Measure Protein Side-Chain Mobility Using NMR Chemical Shifts

Protein side-chain motions are involved in many important biological processes including enzymatic catalysis, allosteric regulation, and the mediation of protein–protein, protein–DNA, protein–RNA, and protein–cofactor interactions. NMR spectroscopy has long been used to provide insights into the motions of side-chain groups. Currently, the method of choice for studying side-chain dynamics by NMR is the measurement of methyl ²H autorelaxation. Methyl ²H autorelaxation exhibits simple relaxation mechanisms and can be straightforwardly converted to meaningful dynamic parameters. However, methyl groups can only be found in 6 of 19 side-chain bearing amino acids. Consequently, only a sparse assessment of protein side-chain dynamics is possible. Therefore, there is a significant interest in developing novel methods of studying side-chain motions that can be applied to all types of side-chains. Here, we show how side-chain chemical shifts can be used to determine the magnitude of fast side-chain motions in proteins. The chemical shift method is applicable to all side-chain bearing residues and does not require any additional measurements beyond standard NMR experiments for backbone and side-chain assignments

Solution NMR of a 463-Residue Phosphohexomutase: Domain 4 Mobility, Substates, and Phosphoryl Transfer Defect

Phosphomannomutase/phosphoglucomutase contributes to the infectivity of <i>Pseudomonas aeruginosa</i>, retains and reorients its intermediate by 180°, and rotates domain 4 to close the deep catalytic cleft. Nuclear magnetic resonance (NMR) spectra of the backbone of wild-type and S108C-inactivated enzymes were assigned to at least 90%. ¹³C secondary chemical shifts report excellent agreement of solution and crystallographic structure over the 14 α-helices, C-capping motifs, and 20 of the 22 β-strands. Major and minor NMR peaks implicate substates affecting 28% of assigned residues. These can be attributed to the phosphorylation state and possibly to conformational interconversions. The S108C substitution of the phosphoryl donor and acceptor slowed transformation of the glucose 1-phosphate substrate by impairing <i>k</i>_cat. Addition of the glucose 1,6-bisphosphate intermediate accelerated this reaction by 2–3 orders of magnitude, somewhat bypassing the defect and apparently relieving substrate inhibition. The S108C mutation perturbs the NMR spectra and electron density map around the catalytic cleft while preserving the secondary structure in solution. Diminished peak heights and faster ¹⁵N relaxation suggest line broadening and millisecond fluctuations within four loops that can contact phosphosugars. ¹⁵N NMR relaxation and peak heights suggest that domain 4 reorients slightly faster in solution than domains 1–3, and with a different principal axis of diffusion. This adds to the crystallographic evidence of domain 4 rotations in the enzyme, which were previously suggested to couple to reorientation of the intermediate, substrate binding, and product release

Solution NMR of a 463-Residue Phosphohexomutase: Domain 4 Mobility, Substates, and Phosphoryl Transfer Defect

Phosphomannomutase/phosphoglucomutase contributes to the infectivity of <i>Pseudomonas aeruginosa</i>, retains and reorients its intermediate by 180°, and rotates domain 4 to close the deep catalytic cleft. Nuclear magnetic resonance (NMR) spectra of the backbone of wild-type and S108C-inactivated enzymes were assigned to at least 90%. ¹³C secondary chemical shifts report excellent agreement of solution and crystallographic structure over the 14 α-helices, C-capping motifs, and 20 of the 22 β-strands. Major and minor NMR peaks implicate substates affecting 28% of assigned residues. These can be attributed to the phosphorylation state and possibly to conformational interconversions. The S108C substitution of the phosphoryl donor and acceptor slowed transformation of the glucose 1-phosphate substrate by impairing <i>k</i>_cat. Addition of the glucose 1,6-bisphosphate intermediate accelerated this reaction by 2–3 orders of magnitude, somewhat bypassing the defect and apparently relieving substrate inhibition. The S108C mutation perturbs the NMR spectra and electron density map around the catalytic cleft while preserving the secondary structure in solution. Diminished peak heights and faster ¹⁵N relaxation suggest line broadening and millisecond fluctuations within four loops that can contact phosphosugars. ¹⁵N NMR relaxation and peak heights suggest that domain 4 reorients slightly faster in solution than domains 1–3, and with a different principal axis of diffusion. This adds to the crystallographic evidence of domain 4 rotations in the enzyme, which were previously suggested to couple to reorientation of the intermediate, substrate binding, and product release

Comparative analysis of essential collective dynamics and NMR-derived flexibility profiles in evolutionarily diverse prion proteins

Collective motions on ns-µs time scales are known to have a major impact on protein folding, stability, binding and enzymatic efficiency. It is also believed that these motions may have an important role in the early stages of prion protein misfolding and prion disease. In an effort to accurately characterize these motions and their potential influence on the misfolding and prion disease transmissibility we have conducted a combined analysis of molecular dynamic simulations and NMR-derived flexibility measurements over a diverse range of prion proteins. Using a recently developed numerical formalism, we have analyzed the essential collective dynamics (ECD) for prion proteins from eight different species including human, cow, elk, cat, hamster, chicken, turtle and frog. We also compared the numerical results with flexibility profiles generated by the random coil index (RCI) from NMR chemical shifts. Prion protein backbone flexibility derived from experimental NMR data and from theoretical computations show strong agreement with each other, demonstrating that it is possible to predict the observed RCI profiles employing the numerical ECD formalism. Interestingly, flexibility differences in the loop between second b strand (S2) and the second a helix (HB) appear to distinguish prion proteins from species that are susceptible to prion disease and those that are resistant. Our results show that the different levels of flexibility in the S2-HB loop in various species are predictable via the ECD method, indicating that ECD may be used to identify disease resistant variants of prion proteins, as well as the influence of prion proteins mutations on disease susceptibility or misfolding propensity