Diffraction and NMR

In 1971 the Protein Data Bank - PDB [146] (see Section 5.8 for a complete story and description) - was established at Brookhaven National Laboratories - BNL -as an archive for biological macromolccular cr7stal structures. This database moved in 1998 to the Research Collaboratory for Structural Bioinformatics -RCSB. A key component in the creation of such a public archive of information was the development of a method for effreient and uniform capture and curation of the data [147], The result of the effort was the PDB file format [53], which evolved over time through several different and non-uniform versions. Nevertheless, the PDB file format has become the standard representation for exchanging inacromolecular information derived from X-ray diffraction and NMR studies, primarily for proteins and nucleic acids. In 1998 the database was moved to the Research Collaboratory for Structural Bioinformatics - RCSB. 

Amphiphiles often have a complex phase behaviour with several liquid crystalline phases These liquid crystalline phases are often characterised by long-range order in one directior together with the formation of a layer structure. The molecules may nevertheless be able tc move laterally within the layer and perpendicular to the surface of the layer. Structura information can be obtained using spectroscopic techniques including X-ray and neutror diffraction and NMR. The quadrupolar splitting in the deuterium NMR spectrum can be... 

The situation is different for other examples—for example, the peptide hormone glucagon and a small peptide, metallothionein, which binds seven cadmium or zinc atoms. Here large discrepancies were found between the structures determined by x-ray diffraction and NMR methods. The differences in the case of glucagon can be attributed to genuine conformational variability under different experimental conditions, whereas the disagreement in the metallothionein case was later shown to be due to an incorrectly determined x-ray structure. A re-examination of the x-ray data of metallothionein gave a structure very similar to that determined by NMR. 

R = CH3, CH3, CD3), which were characterized by single-crystal X-ray diffraction and NMR and IR spectroscopy These complexes are rare examples of first-row transition metal alkyl-hydrido species. ... 

There are three potential methods by which a protein s three-dimensional structure can be visualized X-ray diffraction, NMR and electron microscopy. The latter method reveals structural information at low resolution, giving little or no atomic detail. It is used mainly to obtain the gross three-dimensional shape of very large (multi-polypeptide) proteins, or of protein aggregates such as the outer viral caspid. X-ray diffraction and NMR are the techniques most widely used to obtain high-resolution protein structural information, and details of both the principles and practice of these techniques may be sourced from selected references provided at the end of this chapter. The experimentally determined three-dimensional structures of some polypeptides are presented in Figure 2.8. 

Introduce instrumental techniques used in analysis of the bioinorganic systems I will lecture on (Chapter 3 Instrumental and Computer-Based Methods). Typically, these would be electron paramagnetic resonance (EPR) and Mossbauer spectroscopies not often covered in undergraduate instrumental analysis courses plus X-ray diffraction and NMR techniques used for structural analyses of metalloproteins and their small molecule model compounds. 

X-ray diffraction and NMR studies have shown that metal fragments add exclusively across the [6,6] fusions to give rj2-type adducts. In contrast, organic derivatives add either to the [6,6] to form a closed adduct, or to the [6,5] fusions to form either an open or closed adduct (66). The ease with which some metal fragments dissociate off and on to the cage, in contrast to the organic derivatives, may explain the exclusive formation of the thermodynamically most stable [6,61-adducts. 

A straightforward approach is to hunt for short polypeptides that meet the specificity requirement of an enzyme but which, because of peculiarities of the sequence, are acted upon very slowly. Such a peptide may contain unusual or chemically modified amino acids. For example, the peptide Thr-Pro-nVal-NMeLeu-Tyr-Thr (nVal=norvaline NMeLeu = N-methylleucine) is a very slow elastase substrate whose binding can be studied by X-ray diffraction and NMR spectroscopy.6 Thiol proteases are inhibited by succinyl-Gln-Val-Val-Ala-Ala-p-nitroanilide, which includes a sequence common to a number of naturally occurring peptide inhibitors called cystatins.f They are found in various animal tissues where they inhibit cysteine proteases. 

Further constraint of the aminomethyl chain has been performed by incorporating it into 5,6,7,8-tetrahydronaphthoic acids 19 and 20 (Scheme 7) 5961 731 X-ray diffraction and NMR analysis of the TV-acetyl-jV -isopropylamide 21 revealed that the compound induces a turn structure without the formation of an intramolecular hydrogen bond. However, molecular modeling studies indicate that this amino acid derivative prefers a partially extended conformation 71,72 ... 

The mechanism of an enzymatic reaction is ultimately defined when all the intermediates, complexes, and conformational states of the enzyme are characterized and the rate constants for their interconversion are determined. The task of the kineticist in this elucidation is to detect the number and sequence of these intermediates and processes, define their approximate nature (that is, whether covalent intermediates are formed or conformational changes occur), measure the rate constants, and, from studying pH dependence, search for the participation of acidic and basic groups. The chemist seeks to identify the chemical nature of the intermediates, by what chemical paths they form and decay, and the types of catalysis that are involved. These results can then be combined with those from x-ray diffraction and NMR studies and calculations by theoretical chemists to give a complete description of the mechanism. 

Direct evidence of ocean hydrates has been obtained using in situ Raman measurements in Barkley Canyon, off Vancouver Island (Hester, 2007). The occurrence of sH in natural hydrate deposits has been inferred from gas analyses of recovered core samples from the Gulf of Mexico (Sassen and Macdonald, 1994 Yousuf et al., 2004) and confirmed by diffraction and NMR for samples from Barkley Canyon (Pohlman et al., 2005 Lu et al., 2007). 

There are comparatively few measurements of the hydrate phase composition, due to experimental difficulty. Hydrate phase difficulties arise because water is often occluded in the hydrate mass, separation of hydrate and water is difficult, and the hydrate phase of mixtures is often inhomogeneous in experiments. Consequently, the ratio of water to hydrocarbon is often inaccurate. As discussed in Chapter 6, only over the last two decades have experimental techniques (e.g., diffraction and NMR and Raman spectroscopy) become accurate enough to determine the degree of filling of hydrate cavities with different types of molecules. 

In addition to X-ray diffraction and NMR, which are direct techniques, methods based on the calculation of predicted three-dimensional structures of molecules in the range of 3 to 50 amino acids based on energy considerations are under rapid development. These approaches use what are commonly called molecular dynamics and energy minimization equations to specify the most probable conformation of polypeptides and small proteins. Often, when combined with information from other sources, such as X-ray crystallography or NMR studies, they have been demonstrated to be quite useful. However, when standing alone, their power and the accuracy of their predictive capability remains to be seen. 

The cellulose oligomers, beginning with methyl cellotrioside, yield powder diffraction patterns that are very similar to those of cellulose II. The NMR studies of the cellulose oligomers further establish the extensive analogy between cellotetraose and cellulose II. Work by both Gessler et al. [222] and Raymond et al. [223] has shown that the 06 atoms in cellotetraose and methyl cellotrioside [224] all take the gt position, consistent with the diffraction and NMR results for cellulose II. Because the chains in the methyl cellotrioside and cellotetraose are antiparallel, this work adds support to the above results on cellulose II. On the other hand, molecules in crystalline a-lactose, a related disaccharide, have parallel packing [225]. 

Only recently was the first higfr-resolution atomic structure of a G-protein-coupled receptor solved, namely that of rhodopsin, although lower-resolution spatial structural information based on two-dimensional crystals and electron diffraction and NMR structures was available.3.4 This information makes it certain that all heptahelical receptors have the same topological arrangement of the polypeptide chains. The amino- and carboxy-termini are oriented in the same way, with the amino-terminus outside and carboxy-terminus on the cytoplasmic side. Valuable structural relationships between different G-protein-coupled receptors for hormones have also come to light, mainly thanks to comparisons of cDNA-derived sequences. ... 

FIGURE 14 Schematic molecular structure of the (Sc2C2 C84) carbide metallofullerene based on the synchrotron X-ray powder diffraction and NMR experiments. The two (top and bottom) spheres in the fullerene correspond to Sc atoms, whereas the C2 molecules are depicted between the Sc atoms. 

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