Distance restraints conformers

DG was primarily developed as a mathematical tool for obtaining spahal structures when pairwise distance information is given [118]. The DG method does not use any classical force fields. Thus, the conformational energy of a molecule is neglected and all 3D structures which are compatible with the distance restraints are presented. Nowadays, it is often used in the determination of 3D structures of small and medium-sized organic molecules. Gompared to force field-based methods, DG is a fast computational technique in order to scan the global conformational space. To get optimized structures, DG mostly has to be followed by various molecular dynamic simulation. 

The first step in the DG calculations is the generation of the holonomic distance matrix for aU pairwise atom distances of a molecule [121]. Holonomic constraints are expressed in terms of equations which restrict the atom coordinates of a molecule. For example, hydrogen atoms bound to neighboring carbon atoms have a maximum distance of 3.1 A. As a result, parts of the coordinates become interdependent and the degrees of freedom of the molecular system are confined. The acquisition of these distance restraints is based on the topology of a model structure with an arbitrary, but energetically optimized conformation. 

As it was mentioned in Section 9.4.1, 3D structures generated by DG have to be optimized. For this purpose, MD is a well-suited tool. In addition, MD structure calculations can also be performed if no coarse structural model exists. In both cases, pairwise atom distances obtained from NMR measurements are directly used in the MD computations in order to restrain the degrees of motional freedom of defined atoms (rMD Section 9.4.2.4). To make sure that a calculated molecular conformation is rehable, the time-averaged 3D structure must be stable in a free MD run (fMD Sechon 9.4.2.5J where the distance restraints are removed and the molecule is surrounded by expMcit solvent which was also used in the NMR measurement Before both procedures are described in detail the general preparation of an MD run (Section 9.4.2.1), simulations in vacuo (Section 9.4.2.2) and the handling of distance restraints in a MD calculation (Section 9.4.2.3) are treated. Finally, a short overview of the SA technique as a special M D method is given in Sechon 9.4.2.6. 

Using 18 trNOE-derived distance restraints and 13 trCCR-derived backbone torsion angle restraints, structure calculations using distance geometry and simulated annealing [48] resulted in a well-defined structure of the IKK/1-derived peptide bound to NEMO. The backbone structure is displayed in Fig. 7B and is compared with the result of the calculation carried out using the trNOE-derived distance restraints alone. It is obvious from Eig. 7 that only the combination of the trNOE- and trCCR-derived restraints results in the structure elucidation of the bound conformation of this peptide. 

Fig. 7 Solution of structure calculations of the NBD-peptide bound to NEMO. A Only trNOE-derived distance restraints were used in the calculation. The structure is not defined by the distance restraints alone. B trNOE-derived distance restraints and trCCR-derived torsion angle restraints were combined. These restraints complement each other and are sufficient to define the NEMO-bound conformation of the IKK/8-derived peptide. In both cases ten solutions were superimposed...

Two conformations of EpoA in complex with tubulin have been proposed on the basis of EC [26] and NMR [76, 96] data, respectively (Fig. 11). The tubulin-bound conformation of EpoA was determined by solution NMR spectroscopy [96] before the EC structure of EpoA bound to tubulin was available. The observation that, in a 100 1 mixture with tubulin, NOE cross-peaks of EpoA have negative sign, indicated that there is a fast exchange equilibrium in solution. This offered the opportunity to measure transferred NMR experiments, that report on the bound conformation of the ligand. A total of 46 interproton distances were derived from cross-peak volumes in tr-NOE spectra. However, these distance restraints did not suffice to define a unique conformation, as several distinct structures were consistent with them. Transferred cross-correlated relaxation (Sect. 2.2.1.3) provided the additional dihedral restraints that were crucial to define the bound conformation [96, 97], One requirement to measure CH-CH dipolar and CH-CO dipolar-CSA CCR rates is that the carbon atoms involved in the interaction are labeled with 13C. The availability of a 13C-labeled sample of EpoA offered the opportunity to derive seven of these dihedral angle restraints from tr-CCR measurements (Fig. 12). 

The tubulin-bound conformation of DDM in solution has been determined from tr-NOE data [112], Sample conditions were similar to those used previously to determine the bioactive conformation of EpoA. Distance restraints were obtained from a series of tr-NOE spectra recorded at several mixing times and were used in the structure calculation based on the complete relaxation matrix methodology [37], The NMR-derived bioactive conformation is quite similar to the crystal structure except for the conformation of the 8 lactone ring, that is close to a flattened chair in solution but a twisted boat in the crystal (Fig. 18). 

The structure determination of biopolymers using NMR spectroscopy usually involves interactions of protons[216,33. Typically, interactions of protons (nuclear Overhauser effect, NOE) that are close in space but separated by several subunits of the biopolymer are used to establish the folding of the backbone. Distance restraints are then used to compute a structure which is checked by back-calculation of the NOE spectra and comparison with experimental results 361. For large and highly flexible systems molecular dynamics is invaluable for scanning the conformational space. 

The most important NMR parameters obtained for the hydroxyl protons are chemical shifts (6), vicinal proton-proton coupling constants (3J7hc,Oh), temperature coefficients (AS/7), deuterium-induced differential isotope shifts, and exchange rates ( ex)-119-123 These parameters may provide information on hydrogen bond interactions and hydration as well. Moreover NOEs and chemical exchanges involving hydroxyl groups observed by NOESY and ROESY experiments also add to the number of distance restraints used in conformational analysis. 

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