Protein neutron diffraction

Brunger, A. T., Karplus, M. Polar hydrogen positions in proteins Empirical energy placement and neutron diffraction comparison. Proteins Struct. Func. Genet. 4 (1988) 148-156. 

Protein crystals contain between 25 and 65 vol% water, which is essential for the crystallisation of these biopolymers. A typical value for the water content of protein crystals is 45% according to Matthews et al. l49,150). For this reason it is possible to study the arrangement of water molecules in the hydration-shell by protein-water and water-water interactions near the protein surface, if one can solve the structure of the crystal by X-ray or neutron diffraction to a sufficiently high resolution151 -153). 

According to X-ray and neutron diffraction structures [3, 4] the binding of CO to the heme leads to a bent FeCO unit. The Fe-C-O angle is, however, found to be linear in synthetic models of the protein (hiomimetic molecules). Because of this, it was originally thought that the FeCO distortion was responsible for the well known discrimination of the protein against CO - the affinity ratio C0/02 is lower in the protein than in biomimetic systems [1]. In... 

The mechanism by which serine peptidases, particularly serine endopep-tidases (EC 3.4.21), hydrolyze peptide bonds in peptides and proteins has been extensively investigated by X-ray crystallography, site-directed mutagenesis, detection of intermediates, chemical modification, H-NMR spectroscopy, and neutron diffraction [2-14], These studies revealed that all serine peptidases possess a catalytic triad, composed of a serine, a histidine, and an aspartate residue, and a so-called oxyanion hole formed by backbone NH groups. 

Furthermore, there is a striking parallelism between these data and the neutron diffraction data from nucleosomes in 100% D 0 (Pardon et al., 1977 Suau et al., 1977), where scattering from the histone protein dominates, and from core protein in 2 M NaCl solution (Pardon et al., 1978). The above interference phenomenon may well be the explanation for the protein-dominated scattering maximum between 35 and 37 A observed for chromatin and nucleosomes in solution (Pardon et al., 1977 Suau et al., 1977). 

Even in the crystalline state there is evidence of movement. In the images constructed from X-ray or neutron diffraction experiments side chains on the surfaces of protein molecules are often not clearly visible because of rapid rotational movement. Some segments of the polypeptide chain may be missing from the image. However, side chain groups within the core of a domain are usually seen clearly. They probably move only in discrete steps. However, they may sometimes shift rapidly between different conformations, all of which maintain a close-packed interior.310-312... 

Because hydrogen atoms contain only one electron, and therefore scatter X-rays very weakly, they are usually not seen at all in X-ray structures of proteins. However, neutrons are scattered strongly by hydrogen atoms and neutron diffraction is a useful tool in protein structure determination.414 415 It has been used to locate tightly bonded protons that do not exchange with 2H20 as well as bound water (2H20). 

The relative location of individual ribosomal proteins within the two subunits has been examined in two ways. One method involves determining which ribosomal proteins can be chemically cross-linked to each other and has yielded an elaborate grid of spatial relationships based on the frequency of cross-linking. The other method relies on neutron diffraction whereby the individually deuterated ribosomal proteins are located within the ribosomal subunit. The two methods of determining the location of ribosomal proteins have yielded a consistent spatial picture. 

The great advantage of neutron diffraction is that small nuclei like hydrogen are readily observed. By comparison with carbon and larger elements, hydrogen is a very weak X-ray diffractor and is typically not observable in electron-density maps of proteins. But hydrogen and its isotope deuterium (2H or D) diffract neutrons very efficiently in comparison with larger elements. 

Protein structures determined by X-ray or neutron diffraction or NMR which may include co-factors, substrates, inhibitors, or other ligands... 

X-ray and neutron diffraction patterns can be detected when a wave is scattered by a periodic structure of atoms in an ordered array such as a crystal or a fiber. The diffraction patterns can be interpreted directly to give information about the size of the unit cell, information about the symmetry of the molecule, and, in the case of fibers, information about periodicity. The determination of the complete structure of a molecule requires the phase information as well as the intensity and frequency information. The phase can be determined using the method of multiple isomor-phous replacement where heavy metals or groups containing heavy element are incorporated into the diffracting crystals. The final coordinates of biomacromolecules are then deduced using knowledge about the primary structure and are refined by processes that include comparisons of calculated and observed diffraction patterns. Three-dimensional structures of proteins and their complexes (Blundell and Johnson, 1976), nucleic acids, and viruses have been determined by X-ray and neutron diffractions. 

The second part of the model assumes that an influence on the functioning of the membrane-integrated proteins is possible even without a specific drag-protein interaction. This is understandable if one considers another parameter that characterizes cells or vesicles, namely their curvature. This is a measure of internal stress and depends on the tendency of bilayers to assume a non-bilayer conformation, for instance a hexagonal or cubic phase (see Section 1.1.3.1 and Figure 1.9). This transformation can, for example, be detected and measured by X-ray or neutron diffraction techniques. 

It is not necessary to emphasize how important water is for living systems to maintain their life [24-26], No wonder that many scientists in the field of X-ray and neutron diffraction measurement have been trying to determine positions and orientations of water molecules around and inside biomolecules, or protein and DNA [27,28], However, it is not so easy even for modern experimental technology to locate the position of water molecules, partly due to the limited resolution of diffraction measurements in space as well as in time. This is because water molecules at the surface of protein are not necessarily bound firmly to some particular site of biomolecules, but exchange their positions quite frequently. Actually this flexibility and fluctuation of water molecules are essential for living systems to control their life. The diffraction measurement can identify only some water molecules that have long residence time at some particular position of the biomolecules. 

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