Neutron Laue macromolecular crystallography

Recent progress in neutron protein crystallography such as the use of the Laue technique and improved neutron optics and detector technologies have dramatically improved the speed and precision with which neutron protein structures can now be determined. These studies are providing unique and complementary insights on hydrogen and hydration in protein crystal structures that are not available from X-ray structures alone. Parallel improvements in modern molecular biology now allow fully (per)deuterated protein samples to be produced for neutron scattering that essentially eradicate the large-and ultimately limiting-hydrogen incoherent scattering background that has hampered such studies in the past. High quality neutron data can now be collected to near atomic resolution (approximately 2.0 A) for proteins of up to approximately 50 kDa molecular weight using crystals of volume approximately 0.1 mm3 on the Laue diffractometer at ILL. The ability to flash-cool and collect high resolution neutron data from protein crystals at cryogenic temperature (15 K) has opened the way for kinetic crystallography on freeze trapped systems. Current instrument developments now promise to reduce crystal volume requirements by a further order of magnitude, making neutron protein crystallography a more accessible and routine technique.     

Hydration in proteins observed by high-resolution neutron crystallography

It is well known that water molecules surrounding a protein play important roles in maintaining its structural stability. Water molecules are known to participate in several physiological processes through the formation of hydrogen bonds. However, the hydration structures of most proteins are not known well at an atomic level at present because X-ray protein crystallography has difficulties to localize hydrogen atoms. In contrast, neutron crystallography has no problem in determining the position of hydrogens with high accuracy.1 In this article, the hydration structures of three proteins are described- myoglobin, wild-type rubredoxin, and a mutant rubredoxin-the structures of which were solved at 1.5- or 1.6-A resolution by neutron structure determination. These hydration patterns show fascinating features and the water molecules adopt a variety of shapes in the neutron Fourier maps, revealing details of intermolecular hydrogen bond formation and dynamics of hydration. Our results further show that there are strong relationships between these shapes and the water environments.     

Production and characterization of recombinant perdeuterated cholesterol oxidase

Cholesterol oxidase (CO) is a FAD (flavin adenine dinucleotide) containing enzyme that catalyzes the oxidization and isomerization of cholesterol. Studies directed toward elucidating the catalytic mechanism of CO will provide an important general understanding of Flavin-assisted redox catalysis. Hydrogen atoms play an important role in enzyme catalysis; however, they are not readily visualized in protein X-ray diffraction structures. Neutron crystallography is an ideal method for directly visualizing hydrogen positions at moderate resolutions because hydrogen and deuterium have comparable neutron scattering lengths to other heavy atoms present in proteins. The negative coherent and large incoherent scattering lengths of hydrogen atoms in neutron diffraction experiments can be circumvented by replacing hydrogen atoms with its isotope, deuterium. The perdeuterated form of CO was successfully expressed from minimal medium, purified, and crystallized. X-ray crystallographic structures of the enzyme in the perdeuterated and hydrogenated states confirm that there are no apparent structural differences between the two enzyme forms. Kinetic assays demonstrate that perdeuterated and hydrogenated enzymes are functionally identical. Together, structural and functional studies indicate that the perdeuterated protein is suitable for structural studies by neutron crystallography directed at understanding the role of hydrogen atoms in enzyme catalysis.     

Solvent structure in crystals of trypsin determined by X-ray and neutron diffraction.

The solvent structure in orthorhombic crystals of bovine trypsin has been independently determined by X-ray diffraction to 1.35 A resolution and by neutron diffraction to 2.1 A resolution. A consensus model of the water molecule positions was obtained using oxygen positions identified in the electron density map determined by X-ray diffraction, which were verified by comparison to D2O-H2O difference neutron scattering density. Six of 184 water molecules in the X-ray structure, all with B-factors greater than 50 A2, were found to be spurious after comparison with neutron results. Roughly two-thirds of the water of hydration expected from thermodynamic data for proteins was localized by neutron diffraction; approximately one-half of the water of hydration was located by X-ray diffraction. Polar regions of the protein are well hydrated, and significant D2O-H2O difference density is seen for a small number of water molecules in a second shell of hydration. Hydrogen bond lengths and angles calculated from unconstrained refinement of water positions are distributed about values typically seen in small molecule structures. Solvent models found in seven other bovine trypsin and trypsinogen and rat trypsin structures determined by X-ray diffraction were compared. Internal water molecules are well conserved in all trypsin structures including anionic rat trypsin, which is 65% homologous to bovine trypsin. Of the 22 conserved waters in trypsin, 19 were also found in trypsinogen, suggesting that they are located in regions of the apoprotein that are structurally conserved in the transition to the mature protein. Seven waters were displaced upon activation of trypsinogen. Water structure at crystal contacts is not generally conserved in different crystal forms. Three groups of integral structural water molecules are highly conserved in all solvent structures, including a spline of water molecules inserted between two beta-strands, which may resemble an intermediate in the formation of beta sheets during the folding of a protein.     

Damage susceptibility of human lymphocyte chromosomes at different stages of the mitotic cycle in combined exposure to fast neutrons and postradiation hyperthermia

The combined action of gamma rays (1-4 Gy) and postradiation hyperthermia on the human lymphocyte culture induces intensification of the damaging effect at the stage of DNA synthesis, i.e. at the most radioresistant stage of the mitotic cycle, the thermal intensification factor (TIF) being 1.7-2.0. After the neutron action (0.5-1.5 Gy) the hyperthermia has no effect on the chromosome aberration spectrum, the TIF being 1.8-2.5 at G1 phase and 1.8-2.4 at S phase, which testifies to the possible modification of neutron irradiation.     

A quasi-Laue neutron crystallographic study of d-xylose isomerase

The location of hydrogen atoms in enzyme structures can bring critical understanding of catalytic mechanism. However, whilst it is often difficult to determine the position of hydrogen atoms using X-ray crystallography even with subatomic (<1.0 A) resolution data available, neutron crystallography provides an experimental tool to directly localize hydrogen/deuterium atoms in biological macromolecules at resolution of 1.5-2.0 A. D-Xylose isomerase (D-xylose ketol-isomerase, EC 5.3.1.5) is a 43 kDa enzyme that catalyses the first reaction in the catabolism of D-xylose. Linearization and isomerization of D-xylose at the active site of D-xylose isomerase rely upon a complex hydrogen transfer. Neutron quasi-Laue data at 2.2 A resolution were collected at room temperature on a partially deuterated Streptomyces rubiginosus D-xylose isomerase crystal using the LADI instrument at ILL with the objective to provide insight into the enzymatic mechanism. The neutron structure shows unambiguously that residue His 53 is doubly protonated at the active site of the enzyme. This suggests that the reaction proceeds through an acid catalyzed opening of the sugar ring, which is in accord with the mechanism suggested by Fenn et al. (Biochemistry 43(21): 6464-6474, 2004). This is the first report of direct observation of double protonation of His 53 and the first validation of the ring opening mechanism at the active site of D-xylose isomerase.     

Comparison of hydrogen determination with X-ray and neutron crystallography in a human aldose reductase–inhibitor complex

Protonation states determination by neutron (2.2 A at room temperature) and X-ray (0.66 A at 100 K) crystallographic studies were compared for a medium size enzyme, human aldose reductase (MW=36 kDa), complexed with its NADP+ coenzyme and a selected inhibitor of therapeutic interest. The neutron resolution could be achieved only with the ab initio fully deuterated protein and the subsequent crystallization in D2O of the complex. We used the largest good-quality crystal (1.00x0.67x0.23 mm, i.e. volume of 0.15 mm3) that we were able to grow so far. Both studies enable the determination of protonation states, with a clear advantage for neutrons in the case of less-ordered atoms (B>5 A2). Hydrogen atoms are best determined by a complementary analysis of the Fourier maps obtained from both methods.     

The extended multidomain solution structures of the complement protein Crry and its chimeric conjugate Crry-Ig by scattering,analytical ultracentrifugation and constrained modelling: implications for function and therapy

Complement receptor-related gene/protein y (Crry) is a cell membrane-bound regulator of complement activation found in mouse and rat. Crry contains only short complement/consensus repeat (SCR) domains. X-ray and neutron scattering was performed on recombinant rat Crry containing the first five SCR domains (rCrry) and mouse Crry with five SCR domains conjugated to the Fc fragment of mouse IgG1 (mCrry-Ig) in order to determine their solution structures at medium resolution. The radius of gyration R(G) of rCrry was determined to be 4.9-5.0 nm, and the R(G) of the cross-section was 1.2-1.5 nm as determined by X-ray and neutron scattering. The R(G) of mCrry-Ig was 6.6-6.7 nm, and the R(G) of the cross-section were 2.3-2.4 nm and 1.3 nm. The maximum dimension of rCrry was 18 nm and that for mCrry-Ig was 26 nm. The neutron data indicated that rCrry and mCrry-Ig have molecular mass values of 45,000 Da and 140,000 Da, respectively, in agreement with their sequences, and sedimentation equilibrium data supported these determinations. Time-derivative velocity experiments gave sedimentation coefficients of 2.4S for rCrry and 5.4S for mCrry-Ig. A medium-resolution model of rCrry was determined using homology models that were constructed for the first five SCR domains of Crry from known crystal and NMR structures, and linked by randomly generated linker peptide conformations. These trial-and-error calculations revealed a small family of extended rCrry structures that best accounted for the scattering and ultracentrifugation data. These were shorter than the most extended rCrry models as the result of minor bends in the inter-SCR orientations. The mCrry-Ig solution data were modelled starting from a fixed structure for rCrry and the crystal structure of mouse IgG1, and was based on conformational searches of the hinge peptide joining the mCrry and Fc fragments. The best-fit models showed that the two mCrry antennae in mCrry-Ig were extended from the Fc fragment. No preferred orientation of the antennae was identified, and this indicated that the accessibility of the antennae for the molecular targets C4b and C3b was not affected by the covalent link to Fc. A structural comparison between Crry and complement receptor type 1 indicated that the domain arrangement of Crry SCR 1-3 is as extended as that of the CR1 SCR 15-17 NMR structure.     

Neutron and X-ray crystallographic analysis of the human α-thrombin–bivalirudin complex at pD 5.0: Protonation states and hydration structure of the enzyme–product complex

The protonation states and hydration structures of the α-thrombin–bivalirudin complex were studied by joint XN refinement of the single crystal X-ray and neutron diffraction data at resolutions of 1.6 and 2.8 Å, respectively. The atomic distances were estimated by carrying out X-ray crystallographic analysis at 1.25 Å resolution. The complex represents a model of the enzyme-product (EP) complex of α-thrombin. The neutron scattering length maps around the active site suggest that the side chain of H57/H was deuterated. The joint XN refinement showed that occupancies for Dδ1 and Dε2 of H57/H were 1.0 and 0.7, respectively. However, no significant neutron scattering length density was observed around the hydroxyl oxygen Oγ of S195/H, which was close to the carboxylic carbon atom of dFPR-COOH. These observations suggest that the Oγ atom of S195/H is deprotonated and maintains its nucleophilicity in the EP complex. In addition to the active site, the hydration structures of the S1 subsite and the Exosite I, which are involved in the recognition of bivalirudin, are presented.     

Relative cataractogenic effects of X rays, fission-spectrum neutrons, and 56Fe particles: a comparison with mitotic effects

The eyes of Sprague-Dawley rats were irradiated with doses of 2.5-10 Gy 250-kVp X rays, 1.25-2.25 Gy fission-spectrum neutrons (approximately 0.85 MeV), or 0.1-2.0 Gy 600-MeV/A 56Fe particles. Lens opacifications were evaluated for 51-61 weeks following X and neutron irradiations and for 87 weeks following X and 56Fe-particle irradiations. Average stage of opacification was determined relative to time after irradiation, and the time required for 50% of the irradiated lenses to achieve various stages (T50) was determined as a function of radiation dose. Data from two experiments were combined in dose-effect curves as T50 experimental values taken as percentages of the respective T50 control values (T50-% control). Simple exponential curves best describe dose responsiveness for both high-LET radiations. For X rays, a shallow dose-effect relationship (shoulder) up to 4.5 Gy was followed at higher doses by a steeper exponential dose-effect relationship. As a consequence, RBE values for the high-LET radiations are dose dependent. Dose-effect curves for cataracts were compared to those for mitotic abnormalities observed when quiescent lens epithelial cells were stimulated mechanically to proliferate at various intervals after irradiation. Neutrons were about 1.6-1.8 times more effective than 56Fe particles for inducing both cataracts and mitotic abnormalities. For stage 1 and 2 cataracts, the X-ray Dq was 10-fold greater and the D0 was similar to those for mitotic abnormalities initially expressed after irradiation.     

Neutron emission during the implosion of a wire array onto a deuterated fiber

Results are presented from Z-pinch experiments performed in the S-300 facility (Kurchatov Institute) at a maximum current of 2 MA and current rise time of 100 ns. The Z-pinch load was a 1-cm-long 1-cmdiameter cylindrical array made of 40 tungsten wires with a total mass of 160 μg, at the axis of which a 100-μm-diameter (CD₂) _n deuterated fiber was installed. Hard X-ray and neutron signals were recorded using five scintillation detectors oriented in one radial and two axial directions. The maximum neutron yield from the DD reaction reached 3 × 10⁹ neutrons per shot. The average neutron energy was determined from time-of-flight measurements and Monte Carlo simulations under the assumption that the neutron emission time was independent of the neutron energy. The average neutron energy in different experiments was found to vary within the range 2.5–2.7 MeV. The fact that the average neutron energy was higher than 2.45 MeV (the energy corresponding to the DD reaction) is attributed to the beam-target collisional mechanism for the acceleration of deuterons to 100–500 keV.     

Comparison of the solution and X-ray structures of barley serine proteinase inhibitor 2

A comparison of the solution n.m.r. structures of barley serine protease inhibitor 2 (BSPI-2) with the X-ray structures of both subtilisin complexed and native BSPI-2 is presented. It is shown that the n.m.r. and X-ray structures are very similar in terms of overall shape, size, polypeptide fold and secondary structure. The average atomic rms difference between the 11 restrained dynamics structures on the one hand and the two X-ray structures on the other is 1.9 +/- 0.2 A for the backbone atoms and 3.0 +/- 0.3 A for all atoms. The corresponding values for the restrained energy minimized mean dynamics structure are 1.5 and 2.4 A, respectively.     

Microdosimetry for boron neutron capture therapy.

Preclinical studies for boron neutron capture therapy (BNCT) using epithermal neutrons are ongoing at several laboratories. The absorbed dose in tumor cells is a function of the thermal neutron flux at depth, the microscopic boron concentration, and the size of the cell. Dosimetry is therefore complicated by the admixture of thermal, epithermal, and fast neutrons, plus gamma rays, and the array of secondary high-linear-energy-transfer particles produced within the patient from neutron interactions. Microdosimetry can be a viable technique for determining absorbed dose and radiation quality. A 2.5-cm-diameter tissue-equivalent gas proportional counter has been built with 50 parts per million (ppm) 10B incorporated into the walls and counting gas to simulate the boron uptake anticipated in tumors. Measurements of lineal energy (y) spectra for BNCT in simulated volumes of 1-10 microns diameter show a dose enhancement factor of 4.3 for 30 ppm boron, and a "y" of 250 keV/microns for the boron capture process. Chamber design plus details of experimental and calculated linear energy spectra will be presented.     

Fully reduced ribonuclease A does not expand at high denaturant concentration or temperature

The dimensions of a denatured protein, fully reduced ribonuclease A (r-RNase A), have been measured using synchrotron-based small angle X-ray scattering. The radius of gyration, 34-35 A, is unchanged from 0-6 M guanidinium chloride and from 20-90 degrees C at pH 2.5, and agrees with the known scaling behavior for a multitude of chemically denatured states. The polypeptide is behaving as a statistical coil in the non-interacting, high-temperature limit.     

Study of X-ray and neutron emission in experiments with Al wires in an MA plasma focus

Results are presented from experimental studies of the correlation between X-ray and neutron emissions generated in the implosion of a deuteron plasma shell onto an Al wire. The experiments were carried out on the PF-1000 facility at currents of 1.5–1.8 MA. An Al wire 80 μm in diameter and 7–9 cm in length was placed at the end of the inner electrode. During the implosion of the plasma shell, Al K-shell X-rays were first emitted at the dip of the current derivative. After the X-ray pulse, a relatively stable corona with a diameter of 2–3 mm and lifetime of a few hundred nanoseconds formed around the wire. The presence of the wire did not considerably reduce the total neutron yield (at most 10¹¹ neutrons per shot) in comparison to discharges without a wire. As a rule, the intensity of neutron emission was maximal a few tens of nanoseconds after the peak of X-ray emission. A detailed comparison of two shots with low and high neutron yields have shown that the neutron yield depends on the configuration and dynamics of the discharge. The possible influence of the self-generated axial component of the magnetic field on the development of the plasma focus and the acceleration of fast deuterons is discussed.     

Molecular modeling of the multidomain structures of the proteoglycan binding region and the link protein of cartilage by neutron and synchrotron X-ray scattering

The interaction of proteoglycan monomers with hyaluronate in cartilage is mediated by a globular binding region at the N-terminus of the proteoglycan monomer; this interaction is stabilized by link protein. Sequences show that both the binding region (27% carbohydrate) and the link protein (6% carbohydrate) contain an immunoglobulin (Ig) fold domain and two proteoglycan tandem repeat (PTR) domains. Both proteins were investigated by neutron and synchrotron X-ray solution scattering, in which nonspecific aggregate formation was reduced by the use of citraconylation to modify surface lysine residues. The neutron and X-ray radius of gyration RG of native and citraconylated binding region is 5.1 nm, and the cross-sectional RG (RXS) is 1.9-2.0 nm. No neutron contrast dependence of the RG values was observed; however, a large contrast dependence was seen for the RXS values which is attributed to the high carbohydrate content of the binding region. The neutron RG for citraconylated link protein is 2.9 nm, its RXS is 0.8 nm, and these data are also independent of the neutron contrast. The scattering curves of binding region and link protein were modeled using small spheres. Both protein structures were defined initially by the representation of one domain by a crystal structure for a variable Ig fold and a fixed volume for the two PTR domains calculated from sequence data. The final models showed that the different dimensions and neutron contrast properties of binding region compared to link protein could be attributed to an extended glycosylated C-terminal peptide with extended carbohydrate structures in the binding region.(ABSTRACT TRUNCATED AT 250 WORDS)     

Application of molecular dynamics with interproton distance restraints to three-dimensional protein structure determination. A model study of crambin

The applicability of restrained molecular dynamics for the determination of three-dimensional protein structures on the basis of short interproton distances (less than 4 A) that can be realistically determined from nuclear magnetic resonance measurements in solution is assessed. The model system used is the 1.2 A resolution crystal structure of the 46 residue protein crambin, from which a set of 240 approximate distance restraints, divided into three ranges (2.5 +/- 0.5, 3.0+0.5(-1.0) and 4 +/- 1 A), is derived. This interproton distance set comprises 159 short-range ([i-j] less than or equal to 5) and 56 ([i-j] greater than 5) long-range inter-residue distances and 25 intra-residue distances. Restrained molecular dynamics are carried out using a number of different protocols starting from two initial structures: a completely extended beta-strand; and an extended structure with two alpha-helices in the same positions as in the crystal structure (residues 7 to 19, and 23 to 30) and all other residues in the form of extended beta-strands. The root-mean-square (r.m.s.) atomic differences between these two initial structures and the crystal structure are 43 A and 23 A, respectively. It is shown that, provided protocols are used that permit the secondary structure elements to form at least partially prior to folding into a tertiary structure, convergence to the correct final structure, both globally and locally, is achieved. The r.m.s. atomic differences between the converged restrained dynamics structures and the crystal structure range from 1.5 to 2.2 A for the backbone atoms and from 2.0 to 2.8 A for all atoms. The r.m.s. atomic difference between the X-ray structure and the structure obtained by first averaging the co-ordinates of the converged restrained dynamics structures is even smaller: 1.0 A for the backbone atoms and 1.6 A for all atoms. These results provide a measure with which to judge future experimental results on proteins whose crystal structures are unknown. In addition, from an examination of the dynamics trajectories, it is shown that the convergence pathways followed by the various simulations are different.     

Structural comparisons of the native and reactive-centre-cleaved forms of alpha 1-antitrypsin by neutron- and X-ray-scattering in solution.

alpha 1-Antitrypsin is the best-characterized member of the serpin (serine-proteinase inhibitor) superfamily. Its solution structure was studied by high-flux neutron-scattering and synchrotron X-ray-scattering. Neutron data show that its absorption coefficient A1% 280,1cm is 5.4. The neutron radius of gyration RG at infinite contrast for native alpha 1-antitrypsin is 2.61 nm, characteristic of a moderately elongated structure, and its cross-sectional RG is 1.34 nm. The internal inhomogeneity of scattering densities within alpha 1-antitrypsin is high at 29 x 10(-5). The X-ray RG is 2.91 nm, in good agreement with the neutron RG of 2.82 nm in 1H2O. This RG is unchanged in reactive-centre-cleaved alpha 1-antitrypsin. These parameters are also unchanged at pH 8 in sodium/potassium phosphate buffers up to 0.6 M. The neutron and X-ray curves for native alpha 1-antitrypsin were compared with Debye simulation based on the crystal structure of reactive-centre-cleaved (papain) alpha 1-antitrypsin. After allowance for residues not visible in the crystallographic electron-density map, and rejoining the proteolysed site between Met-358 and Ser-359 by means of a relatively minor conformational re-arrangement, good agreement to a structural resolution of 4 nm is obtained with the neutron data in two contrasts and with the X-ray data. The structures of the native and cleaved forms of alpha 1-antitrypsin are thus similar within the resolution of solution scattering. This places an upper limit on the magnitude of the presumed conformational changes that occur in alpha 1-antitrypsin on reactive-centre cleavage, as indicated in earlier spectroscopic investigations of the Met-358-Ser-359 peptide-bond cleavage. Methods for scattering-curve simulations from crystal structures are critically assessed. The RG data lead to dimensions of 7.8 nm x 4.9 nm x 2.2 nm for native alpha 1-antitrypsin. The high internal inhomogeneity and the asymmetric shorter semi-axes of 4.9 nm and 2.2 nm suggest that the three oligosaccharide chains of alpha 1-antitrypsin are essentially freely extended into solvent in physiological conditions. This conclusion is also supported by the Debye simulations, and by modelling based on hydrodynamic parameters.     

Renal damage in the mouse: the effect of d(4)-Be neutrons

A further study on the response of the mouse kidney to d(4)-Be neutrons (EN = 2.3 MeV) is described. The results confirm and augment the work published previously by Stewart et al. [Br. J. Radiol. 57, 1009-1021 (1984)]; the present paper includes the data from a "top-up" design of experiment which extends the measurements of neutron RBE (relative to 240 kVp X rays) down to X-ray doses of 0.75 Gy per fraction. The mean RBE for these neutrons increases from 5.8 to 7.3 as X-ray dose per fraction decreases from 3.0 to 1.5 Gy in the kidney. This agrees with the predictions from the linear quadratic (LQ) model, based on the renal response to X-ray doses above 4 Gy per fraction. The mean RBE estimate from a single dose group at 0.75 Gy per fraction of X rays is, however, 3.9. This is below the LQ prediction and may indicate increasing X-ray sensitivity at low doses. Data from this study and from those published previously have been used to determine more accurately the shape of the underlying response to d(4)-Be neutrons; an alpha/beta ratio of 20.5 +/- 3.7 Gy was found. The best value of alpha/beta for X rays determined from these experiments was 3.04 +/- 0.35 Gy, in agreement with previous values.     

The Neutron Structure of Urate Oxidase Resolves a Long-Standing Mechanistic Conundrum and Reveals Unexpected Changes in Protonation

Urate oxidase transforms uric acid to 5-hydroxyisourate without the help of cofactors, but the catalytic mechanism has remained enigmatic, as the protonation state of the substrate could not be reliably deduced. We have determined the neutron structure of urate oxidase, providing unique information on the proton positions. A neutron crystal structure inhibited by a chloride anion at 2.3 Å resolution shows that the substrate is in fact 8-hydroxyxanthine, the enol tautomer of urate. We have also determined the neutron structure of the complex with the inhibitor 8-azaxanthine at 1.9 Å resolution, showing the protonation states of the K10–T57–H256 catalytic triad. Together with X-ray data and quantum chemical calculations, these structures allow us to identify the site of the initial substrate protonation and elucidate why the enzyme is inhibited by a chloride anion.