X-ray diffraction fiber

Complementary polynucleotide strands can be separated in alkaline CS2SO4 or CsCI gradients (6,17) (Fig. 2.4). 

4 If a parallel monochromatic light beam is incident upon a 

X-Ray Fiber slit, a Fraunhofer diffraction pattern will be observed through 

Diffraction a lens in the focal plane of the lens. An infinite array of parallel slits (or a grating) will give a discrete spectrum of lines the distance between these lines is inversely proportional to the distance between the slits, and the amplitude of the lines will depend on the slit width. This is an example of reciprocal space.  

If the slits are replaced by a regular array of atoms, e.g., a crystal or a periodic polymer, and if the light beam is replaced by a monochromatic X-ray beam, similar diffraction patterns will be obtained. Again, the distances in the pattern obtained will be Inversely proportional to the distances in the atomic array. Since the wavelength of X-rays is in the Angstrom range, the diffracting units will be atoms. 

---

The F-actin helix has 13 molecules of G-actin in six turns of the helix, repeating every 360 A. Oriented gels of actin fibers yield x-ray fiber diffraction patterns to about 6 A resolution. Knowing the atomic structure of G-actin it was possible for the group of Ken Holmes to determine its orientation in the F-actin fiber, and thus arrive at an atomic model of the actin filament that best accounted for the fiber diffraction pattern. 

Five articles on polysaccharide helices solved prior to 1979 have appeared in the volumes published between 1967 and 1982.2-6 The first was a review on X-ray fiber diffraction and its application to cellulose, chitin, amylose, and related structures, and the rest were bibliographic accounts. Since then, X-ray structures of several new polysaccharides composed of simple to complex repeating units have been successfully determined, thanks to technological advances in fiber-diffraction techniques, the availability of fast and powerful computers, and the development of sophisticated software. Also, some old models have been either re-... 

The Pn conformation of poly-L-proline (PP) or collagen in the solid state could be identified from X-ray fiber diffraction results (Cowan and McGavin, 1955). Persistence of this basic structure in solution was inferred from the resemblance between the CD spectra of solutions and films of the polypeptide. The CD spectra of the charged forms of PGA and PL closely resemble that of Pn (compare Fig. IB, 1C, and ID) however, these spectra differ significantly from those of PP peptides at high temperature or in the presence of high concentration of salts... 

Different kinds of disorder may affect differently the X-ray diffraction pattern of the crystals. Depending on the features present in the X-ray fiber diffraction pattern, it is useful to distinguish three main classes of disordered structures170 ... 

Mesomorphic forms characterized by conformationally ordered polymer chains packed in lattices with different kinds of lateral disorder have been described for various isotactic and syndiotactic polymers. For instance, for iPP,706 sPP,201 sPS,202 syndiotactic poly(p-methylstyrene) (sPPMS),203 and syndiotactic poly(m -methylstyrene),204 mesomorphic forms have been found. In all of these cases the X-ray fiber diffraction patterns show diffraction confined in well-defined layer lines, indicating order in the conformation of the chains, but broad reflections and diffuse haloes on the equator and on the other layer lines, indicating the presence of disorder in the arrangement of the chain axes as well as the absence of long-range lateral correlations between the chains. 

Amyloid fibrils form from a variety of native proteins with diverse sequences and folds. The classic method for the structural analysis of amyloid has been X-ray fiber diffraction amyloid fibrils exhibit a characteristic diffraction signature, called the cross-/) pattern. This cross-/ pattern suggested a repeating structure in which /1-sheets run parallel to the fiber axis with their constituent /1-strands perpendicular to that direction (Sunde and Blake, 1997). This diffraction signature pointed to an underlying common core molecular structure for the amyloid fibril that could accommodate diverse sequences and folds. A number of groups have proposed amyloid folds that are consistent with the experimental data and these can be linked to repeating /1-structured units. 

Cross-/] structure has been demonstrated for Sup35pNM filaments. Serio et al. (2000) observed a 0.47-nm reflection by X-ray diffraction, and subsequently this reflection was shown to be meridional both by X-ray fiber diffraction (Kishimoto et al., 2004) and electron diffraction (King and Diaz-Avalos, 2004). In the Ure2p system, cross-/ structure has been established by electron diffraction from prion domain filaments preserved in vitreous ice (Fig. 7 Baxa et al, 2005). In addition, a 0.47-nm reflection was detected by both X-ray diffraction and electron diffraction from filament preparations of full-length Ure2p and the Ure2p1 65-GFP fusion, indicating that they contain the same structure (Fig. 7 Baxa et al, 2005). 

The notion of a common core structure has been further supported by synchrotron X-ray fiber diffraction patterns of several amyloid fibrils the patterns show common reflections in addition to those at 4.7 and 10 A (Sunde et al., 1997). Although these data give some insight into the arrangement of the amyloid fibril core, the exact molecular structure and organization of the proteins making up this common core have yet to be uniquely defined. The inherently noncrystalline, insoluble nature of the fibrils makes their structures difficult to study via traditional techniques of X-ray crystallography and solution NMR. An impressive breadth of biochemical and biophysical techniques has therefore been employed to illuminate additional features of amyloid fibril structure. 

Using computer modeling, jointly with x-ray fiber diffraction data, the molecular architectures of two different gel-forming polysaccharides have been examined. Preliminary results indicate that the neutral and doubly branched capsular polysaccharide from Rhizobium trifolii can form a 2-fold single helix of pitch 1.96 nm or a half-staggered, 4-fold doublehelix of pitch 3.92 nm. The molecules are likely to be stabilized by main chain — side chain interactions. 

Structural information at the molecular level can be extracted using a number of experimental techniques which include, but are not restricted to, optical rotation, infra-red and ultra-violet spectroscopy, nuclear magnetic resonance in the solid state and in solution, diffraction using electrons, neutrons or x-rays. Not all of them, however, are capable of yielding structural details to the same desirable extent. By far, experience shows that x-ray fiber diffraction (2), in conjunction with computer model building, is the most powerful tool which enables to establish the spatial arrangement of atoms in polymer molecules. 

X-ray fiber diffraction can be used to visualize ordered structures of polysaccharides at atomic resolution. The structures provide information on structure/property relationships in these systems. X-ray fiber diffraction techniques are outlined and are illustrated with applications to typical polysaccharides. 

Figure 2. X-ray fiber diffraction pattern from K " chondroitin 4-sulfate. (Reproduced with permission from ref. 18. Copyright 1983 Academic Press Inc.)...

The structures of agarose, iota- and kappa-carrageenan have been determined by x-ray fiber diffraction (24-27). The quality of the diffraction data obtained from each of these three specimens varies considerably and the way in which these data are used in structure determination is outlined here. Diffraction patterns from oriented specimens of agarose, and kappa- and iota-carrageenan are shown in Figure 6 (28). The molecular repeat distances derived from these patterns are listed in Table I. 

X-ray fiber diffraction can be used to visualize highly hydrated polymer specimens at atomic resolution. An essential part of such an analysis is the inclusion of reliable stereochemical information to supplement the diffraction data. Structure determination involves modelling and refinement of putative structures, and adjudication amongst the optimized models. This technique has been successfully applied to a number of polysaccharides. The precision of resulting structures is often sufficient to identify the critical interactions within and between molecules, that are responsible for the unique properties of these materials. 

---