V.T. Forsyth

Metal Ion Roles and the Movement of Hydrogen during Reaction Catalyzed by D-Xylose Isomerase: A Joint X-Ray and Neutron Diffraction Study

Conversion of aldo to keto sugars by the metalloenzyme D-xylose isomerase (XI) is a multistep reaction that involves hydrogen transfer. We have determined the structure of this enzyme by neutron diffraction in order to locate H atoms (or their isotope D). Two studies are presented, one of XI containing cadmium and cyclic D-glucose (before sugar ring opening has occurred), and the other containing nickel and linear D-glucose (after ring opening has occurred but before isomerization). Previously we reported the neutron structures of ligand-free enzyme and enzyme with bound product. The data show that His54 is doubly protonated on the ring N in all four structures. Lys289 is neutral before ring opening and gains a proton after this; the catalytic metal-bound water is deprotonated to hydroxyl during isomerization and O5 is deprotonated. These results lead to new suggestions as to how changes might take place over the course of the reaction.

Looking at hydrogen bonds in cellulose.

A series of cellulose crystal allomorphs has been studied using high-resolution X-ray and neutron fibre diffraction to locate the positions of H atoms involved in hydrogen bonding. One type of position was always clearly observed in the Fourier difference map (F(d)-F(h)), while the positions of other H atoms appeared to be less well established. Despite the high crystallinity of the chosen samples, neutron diffraction data favoured some hydrogen-bonding disorder in native cellulose. The presence of disorder and a comparison of hydrogen-bond geometries in different allomorphs suggests that although hydrogen bonding may not be the most important factor in the stabilization of cellulose I, it is essential for stabilizing cellulose III, which is the activated form, and preventing it from collapsing back to the more stable cellulose I.

Neutron fibre diffraction study of DNA hydration

Interactions with water are crucial to the conformation assumed by the DNA double helix. The location of water around the D conformation has been investigated in a neutron fibre diffraction study which shows that water is ordered in the minor groove of the DNA. The D conformation is important since its occurrence is limited to specific DNA base pair sequences which have been identified as functionally significant. This study is of particular interest because the D conformation has not been reported in single crystal studies of oligonucleotides.

A high angle neutron fibre diffraction study of the hydration of the A conformation of the DNA double helix.

A high angle neutron fibre diffraction study of the distribution of water around the A-form of DNA has been performed using the diffractometer D19 at the Institut Laue-Langevin, Grenoble. These experiments have exploited the ability to replace H2O surrounding the DNA by D2O so that isotopic difference Fourier maps can be computed in which peaks are identified with the distribution of water in the unit cell. All peaks of significant height have been accounted for by four families of water molecules whose positions and occupancies have been determined using least squares refinement. The coordinates of the water peaks making up each family do not deviate significantly from a regular helical arrangement with the same parameters as the DNA. Two of these families are of particular interest. The first consists of water molecules in the major groove linking successive charged phosphate oxygens along the polynucleotide chains. The second is associated with bases in the major groove and forms a central core of density along the helix axis. These two families provide a layer of hydration lining the interior wall of the major groove leaving a central channel to accommodate cations. The relationship between these observations and conformational stability is discussed.

X‐ray camera for high‐ and small‐angle x‐ray diffraction studies of the drawing and annealing of polymers at Daresbury Synchrotron Radiation Source

A purpose‐designed x‐ray fiber diffraction camera has been constructed in the Keele University Physics Department to be used at the SERC Daresbury Laboratory Synchrotron Radiation Source. The camera allows time‐resolved studies of the change in both the high‐ and low‐angle diffraction patterns during drawing and annealing of polymer films to be recorded. Drawing of the films is achieved by two opposed stepper motors which allow films to be drawn uniaxially in both directions. The temperature of the sample environment can be controlled to within 1 °C by a radio spares proportional, integral, and derivative (PID) controller. Diffraction patterns can be recorded on the Enraf‐Nonius TV FAST detector or on photographic film. Exposure times using the FAST detector are typically 5 s, representing a gain of approximately a factor of 5 over photographic film. The FAST detector has a further advantage over photographic film in that essentially an unlimited number of diffraction patterns can be recorded end‐to‐end w...

SEC-SANS: size exclusion chromatography combined in situ with small-angle neutron scattering

This publication describes the combination of size exclusion chromatography with small-angle neutron scattering in situ. The need for this technique and the progress achieved thanks to it are illustrated by relevant examples.

THE LOCATION OF WATER AROUND THE DNA DOUBLE-HELIX

Abstract The diffractometer D19 at the ILL has been used to locate water around the DNA double-helix in fibres of the polynucleotide poly[d(A-T)].poly[d(A-T)]. Neutron fibre diffraction data were recorded to a resolution of 3 A first when the DNA was surrounded by D 2 O and second when it was surrounded by H 2 O.

Binding site asymmetry in human transthyretin: insights from a joint neutron and X-ray crystallographic analysis using perdeuterated protein

A neutron crystallographic study of perdeuterated transthyretin reveals important aspects of the structure relating to its stability and its propensity to form fibrils, as well as evidence of a single water molecule that affects the symmetry of the two binding pockets.

Inhibition of d-xylose isomerase by polyols: atomic details by joint X-ray/neutron crystallography

D-Xylose isomerase (XI) converts the aldo-sugars xylose and glucose to their keto analogs xylulose and fructose, but is strongly inhibited by the polyols xylitol and sorbitol, especially at acidic pH. In order to understand the atomic details of polyol binding to the XI active site, a 2.0 Å resolution room-temperature joint X-ray/neutron structure of XI in complex with Ni(2+) cofactors and sorbitol inhibitor at pH 5.9 and a room-temperature X-ray structure of XI containing Mg(2+) ions and xylitol at the physiological pH of 7.7 were obtained. The protonation of oxygen O5 of the inhibitor, which was found to be deprotonated and negatively charged in previous structures of XI complexed with linear glucose and xylulose, was directly observed. The Ni(2+) ions occupying the catalytic metal site (M2) were found at two locations, while Mg(2+) in M2 is very mobile and has a high B factor. Under acidic conditions sorbitol gains a water-mediated interaction that connects its O1 hydroxyl to Asp257. This contact is not found in structures at basic pH. The new interaction that is formed may improve the binding of the inhibitor, providing an explanation for the increased affinity of the polyols for XI at low pH.

A neutron diffraction study of the distribution of water in the A form of the DNA double helix

Abstract High angle diffraction data from ordered fibres of DNA in the A form have been collected on diffractometer D19 at the Institute Laue-Langevin. By exploiting the ability to isotopically replace H 2 O surrounding the DNA by D 2 O we have been able to locate water around the double helix.