Structural Neutron Research Articles

Temperature-Induced Replacement of Phosphate Proton with Metal Ion Captured in Neutron Structures of A-DNA

Nucleic acids can fold into well-defined 3D structures that help determine their function. Knowing precise nucleic acid structures can also be used for the design of nucleic acid-based therapeutics. However, locations of hydrogen atoms, which are key players of nucleic acid function, are normally not determined with X-ray crystallography. Accurate determination of hydrogen atom positions can provide indispensable information on protonation states, hydrogen bonding, and water architecture in nucleic acids. Here, we used neutron crystallography in combination with X-ray diffraction to obtain joint X-ray/neutron structures at both room and cryo temperatures of a self-complementary A-DNA oligonucleotide d[GTGG(CSe)CAC]2 containing 2'-SeCH3 modification on Cyt5 (CSe) at pH 5.6. We directly observed protonation of a backbone phosphate oxygen of Ade7 at room temperature. The proton is replaced with hydrated Mg2+ upon cooling the crystal to 100 K, indicating that metal binding is favored at low temperature, whereas proton binding is dominant at room temperature.

Intriguing role of water in protein-ligand binding studied by neutron crystallography on trypsin complexes

Hydrogen bonds are key interactions determining protein-ligand binding affinity and therefore fundamental to any biological process. Unfortunately, explicit structural information about hydrogen positions and thus H-bonds in protein-ligand complexes is extremely rare and similarly the important role of water during binding remains poorly understood. Here, we report on neutron structures of trypsin determined at very high resolutions ≤1.5 Å in uncomplexed and inhibited state complemented by X-ray and thermodynamic data and computer simulations. Our structures show the precise geometry of H-bonds between protein and the inhibitors N-amidinopiperidine and benzamidine along with the dynamics of the residual solvation pattern. Prior to binding, the ligand-free binding pocket is occupied by water molecules characterized by a paucity of H-bonds and high mobility resulting in an imperfect hydration of the critical residue Asp189. This phenomenon likely constitutes a key factor fueling ligand binding via water displacement and helps improving our current view on water influencing protein–ligand recognition.

Elucidation of Hydrogen Bonding Patterns in Ligand-Free, Lactose- and Glycerol-Bound Galectin-3C by Neutron Crystallography to Guide Drug Design.

The medically important drug target galectin-3 binds galactose-containing moieties on glycoproteins through an intricate pattern of hydrogen bonds to a largely polar surface-exposed binding site. All successful inhibitors of galectin-3 to date have been based on mono- or disaccharide cores closely resembling natural ligands. A detailed understanding of the H-bonding networks in these natural ligands will provide an improved foundation for the design of novel inhibitors. Neutron crystallography is an ideal technique to reveal the geometry of hydrogen bonds because the positions of hydrogen atoms are directly detected rather than being inferred from the positions of heavier atoms as in X-ray crystallography. We present three neutron crystal structures of the C-terminal carbohydrate recognition domain of galectin-3: the ligand-free form and the complexes with the natural substrate lactose and with glycerol, which mimics important interactions made by lactose. The neutron crystal structures reveal unambiguously the exquisite fine-tuning of the hydrogen bonding pattern in the binding site to the natural disaccharide ligand. The ligand-free structure shows that most of these hydrogen bonds are preserved even when the polar groups of the ligand are replaced by water molecules. The protonation states of all histidine residues in the protein are also revealed and correlate well with NMR observations. The structures give a solid starting point for molecular dynamics simulations and computational estimates of ligand binding affinity that will inform future drug design.

"To Be or Not to Be" Protonated: Atomic Details of Human Carbonic Anhydrase-Clinical Drug Complexes by Neutron Crystallography and Simulation

Human carbonic anhydrases (hCAs) play various roles in cells, and have been drug targets for decades. Sequence similarities of hCA isoforms necessitate designing specific inhibitors, which requires detailed structural information for hCA-inhibitor complexes. We present room temperature neutron structures of hCA II in complex with three clinical drugs that provide in-depth analysis of drug binding, including protonation states of the inhibitors, hydration water structure, and direct visualization of hydrogen-bonding networks in the enzyme's active site. All sulfonamide inhibitors studied bind to the Zn metal center in the deprotonated, anionic, form. Other chemical groups of the drugs can remain neutral or be protonated when bound to hCA II. MD simulations have shown that flexible functional groups of the inhibitors may alter their conformations at room temperature and occupy different sub-sites. This study offers insights into the design of specific drugs to target cancer-related hCA isoform IX.

A single crystal neutron diffraction study on mixed crystal $$\hbox {(K)}_{0.25}(\hbox {NH}_{4})_{0.75}\hbox {H}_{2}\hbox {PO}_{4}$$ (K) 0.25 ( NH 4 ) 0.75 H 2 PO 4 : tuning of short strong hydrogen bonds by ionic interactions

One of the most intriguing facts about hydrogen bonds is that bonds formed between the same couple of donor and acceptor atoms can have large variation in the geometry as well as energies; this is mainly because hydrogen bonds are highly sensitive to their environment. We report here a comparative study of neutron structures of mixed crystals of potassium–ammonium dihydrogen phosphate. Basic structural framework of crystals belonging to potassium dihydrogen phosphate family is constructed by a network of zigzag O–H–O hydrogen bonded chains of \(\hbox {PO}_{2}\hbox {(OH)}_{2}\) anions, with cations occupying the interspaces of the zigzagged anionic chains. These crystals having simple crystal structure primarily determined by the O–H–O hydrogen bonds connecting \(\hbox {PO}_{2}\hbox {(OH)}_{2}\) anions provide us with a unique opportunity to tune the hydrogen bond geometry by varying the cationic occupancy and study the effect of this change on the average structure of the crystal. The cation–anion interaction is found to have a direct influence on the O–H–O hydrogen bonds of the mixed crystals.

The Nature and Reactivity of Ferryl Heme in Compounds I and II.

Aerobic organisms have evolved to activate oxygen from the atmosphere, which allows them to catalyze the oxidation of different kinds of substrates. This activation of oxygen is achieved by a metal center (usually iron or copper) buried within a metalloprotein. In the case of iron-containing heme enzymes, the activation of oxygen is achieved by formation of transient iron-oxo (ferryl) intermediates; these intermediates are called Compound I and Compound II. The Compound I and II intermediates were first discovered in the 1930s in horseradish peroxidase, and it is now known that these same species are used across the family of heme enzymes, which include all of the peroxidases, the heme catalases, the P450s, cytochrome c oxidase, and NO synthase. Many years have passed since the first observations, but establishing the chemical nature of these transient ferryl species remains a fundamental question that is relevant to the reactivity, and therefore the usefulness, of these species in biology. This Account summarizes experiments that were conceived and conducted at Leicester and presents our ideas on the chemical nature, stability, and reactivity of these ferryl heme species. We begin by briefly summarizing the early milestones in the field, from the 1940s and 1950s. We present comparisons between the nature and reactivity of the ferryl species in horseradish peroxidase, cytochrome c peroxidase, and ascorbate peroxidase; and we consider different modes of electron delivery to ferryl heme, from different substrates in different peroxidases. We address the question of whether the ferryl heme is best formulated as an (unprotonated) FeIV═O or as a (protonated) FeIV-OH species. A range of spectroscopic approaches (EXAFS, resonance Raman, Mossbauer, and EPR) have been used over many decades to examine this question, and in the last ten years, X-ray crystallography has also been employed. We describe how information from all of these studies has blended together to create an overall picture, and how the recent application of neutron crystallography has directly identified protonation states and has helped to clarify the precise nature of the ferryl heme in cytochrome c peroxidase and ascorbate peroxidase. We draw comparisons between the Compound I and Compound II species that we have observed in peroxidases with those found in other heme systems, notably the P450s, highlighting possible commonality across these heme ferryl systems. The identification of proton locations from neutron structures of these ferryl species opens the door for understanding the proton translocations that need to occur during O-O bond cleavage.

Characterization of the new scintillator Cs2LiYCl6:Ce3+

The first domestic inorganic scintillator, Cs2LiYCl6:Ce3+ (CLYC), was grown at Beijing Glass Research Institute using the vertical Bridgman method. In this work, we evaluated the performance of this new CLYC crystal in terms of its gamma-ray energy resolution and pulse shape discrimination (PSD) capability between neutrons and gamma rays. The decay times associated with different scintillation mechanisms were obtained by fitting decay functions to the neutron and gamma-ray waveform structures. We found an energy resolution of ~ 4.5% for 662-keV gamma rays and efficient neutron/gamma PSD with a figure of merit of ~ 2.6. Under gamma-ray excitation, there is an ultrafast scintillation mechanism in CLYC with a decay time of approximately 2 ns, whereas there is no evidence of ultrafast decay under thermal neutron excitation. This work contributes to the promotion of domestic development of CLYC.

Neutron and Atomic Resolution X-ray Structures of a Lytic Polysaccharide Monooxygenase Reveal Copper-Mediated Dioxygen Binding and Evidence for N-Terminal Deprotonation.

A 1.1 Å resolution, room-temperature X-ray structure and a 2.1 Å resolution neutron structure of a chitin-degrading lytic polysaccharide monooxygenase domain from the bacterium Jonesia denitrificans (JdLPMO10A) show a putative dioxygen species equatorially bound to the active site copper. Both structures show an elongated density for the dioxygen, most consistent with a Cu(II)-bound peroxide. The coordination environment is consistent with Cu(II). In the neutron and X-ray structures, difference maps reveal the N-terminal amino group, involved in copper coordination, is present as a mixed ND2 and ND-, suggesting a role for the copper ion in shifting the pKa of the amino terminus.

Insights into the Catalytic Mechanism and Transition State Stabilization of 5′‐Methylthioadenosine Nucleosidase using Neutron Crystallography

5′‐Methylthioadenosine nucleosidase (MTAN) is an obligatory homodimeric protein that catalyzes the hydrolysis of the N‐ribosidic bond of a variety of adenosine‐containing metabolites. The proposed catalytic mechanism of MTAN is initiated by D198 protonating the N7 position of the adenyl moiety, which consequently causes elongation and ultimate breaking of the N‐ribosidic bond, resulting in the formation of an oxocarbenium ion intermediate. Of particular interest is the pKa elevation of D198 that was proposed to have a value of 8.2, which is significantly higher than the theoretical pKa for an aspartic acid side chain. In this study we present the first joint X‐ray/neutron crystal structures of Helicobacter pylori MTAN (HpMTAN) in complex with a substrate (S‐adenosylhomocysteine), early (Formycin A) and late (p‐ClPh‐Thio‐DADMe‐ImmA) dissociative transition state analogues, as well as with the catalytic products (adenine and S‐ribosylhomocysteine). The solved X‐ray/neutron crystal structures provide the positions of exchangeable hydrogen atoms and protonation states of ionizable groups that further our understanding of the catalytic mechanism as well as the stabilization of the transition state for HpMTAN. These results confirm that the catalytic reaction is initiated by D198 functioning as a general acid through the observation of a shared deuterium atom with unconventional hydrogen bond geometry between the carboxylate group of D198 and the N7 position of adenine. Additionally, the transition state analogues neutron structures illustrate the minor differences in the hydrogen bond interactions resulting in the DADMe‐ImmA analogues being extremely potent inhibitors with Kd values in the picomolar range, despite HpMTAN shown to contain an early dissociative transition state structure.Support or Funding InformationThis work was supported by the Center for the Advancement of Science in Space via a cooperative agreement with National Aeronautics and Space Administration Grant N‐123528‐01 (to D.R.R) and by National Institute of Allergy and Infectious Disease/NIH Grant AI105084 (to D.R.R.).

Study of neutron irradiated structures of ammonothermal GaN

Study of the radiation damage in GaN-based materials becomes an important aspect for possible application of the GaN detectors in the harsh radiation environment at the Large Hadron Collider and at other particle acceleration facilities. Intentionally doped and semi-insulating bulk ammonothermal GaN materials were studied to reveal the dominant defects introduced by reactor neutron irradiations. These radiation defects have been identified by combining electron spin resonance and transmission spectroscopy techniques. Characteristics of carrier lifetime dependence on neutron irradiation fluence were examined. Variations of the response of the capacitor-type sensors with neutron irradiation fluence have been correlated with the carrier lifetime changes. The measurements of the photoconductivity and photoluminescence transients have been used to study the variation of the parameters of radiative and non-radiative recombination. The examined characteristics indicate that AT GaN as a particle sensing material is radiation hard up to high hadron fluences ⩾1016 cm−2.

Neutron Diffraction Studies on Guest-Induced Distortions in Urea Inclusion Compounds

Single crystal neutron structures at several temperatures have been determined for β-phase urea inclusion compounds containing hexadecane, 1,6-dibromohexane, and 2,7-octanedione guests. The neutron structure of the “partial channel” cocrystal of urea and DMF is also reported. Here we present an in-depth discussion and analysis of the structure and bonding of this urea series, in particular, how the guest compound affects the symmetry and hydrogen bonding of the host urea network. Additionally, we address the challenge of obtaining crystals suitable for neutron diffraction and present a new heating/cooling device to aid crystallization.

Neutron structures of the Helicobacter pylori 5′-methylthioadenosine nucleosidase highlight proton sharing and protonation states

MTAN (5'-methylthioadenosine nucleosidase) catalyzes the hydrolysis of the N-ribosidic bond of a variety of adenosine-containing metabolites. The Helicobacter pylori MTAN (HpMTAN) hydrolyzes 6-amino-6-deoxyfutalosine in the second step of the alternative menaquinone biosynthetic pathway. Substrate binding of the adenine moiety is mediated almost exclusively by hydrogen bonds, and the proposed catalytic mechanism requires multiple proton-transfer events. Of particular interest is the protonation state of residue D198, which possesses a pKa above 8 and functions as a general acid to initiate the enzymatic reaction. In this study we present three corefined neutron/X-ray crystal structures of wild-type HpMTAN cocrystallized with S-adenosylhomocysteine (SAH), Formycin A (FMA), and (3R,4S)-4-(4-Chlorophenylthiomethyl)-1-[(9-deaza-adenin-9-yl)methyl]-3-hydroxypyrrolidine (p-ClPh-Thio-DADMe-ImmA) as well as one neutron/X-ray crystal structure of an inactive variant (HpMTAN-D198N) cocrystallized with SAH. These results support a mechanism of D198 pKa elevation through the unexpected sharing of a proton with atom N7 of the adenine moiety possessing unconventional hydrogen-bond geometry. Additionally, the neutron structures also highlight active site features that promote the stabilization of the transition state and slight variations in these interactions that result in 100-fold difference in binding affinities between the DADMe-ImmA and ImmA analogs.

Tolman-Oppenheimer-Volkoff equations and their implications for the structures of relativistic stars in f ( T ) $f(T)$ gravity

We investigate in this paper the structures of neutron and quark stars in f(T) theory of gravity where T denotes the torsion scalar. Attention is attached to the TOV type equations of this theory and numerical integrations of these equations are performed with suitable EoS. We search for the deviation of the mass-radius diagrams for power-law and exponential type correction from the TT gravity. Our results show that for some values of the input parameters appearing in the considered models, f(T) theory promotes more the structures of the relativistic stars, in consistency with the observational data.

Neutron protein crystallography: A complementary tool for locating hydrogens in proteins

Neutron protein crystallography is a powerful tool for investigating protein chemistry because it directly locates hydrogen atom positions in a protein structure. The visibility of hydrogen and deuterium atoms arises from the strong interaction of neutrons with the nuclei of these isotopes. Positions can be unambiguously assigned from diffraction at resolutions typical of protein crystals. Neutrons have the additional benefit to structural biology of not inducing radiation damage in protein crystals. The same crystal could be measured multiple times for parametric studies. Here, we review the basic principles of neutron protein crystallography. The information that can be gained from a neutron structure is presented in balance with practical considerations. Methods to produce isotopically-substituted proteins and to grow large crystals are provided in the context of neutron structures reported in the literature. Available instruments for data collection and software for data processing and structure refinement are described along with technique-specific strategies including joint X-ray/neutron structure refinement. Examples are given to illustrate, ultimately, the unique scientific value of neutron protein crystal structures.

Protonation State and Hydration of Bisphosphonate Bound to Farnesyl Pyrophosphate Synthase.

Farnesyl pyrophosphate synthase (FPPS) catalyzes the condensation of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate to FPP and is known to be a molecular target of osteoporosis drugs, such as risedronate (RIS), which is a nitrogen-containing bisphosphonate. The protonation states and hydration structure of RIS bound to FPPS were determined by neutron protein crystallography, which allows direct visualization of hydrogens and deuteriums. The structure analysis revealed that the phosphate groups of RIS were fully deprotonated with the abnormally decreased pKa, and that the roles of E93 and D264 consisted of canceling the extra negative charges upon the binding of ligands. Collectively, our neutron structures provided insights into the physicochemical properties during the bisphosphonate binding event.

Neutron/X‐ray Joint Refinement of The Active and Inactive Form of Helicobacter pylori 5′‐ Methylthioadenosine Nucleosidase

5′‐Methylthioadenosine nucleosidase (HpMTAN) is an essential Helicobacter pylori enzyme that hydrolyzes N‐ribosidic bonds of a variety of adenosine‐based metabolites. Recently, HpMTAN has become an attractive drug target due to the enzyme's role in the menaquinone pathway that is only specific for H. pylori and Campylobacter. In the proposed catalytic mechanism of HpMTAN, the reaction is initiated by D198 that acts as a general acid to protonate the N7 position of the adenine moiety. The protonation creates a delocalized positive charge in the adenine moiety that promotes stretching of the N‐ribosidic bond to progress the catalytic reaction. Currently, transition state analogs are known to function as powerful inhibitors of MTAN; however, the majority of these compounds exhibit inhibition of the human enzyme 5′‐methylthioadenosine phosphorylase (hMTAP), a homolog to HpMTAN. In this study, we present the first neutron structures of an active and an inactive variant (D198N) of HpMTAN that were co‐crystallized with SAH. The structures were solved using X‐ray crystallography and subjected to joint X‐ray/neutron refinement to allow for direct determination of hydrogen atom positioning throughout the catalytic domain of HpMTAN. The neutron structures confirm the proposed mechanism in which D198 acts as a general acid and illustrates other features of the catalytic reaction. Additionally, key polar interactions are highlighted in the catalytic domain that will aid in structure‐based design of inhibitors that will be selective for HpMTAN and have little to no effect on hMTAP.

Frequent side chain methyl carbon-oxygen hydrogen bonding in proteins revealed by computational and stereochemical analysis of neutron structures.

The propensity of backbone Cα atoms to engage in carbon-oxygen (CH · · · O) hydrogen bonding is well-appreciated in protein structure, but side chain CH · · · O hydrogen bonding remains largely uncharacterized. The extent to which side chain methyl groups in proteins participate in CH · · · O hydrogen bonding is examined through a survey of neutron crystal structures, quantum chemistry calculations, and molecular dynamics simulations. Using these approaches, methyl groups were observed to form stabilizing CH · · · O hydrogen bonds within protein structure that are maintained through protein dynamics and participate in correlated motion. Collectively, these findings illustrate that side chain methyl CH · · · O hydrogen bonding contributes to the energetics of protein structure and folding.

Precise Calculation of the Up and Down Quark Mass Using an Adjusted Compton Wavelength Common Factor Analysis

This paper presents an analytical method used to calculate a precise value of the up and down quark masses. The method used is based on a hypothesis that elementary particles share force carrier particles with precise quantities of energy in order to bond using integer multiples of their adjusted Compton wavelengths in addition to their deBroglie momentum wavelengths. Overlapping wavelengths were found by converting mass and coupling energies into large integers using an integer based derivation of Einstein's energy momentum equation. Both the (uud) proton and (udd) neutron structures were modeled, and the 2010 CODATA neutron-proton mass ratio was used to narrow the range of uncertainty in the quark mass calculations. The up and down quark mass search range extended from 0-6.2 MeV and 0.8-8.8 MeV respectively, with the largest common factor spike appearing within the QCD predicted values suggesting this method is valid.

Accurate Determination of Tautomeric/Protonation States in Quantum-Mechanic Driven Macromolecular Crystallographic Refinement

Structure Based Drug Discovery (SBDD) is employed by virtually all pharmaceutical research and development organizations. Gaining an understanding of the protein:ligand complex structure along with the proper protonation and explicit solvent effects is crucial for obtaining meaningful results from docking, thermodynamic calculations, active site exploration, and ultimately lead optimization. Recently, we incorporated our linear-scaling, quantum mechanics (QM) DivCon tool with Phenix (e.g. Phenix/DivCon) in order to accurately elucidate the protein:ligand complex molecular structure. An intrinsic problem of the X-ray crystallographic data is its inability to detect hydrogen atoms - even at higher resolutions. It is generally extremely difficult to experimentally determine the protonation/tautomeric state of the ligand and the surrounding active site. Traditionally, protonation can be established using the neutron diffraction; however, experimental requirements such as reliance on very large crystals and on deuterium exchange limit the method's suitability in SBDD.In order to address this X-day data deficiency, we have challenged Phenix/DivCon with various protonation candidates and applied rigorous statistical analyses to measure the agreement between the 3D structure of each candidate with electron density. While through the experiment we still cannot directly observe hydrogen atoms, using the accurate QM functional we are able to observe the presence/absence of hydrogen atoms by studying their influences on bound heavy atoms (Carbon, Nitrogen, Oxygen). To evaluate our protocol we have chosen two protein:ligand structures 4N9S and 2JJJ for which both neutron and X-ray structures and data are available in PDB. Ten probable protonation states for the ligands in those structures have been generated, and each of the possible candidates has been refined against X-ray data with Phenix/DivCon. We have found out that the top scored tautomer in each case coincides with the ligand structure revealed by the neutron diffraction.

Insights into the crystallisation process from anhydrous, hydrated and solvated crystal forms of diatrizoic acid.

Diatrizoic acid (DTA), a clinically used X-ray contrast agent, crystallises in two hydrated, three anhydrous and nine solvated solid forms, all of which have been characterised by X-ray crystallography. Single-crystal neutron structures of DTA dihydrate and monosodium DTA tetrahydrate have been determined. All of the solid-state structures have been analysed using partial atomic charges and hardness algorithm (PACHA) calculations. Even though in general all DTA crystal forms reveal similar intermolecular interactions, the overall crystal packing differs considerably from form to form. The water of the dihydrate is encapsulated between a pair of host molecules, which calculations reveal to be an extraordinarily stable motif. DTA presents functionalities that enable hydrogen and halogen bonding, and whilst an extended hydrogen-bonding network is realised in all crystal forms, halogen bonding is not present in the hydrated crystal forms. This is due to the formation of a hydrogen-bonding network based on individual enclosed water squares, which is not amenable to the concomitant formation of halogen bonds. The main interaction in the solvates involves the carboxylic acid, which corroborates the hypothesis that this strong interaction is the last one to be broken during the crystal desolvation and nucleation process.