Benno P. Schoenborn

Protein crystallography with spallation neutrons: the user facility at Los Alamos Neutron Science Center

In this report a neutron protein crystallography station (PCS) is described that has been built at Los Alamos National Laboratory for the study of proteins using the wavelength-resolved Laue technique. This user facility is the first of its kind to be built at a spallation neutron source and the first to use the wavelength-resolved Laue technique.

Rapid determination of hydrogen positions and protonation states of diisopropyl fluorophosphatase by joint neutron and X-ray diffraction refinement

Hydrogen atoms constitute about half of all atoms in proteins and play a critical role in enzyme mechanisms and macromolecular and solvent structure. Hydrogen atom positions can readily be determined by neutron diffraction, and as such, neutron diffraction is an invaluable tool for elucidating molecular mechanisms. Joint refinement of neutron and X-ray diffraction data can lead to improved models compared with the use of neutron data alone and has now been incorporated into modern, maximum-likelihood based crystallographic refinement programs like CNS. Joint refinement has been applied to neutron and X-ray diffraction data collected on crystals of diisopropyl fluorophosphatase (DFPase), a calcium-dependent phosphotriesterase capable of detoxifying organophosphorus nerve agents. Neutron omit maps reveal a number of important features pertaining to the mechanism of DFPase. Solvent molecule W33, coordinating the catalytic calcium, is a water molecule in a strained coordination environment, and not a hydroxide. The smallest Ca–O–H angle is 53°, well beyond the smallest angles previously observed. Residue Asp-229, is deprotonated, supporting a mechanism involving nucleophilic attack by Asp-229, and excluding water activation by the catalytic calcium. The extended network of hydrogen bonding interactions in the central water filled tunnel of DFPase is revealed, showing that internal solvent molecules form an important, integrated part of the overall structure.

Structural characterization of crystals of α-glycine during anomalous electrical behaviour

The crystal structure of α-glycine has been investigated in the temperature range 288–427u2005K using neutron diffraction. The molecular structure does not change significantly and the putative crystallographic phase transition associated with anomalous electrical behaviour in this temperature range is not observed. The unit cell expands anisotropically with increasing temperature, with the unique monoclinic b axis, corresponding to the stacking direction of molecular layers, changing the most. The increasing separation of antiferroelectric molecular layers with increasing temperature is driven by an increase in molecular libration about an axis that lies perpendicular to the b axis. There is also a weakening of the interlayer hydrogen bonds with temperature. These structural and dynamic changes will affect the response of molecular dipoles to an applied electric field and provide a possible mechanism for the anomalous electrical behaviour.

Hydration in protein crystallography.

Water in close proximity to the protein surface is fundamental to protein folding, stability, recognition and activity. Protein structures studied by diffraction methods show ordered water molecules around some charged, polar, and non-polar (hydrophobic) amino acids, although the later are only observed when they are at the interface between symmetry related molecules in the crystal. Water networks surrounding the protein have been observed for small proteins. Crystallographically observed water molecules are referred to as bound structural water molecules. During crystallographic data analysis, bound water molecules are often treated as though they belong to the protein. Recent developments in the treatment of the bulk solvent contribution to the low order diffraction data allow a better evaluation of the surface structure of the protein and a better localization of bound waters. The mobility of bound waters is studied by means of temperature and occupancy factors. The bulk solvent has relatively large disorder (liquid like) which is represented by liquidity factors. Within this context water layers surrounding the protein have little mobility.

Neutron diffraction studies of Escherichia coli dihydrofolate reductase complexed with methotrexate.

Hydrogen atoms play a central role in many biochemical processes yet are difficult to visualize by x-ray crystallography. Spallation neutron sources provide a new arena for protein crystallography with TOF measurements enhancing data collection efficiency and allowing hydrogen atoms to be located in smaller crystals of larger biological macromolecules. Here we report a 2.2-Å resolution neutron structure of Escherichia coli dihydrofolate reductase (DHFR) in complex with methotrexate (MTX). Neutron data were collected on a 0.3-mm3 D2O-soaked crystal at the Los Alamos Neutron Scattering Center. This study provides an example of using spallation neutrons to study protein dynamics, to identify protonation states directly from nuclear density maps, and to analyze solvent structure. Our structure reveals that the occluded loop conformation [monomer (mon.) A] of the DHFR·MTX complex undergoes greater H/D exchange compared with the closed-loop conformer (mon. B), partly because the Met-20 and β(F-G) loops readily exchange in mon. A. The eight-stranded β sheet of both DHFR molecules resists H/D exchange more than the helices and loops. However, the C-terminal strand, βH, in mon. A is almost fully exchanged. Several D2Os form hydrogen bonds with exchanged amides. At the active site, the N1 atom of MTX is protonated and thus charged when bound to DHFR. Several D2Os are observed at hydrophobic surfaces, including two pockets near the MTX-binding site. A previously unidentified D2O hydrogen bonds with the catalytic D27 in mon. B, stabilizing its negative charge.

Hydrogen location in stages of an enzyme-catalyzed reaction: time-of-flight neutron structure of D-xylose isomerase with bound D-xylulose

The time-of-flight neutron Laue technique has been used to determine the location of hydrogen atoms in the enzyme d-xylose isomerase (XI). The neutron structure of crystalline XI with bound product, d-xylulose, shows, unexpectedly, that O5 of d-xylulose is not protonated but is hydrogen-bonded to doubly protonated His54. Also, Lys289, which is neutral in native XI, is protonated (positively charged), while the catalytic water in native XI has become activated to a hydroxyl anion which is in the proximity of C1 and C2, the molecular site of isomerization of xylose. These findings impact our understanding of the reaction mechanism.

Iron–germanium multilayer neutron polarizing monochromators

A new type of thermal neutron polarizing monochromator, consisting of alternate thin layers of iron and germanium, has been constructed and tested. Bragg reflection from such artificial `crystals in the fully magnetized state yields a highly polarized beam with high reflecting efficiency (~0.84) for the reflected spin state. These multilayer monochromators have the additional advantage that higher orders in the reflected beam are almost completely suppressed. Since d spacings are typically large (~100u2005A), they produce a broader wavelength distribution than conventional single-crystal polarizing monochromators. Nevertheless, there are many applications where wavelength resolution is of secondary importance and the large gain in intensity (~40-fold) over conventional polarizing crystals can be a considerable advantage. Multilayers can also be used to advantage in combination with good monochromating crystals such as pyrolytic graphite or beryllium to produce polarized beams of high intensity and good wavelength resolution.

Protein structures by spallation neutron crystallography

The capabilities of the Protein Crystallography Station at Los Alamos Neutron Science Center for determining protein structures by spallation neutron crystallography are illustrated, and the methodological and technological advances that are emerging from the Macromolecular Neutron Crystallography consortium are described.

Protein crystallography with spallation neutrons.

proteins and oriented molecular complexes. With spallation neutrons and their time dependent wavelength structure, one can select data with an optimal wavelength bandwidth and cover the whole Laue spectrum as time (wavelength) resolved diffraction data. This optimizes data quality with best peak to background ratios and provides spatial and energy resolution to eliminate peak overlaps. Such a Protein Crystallography Station (PCS) has been built and tested at Los Alamos Neutron Science Center. A partially coupled moderator is used to increase flux and data are collected by a Cylindrical He3 detector covering 120 with 200mm height. The PCS is described along with examples of data collected from a number of proteins.

A preliminary time-of-flight neutron diffraction study of Streptomyces rubiginosus D-xylose isomerase.

The metalloenzyme D-xylose isomerase forms well ordered crystals that diffract X-rays to ultrahigh resolution (<1 A). However, structural analysis using X-ray diffraction data has as yet been unable to differentiate between several postulated mechanisms that describe the catalytic activity of this enzyme. Neutrons, with their greater scattering sensitivity to H atoms, could help to resolve this by determining the protonation states within the active site of the enzyme. As the first step in the process of investigating the mechanism of action of D-xylose isomerase from Streptomyces rubiginosus using neutron diffraction, data to better than 2.0 A were measured from the unliganded protein at the Los Alamos Neutron Science Center Protein Crystallography Station. Measurement of these neutron diffraction data represents several milestones: this is one of the largest biological molecules (a tetramer, MW approximately 160 000 Da, with unit-cell lengths around 100 A) ever studied at high resolution using neutron diffraction. It is also one of the first proteins to be studied using time-of-flight techniques. The success of the initial diffraction experiments with D-xylose isomerase demonstrate the power of spallation neutrons for protein crystallography and should provide further impetus for neutron diffraction studies of biologically active and significant proteins. Further data will be measured from the enzyme with bound substrates and inhibitors in order to provide the specific information needed to clarify the catalytic mechanism of this enzyme.