Disordered water

Another important reason for the significant deviation between calculated and X-ray structures can be the low resolution (2.9 A) of the X-ray structure. It is well known that X-ray structures may miss disordered water molecules inside the enzyme. The X-ray structure of the bovine erythrocyte GPx has a significantly higher resolution (2.0 A) and that structure contains two water molecules in the active site [63], Unfortunately that X-ray structure is not complete. In order to test for the presence of water molecules at the active site of the mammalian GPx, calculations were performed with two additional water molecules at the active site. This reduced the RMS deviation to from 0.97 to 0.19k for ONIOM(B3LYP/6-31G(d) Amber)-ME and suggests the presence of water molecules also in the active site of mammalian GPx. In our investigation of the reaction mechanism, these water molecules turns out to be critical. 

Fig. 10 Crystal packing of tpcb bta (34 33) cocrystal material disordered water and DMSO molecules have been omitted from supramolecular cavity [57]...

Hydrogen bonding connects the complexes via interactions between the eoordinated water molecule (06) and carbonyl oxygen atoms 03 (2.817(5) A) and 04 (2.764(5) A). The disordered water molecule present in the lattice does not form any significant hydrogen bonds (perhaps accounting for its disorder). ... 

The DNA solvation shell consists of about 20-22 water molecules per nucleotide of these, — 15-17 waters associate with the nucleoside and —5 waters associate with the phosphate group [13,14]. Water outside the solvation layer is termed bulk water. Upon freezing, the DNA solvation water forms two primary phases the ice phase, consisting of one or more of the crystalline forms of ice, and a DNA-associated phase, consisting of ordered water which comes in direct contact with the DNA (primary layer) and disordered water in the secondary layer. DNA hydration is expressed in terms of F, the number of water molecules per nucleotide. 

In the case of the denatured PYP, the chromophore is surrounded by completely disordered environment of the water. The relaxations due to the interactions between the excited chromophore with a large dipole moment and surrounding disordered water environment produce a large extent dynamic Stokes shift of the fluorescence. Nevertheless, it is much slower compared with that taking place in the disordered PNS of the ferulic acid analogue as demonstrated in the analysis of the previous section (3.3). 

Careful analysis of electron-density maps usually reveals many ordered water molecules on the surface of crystalline proteins (Plate 4). Additional disordered water is presumed to occupy regions of low density between the ordered particles. The quantity of water varies among proteins and even among different crystal forms of the same protein. The number of detectable ordered water molecules averages about one per amino-acid residue in the protein. Both the ordered and disordered water are essential to crystal integrity, and drying destroys the crystal structure. For this reason, protein crystals are subjected to X-ray analysis in a very humid atmosphere or in a solution that will not dissolve them, such as the mother liquor. 

Recall that stable protein crystals contain a large amount of both ordered and disordered water molecules. As a result, the proteins in the crystal are still in the aqueous state, subject to the same solvent effects that stabilize the structure in solution. Viewed in this light, it is less surprising that proteins retain their solution structure in the crystal. 

In crystal structure analyses where the hydrogen atoms are not directly observed, as in macromolecules and structures with disordered water molecules, the signature of the three-center hydrogen bond is a triangle of potential donor or acceptor nonhydrogen atoms at distances in the range observed for weak hydrogenbonding interactions, i.e., 2.8 to 3.5 A. 

Of the 7.57 water molecules per asymmetric unit, four are fully ordered and one, W(5), shows two positions at 0.64 and 0.36 occupation. These water molecules are located in intermolecular voids, and the remaining 2.57 waters are statistically distributed over four sites in the a-cyclodextrin cavity (see Fig. 18.6 b). Associated with this disorder in the a-cyclodextrin cavity is a round shape of the macrocycle, where in contrast to the other two a-cyclodextrin hydrate forms, all six intramolecular interglucose 0(2) 0(3 ) hydrogen bonds are formed. Because hydrogen atoms attached to the disordered water molecules in the 7.57 hydrate could... 

The nonbonding of the )C=0 groups may be overestimated, since some may be involved in long three-center bonds. The presence of unidentified disordered water molecules as donors may also reduce this statistic. 

In this crystal structure, the vitamin B12 molecules are well ordered (except for a side chain). Between the molecules there are wide channels which are filled with four acetone and with a total of >140 mostly disordered water molecules in the unit cell. Careful analysis of X-ray and neutron diffraction data suggested that the disorder can be described in terms of a movement of water molecules within the wide channels, as indicated schematically in Fig. 25.1. According to this interpretation, there exist two dynamically related water networks I (atoms in circles) and II (atoms in squares). Starting with network I, a water molecule can be either pushed into the asymmetric unit at position 410 (path A) or pulled out at position 228 (path B). If water molecule 228 is pulled out and moves into position 410 in the adjacent asymmetric unit, water 222 is pulled towards 428 and the void is filled by consecutive movements of water molecules 231, 226, 410 into positions 431, 227, 210. These movements are consistent with a transition from network I into network II. Conversely, if a water molecule enters the asymmetric unit through path A, then water molecules 210, 431, and 428 of network II move into positions... 

In this arrangement, the angle between the central N1-N2 axis of the NDI core and the central axis of the nanotube is 60°, whereas the distance between two sequential aromatic cores is on average 4.8 A. The nanotube has an average inner diameter of 12.4 A and it contains diffuse electron density attributed to disordered water molecules. 

Blake et al. (1983) refined the structures of human lysozyme (HL) and tortoise egg white lysozyme (TEWL) to 1.5 and 1.6 A resolution, respectively. The diffraction was modeled as arising from three components the protein, ordered water, and disordered water. Most of the water in the crystals (i.e., 60—80%) is disordered. The analysis located 143 molecules of ordered water out of about 350 per HL molecule, and 122 molecules out of 650 per TEWL molecule. The ordered water covers 75% of the available surface of the the protein. One-third (TEWL) to one-half (HL) of the total surface is unavailable for analysis of the adjacent water, owing to crystal contacts or disorder in the protein region. Thus, the estimate of surface coverage is in good agreement with the 300 molecules of water estimated by heat capacity measurements as full hydration (0.38 h). The area covered per water molecule is estimated as 18.9... 

About 300 water molecules are sufficient to cover the lysozyme surface. This is a remarkably small amount of water. Calculations based on the crystal structure of lysozyme show that the surface is about 6000 A in area (Lee and Richards, 1971 Shrake and Rupley, 1973). Thus, each water covers, on average, about 20 A, which is twice the projection of a water molecule packed as in the liquid. Since 20 is the most area a water molecule can cover and maintain hydrogen bonding, there can be no multilayer water. Moreover, whatever arrangements there are at the surface must integrate simply into the bulk water namely, there is no B shell of disordered water, required to interface the water adjacent to the protein surface with the bulk solvent. 

The role of disordered water as a tunneling medium has been the subject of several electronic-stmcture computational studies. 

If water replaces dichloroacetic acid or trifluoroacetic acid as the hydrogen-bonding component in mixtures of decreasing polarity, then the helical content of disordered water-soluble polypeptide chains will increase continuously, as is, for example, the case with equimolar copoly-L-lysine-L-... 

Figure 9. Electron density maps of the same sections of tortoise eggwhite lysozyme showing (a) the protein component (b) the ordered water component (c) the electron density of the disordered water component and (d) the model of the ordered water component. From [130].

Consequences of chronic renal failure include disordered water and sodium metabolism, hyperkalaemia, abnormal calcium and phosphate metabolism, and anaemia. 

Figure 5.19 Illustration of the use of a difference Fourier map in locating a deficiency in a structural model. In this case the map reveals a region of missing density at the centre of the cell which is occupied in the true structure by a disordered water molecule.

Ernst, J. A., Clubb, R.T., Zhou, H.-X., Gronenbom, A.M., Clore, G.M. Demonstration of positionally disordered water within a protein hydrophobic cavity by NMR. Science 1995, 267,1813-7. 

The disordered water molecules are not seen in x-ray structure, yet they are critical for proton transport in the enzyme. In MD simulations, we have discovered [21] that water molecules in the catalytic cavity in CcO form two branching chains (Figures 4.5 and 4.6) that connect an experimentally known proton donor Glu242, the Fe-Cu binuclear center, and the putative PLS His291, from which protons can be pumped using our mechanism of proton-proton repulsion. The possibility of such connectivity has been discussed however, they were never observed through computer simulations before. 

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