Neutron structure and mechanistic studies of diisopropyl fluorophosphatase (DFPase).

Diisopropyl fluorophosphatase (DFPase) is a calcium-dependent phosphotriesterase that acts on a variety of highly toxic organophosphorus compounds that act as inhibitors of acetylcholinesterase. The mechanism of DFPase has been probed using a variety of methods, including isotopic labelling, which demonstrated the presence of a phosphoenzyme intermediate in the reaction mechanism. In order to further elucidate the mechanism of DFPase and to ascertain the protonation states of the residues and solvent molecules in the active site, the neutron structure of DFPase was solved at 2.2 Å resolution. The proposed nucleophile Asp229 is deprotonated, while the active-site solvent molecule W33 was identified as water and not hydroxide. These data support a mechanism involving direct nucleophilic attack by Asp229 on the substrate and rule out a mechanism involving metal-assisted water activation. These data also allowed for the re-engineering of DFPase through rational design to bind and productively orient the more toxic S(P) stereoisomers of the nerve agents sarin and cyclosarin, creating a modified enzyme with enhanced overall activity and significantly increased detoxification properties.

A history of neutrons in biology: the development of neutron protein crystallography at BNL and LANL.

The first neutron diffraction data were collected from crystals of myoglobin almost 42 years ago using a step-scan diffractometer with a single detector. Since then, major advances have been made in neutron sources, instrumentation and data collection and analysis, and in biochemistry. Fundamental discoveries about enzyme mechanisms, biological complex structures, protein hydration and H-atom positions have been and continue to be made using neutron diffraction. The promise of neutrons has not changed since the first crystal diffraction data were collected. Today, with the developments of beamlines at spallation neutron sources and the use of the Laue method for data collection, the field of neutrons in structural biology has renewed vitality.

X-ray structure of perdeuterated diisopropyl fluorophosphatase (DFPase): perdeuteration of proteins for neutron diffraction.

The signal-to-noise ratio is one of the limiting factors in neutron macromolecular crystallography. Protein perdeuteration, which replaces all H atoms with deuterium, is a method of improving the signal-to-noise ratio of neutron crystallography experiments by reducing the incoherent scattering of the hydrogen isotope. Detailed analyses of perdeuterated and hydrogenated structures are necessary in order to evaluate the utility of perdeuterated crystals for neutron diffraction studies. The room-temperature X-ray structure of perdeuterated diisopropyl fluorophosphatase (DFPase) is reported at 2.1 A resolution. Comparison with an independently refined hydrogenated room-temperature structure of DFPase revealed no major systematic differences, although the crystals of perdeuterated DFPase did not diffract neutrons. The lack of diffraction is examined with respect to data-collection and crystallographic parameters. The diffraction characteristics of successful neutron structure determinations are presented as a guideline for future neutron diffraction studies of macromolecules. X-ray diffraction to beyond 2.0 A resolution appears to be a strong predictor of successful neutron structures.

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 degrees, 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.

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.

Protein structures by spallation neutron crystallography.

The Protein Crystallography Station at Los Alamos Neutron Science Center is a high-performance beamline that forms the core of a capability for neutron macromolecular structure and function determination. This capability also includes the Macromolecular Neutron Crystallography (MNC) consortium between Los Alamos (LANL) and Lawrence Berkeley National Laboratories for developing computational tools for neutron protein crystallography, a biological deuteration laboratory, the National Stable Isotope Production Facility, and an MNC drug design consortium between LANL and Case Western Reserve University.

Locating active-site hydrogen atoms in D-xylose isomerase: time-of-flight neutron diffraction.

Time-of-flight neutron diffraction has been used to locate hydrogen atoms that define the ionization states of amino acids in crystals of D-xylose isomerase. This enzyme, from Streptomyces rubiginosus, is one of the largest enzymes studied to date at high resolution (1.8 A) by this method. We have determined the position and orientation of a metal ion-bound water molecule that is located in the active site of the enzyme; this water has been thought to be involved in the isomerization step in which D-xylose is converted to D-xylulose or D-glucose to D-fructose. It is shown to be water (rather than a hydroxyl group) under the conditions of measurement (pH 8.0). Our analyses also reveal that one lysine probably has an -NH(2)-terminal group (rather than NH(3)(+)). The ionization state of each histidine residue also was determined. High-resolution x-ray studies (at 0.94 A) indicate disorder in some side chains when a truncated substrate is bound and suggest how some side chains might move during catalysis. This combination of time-of-flight neutron diffraction and x-ray diffraction can contribute greatly to the elucidation of enzyme mechanisms.

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.

W3Y single mutant of rubredoxin from Pyrococcus furiosus: a preliminary time-of-flight neutron study.

Rubredoxin from the hyperthermophilic archaeon Pyrococcus furiosus maintains its native structure at high temperatures (373 K). In order to investigate the role of hydrogen bonding, hydration and chain dynamics in this thermostability, wavelength-resolved Laue neutron diffraction data have been collected from the W3Y single mutant (Trp3-->Tyr3) on the spallation neutron protein crystallography station (PCS) at Los Alamos Neutron Science Center. Data were measured at room temperature from nine crystal settings, each of approximately 12 h duration. The total data-measurement period was less than 5 d from a single crystal that had undergone H(2)O/D(2)O exchange. The nominal resolution of the data is 2.1 A.

Protein crystallography with spallation neutrons.

Spallation neutrons are ideal for diffraction studies of proteins and oriented molecular complexes. With spallation neutrons and their time-dependent wavelength structure, one can select data with an optimal wavelength band and cover the whole Laue spectrum as time (wavelength) resolved diffraction data. This optimises 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 Centre. A partially coupled moderator is used to increase flux and data are collected by a cylindrical He3 detector covering 120 degrees with 200 mm height. The PCS is described along with some examples of data collected from proteins.

Structural characterization of crystals of alpha-glycine during anomalous electrical behaviour.

The crystal structure of alpha-glycine has been investigated in the temperature range 288-427 K 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.