NMR distance constraints

In order to integrate the NMR distance constraint data into a molecular mechanics minimization routine, we have chosen a smooth approximation of the above function... 

Another globular protein, bovine pancreatic trypsin inhibitor (BPTI), has also been treated by the build-up procedure however, because of a limitation on computer time when the calculation on this protein was carried out, a limited set of simulated NMR distance constraints (taken from the known X-ray structure225) was used226-227 to reduce the number of conformations... 

MAS dipolar recoupling methods, (b) Determination of the cimetidine crystal structure (inset) from XRPD data using a simulated annealing (S A) algorithm, comparing the number of steps required and success rate to achieve an accurate determination when starting with an unconstrained cimetidine molecule and when incorporating the NMR distance constraints. 

The amount of computation necessary to try many conformers can be greatly reduced if a portion of the structure is known. One way to determine a portion of the structure experimentally is to obtain some of the internuclear distances from two-dimensional NMR experiments, as predicted by the nuclear Over-hauser effect (NOE). Once a set of distances are determined, they can be used as constraints within a conformation search. This has been particularly effective for predicting protein structure since it is very difficult to obtain crystallographic structures of proteins. It is also possible to define distance constraints based on the average bond lengths and angles, if we assume these are fairly rigid while all conformations are accessible. 

Figure 18.20 The two-dimensional NMR spectrum shown in Figure 18.17 was used to derive a number of distance constraints for different hydrogen atoms along the polypeptide chain of the C-terminal domain of a cellulase. The diagram shows 10 superimposed structures that all satisfy the distance constraints equally well. These structures are all quite similar since a large number of constraints were experimentally obtained. (Courtesy of P. Kraulis, Uppsala, from data published in P. Kraulis et ah. Biochemistry 28 7241-7257, 1989, by copyright permission of the American Chemical Society.)...

In NMR the magnetic-spin properties of atomic nuclei within a molecule are used to obtain a list of distance constraints between those atoms in the molecule, from which a three-dimensional structure of the protein molecule can be obtained. The method does not require protein crystals and can be used on protein molecules in concentrated solutions. It is, however, restricted in its use to small protein molecules. 

Salgado J, Grage SL, Kondejewski LH, Hodges RS, McElhaney RN, Ulrich AS (2001) Membrane-bound structure and alignment of the antimicrobial beta-sheet peptide gramicidin S derived from angular and distance constraints by solid state F-19-NMR. J Biomol NMR 21 191-208... 

On the basis of Eq. (1), NOEs are usually treated as upper bounds on interatomic distances rather than as precise distance constraints, because the presence of internal motions and, possibly, chemical exchange may diminish the strength of an NOE [23]. In fact, much of the robustness of the NMR structure determination method is due to the use of upper distance bounds instead of exact distance constraints in conjunction with the observation that internal motions and exchange effects usually reduce rather than increase the NOEs [5]. For the same reason, the absence of an NOE is in general not interpreted as a lower bound on the distance between the two interacting spins. 

In de novo three-dimensional structure determinations of proteins in solution by NMR spectroscopy, the key conformational data are upper distance limits derived from nuclear Overhauser effects (NOEs) [11, 14]. In order to extract distance constraints from a NOESY spectrum, its cross peaks have to be assigned, i.e. the pairs of hydrogen atoms that give rise to cross peaks have to be identified. The basis for the NOESY assignment... 

Candid [26] combines features from Noah and Aria, such as the use of three-dimensional structure-based filters and ambiguous distance constraints, with the new concepts of network-anchoring and constraint combination that further enable an efficient and reliable search for the correct fold in the initial cycle of de novo NMR structure determinations. A flowchart of the Candid algorithm is given in Fig. 2.3. 

Structural information on aromatic donor molecule binding was obtained initially by using H NMR relaxation measurements to give distances from the heme iron atom to protons of the bound molecule. For example, indole-3-propionic acid, a structural homologue of the plant hormone indole-3-acetic acid, was found to bind approximately 9-10 A from the heme iron atom and at a particular angle to the heme plane (234). The disadvantage of this method is that the orientation with respect to the polypeptide chain cannot be defined. Other donor molecules examined include 4-methylphenol (p-cresol) (235), 3-hydroxyphenol (resorcinol), 2-methoxy-4-methylphenol and benzhydroxamic acid (236), methyl 2-pyridyl sulfide and methylp-tolyl sulfide (237), and L-tyrosine and D-tyrosine (238). Distance constraints of between 8.4 and 12.0 A have been reported (235-238). Aromatic donor proton to heme iron distances of 6 A reported earlier for aminotriazole and 3-hydroxyphenol (resorcinol) are too short because of an inappropriate estimate of the molecular correlation time (239), a parameter required for the calculations. Distance information for a series of aromatic phenols and amines bound to Mn(III)-substituted HRP C has been published (240). 

To generate the distance constraints to be used in the constraint energy function, E(NOE), the spatial contacts were established on the basis of a 2D-N0ESY experiment with a 400 ms mixing time performed on a Bruker VTH-400 NMR spectrometer. The following... 

The basis for the determination of solution conformation from NMR data lies in the determination of cross relaxation rates between pairs of protons from cross peak intensities in two-dimensional nuclear Overhauser effect (NOE) experiments. In the event that pairs of protons may be assumed to be rigidly fixed in an isotopically tumbling sphere, a simple inverse sixth power relationship between interproton distances and cross relaxation rates permits the accurate determination of distances. Determination of a sufficient number of interproton distance constraints can lead to the unambiguous determination of solution conformation, as illustrated in the early work of Kuntz, et al. (25). While distance geometry algorithms remain the basis of much structural work done today (1-4), other approaches exist. For instance, those we intend to apply here represent NMR constraints as pseudoenergies for use in molecular dynamics or molecular mechanics programs (5-9). 

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