Site-specific relaxation of peptide bond planarity induced by electrically attracted proton/deuteron observed by neutron crystallography.

In structural biology, peptide bonds, fundamental linkages between hundreds of amino acids, of which a protein molecule is composed, have been commonly treated as a plane structure just as Linus Pauling et al. proposed. In this paper, a site-specific peptide bond relaxation mechanism by deuterons whose localization has been suggested by neutron crystallography is proposed. Such deuteron was observed as an arm of neutron scattering length density protruding from the carbonyl oxygen atoms in the main chain in the omit map drawn by neutron crystallography of human lysozyme. Our comprehensive study using x-ray and neutron diffraction and 15 N chemical shifts of individual amide nitrogen atoms within the same peptide bond strongly suggests the relaxation of the electronic resonance structure because of site-specific modulation by protons/deuterons localized on the electron orbital of the carbonyl oxygen. All experimental data used in this examination were obtained at room temperature, which is preferable for enzymatic activity. Such a close interaction between the electron resonance structure of a peptide bond and the exchangeable protons/deuterons well agreed with that observed in an intermediate state in an amide hydrolytic reaction simulated by the ab-initio calculation including water molecules.

Characterization of K-binding factor involved in water-soluble complex of menaquinone-7 produced by Bacillus subtilis natto.

Vitamin Ks are expected to contribute bone and cardiovascular health. Especially, menaquinone-7 has a higher bioavailability and a longer half-life than other vitamin Ks in the human body. However, their low water-solubility limits their application. On the other hand, Bacillus subtilis natto produces a water-soluble complex, which comprises menaquinone-7 and peptides. The peptide named K-binding factor (KBF) has been reported as the main component of the complex. In the present, the structural characteristics of KBF were studied. Mass spectrometry showed significant peaks at m/z = 1050, while the previous PAGE suggested that molecular weight of KBF was ~ 3k. Amino acid analysis revealed that the 1k peptides were the various combinations of nine amino acids, among which Asx, Glx, Val, Leu and Met were found to be the most abundant. The peptides could serve as detergent properties. The 1k peptides could be isolated by reverse-phase high performance liquid chromatography. The bundle of three 1k detergent-like peptides would participate to the micelle structure containing menqauinone-7 inside. In conclusion, a basic unit of KBF would be the ~ 1k peptides, and the three basic unit assemble to the ~ 3k bundle, then the bundle form a water-soluble micelle including menqauinone-7 inside.

Protonation states of hen egg-white lysozyme observed using D/H contrast neutron crystallography.

Hen egg-white lysozyme (HEWL) is an enzymatic protein with two acidic amino acids, Glu35 and Asp52, in its active site. Glu35 acts as a proton donor to the substrate and Asp52 interacts with the positively charged substrate, suggesting different protonation states of these residues. However, neutron crystallographic studies thus far have not provided a consistent picture of the protonation states of these residues. Only one study succeeded in observing the active protonation states of Glu35 and Asp52 in the triclinic crystal system. However, their active states in the most widely studied tetragonal crystal system are still unknown. The application of the D/H contrast technique in neutron crystallography improves the ability to locate exchangeable D/H atoms in proteins. In the present study, D2O and H2O solvent crystals were prepared. Each neutron data set was collected for only five days by combining a time-of-flight diffractometer (iBIX) and the spallation neutron source at the Japan Proton Accelerator Research Complex. The D/H contrast map provided better visualization of the D/H atoms in HEWL than the conventional neutron scattering length density map. The neutron D/H contrast map demonstrated the alternative protonation of the OE1 and OE2 atoms in the carboxyl group of Glu35. This alternative protonation occurs in the absence of a substrate, where high selectivity of the protonation site does not occur. In this case, only the OE1-HE1 bond attacks the substrate in an equilibrium between OE1-HE1 and OE2-HE2, or the H+ ion of the OE2-HE2 bond moves to the OE1 atom just before or after substrate binding to initiate the catalytic reaction. In contrast, the carboxyl group of Asp52 is not protonated. Protonation of the carboxyl group was not observed for other Asp and Glu residues. These results are consistent with results from NMR spectroscopy and explain the protonation states at the active site in the apo form of HEWL.

Recent structural insights into the mechanism of lysozyme hydrolysis.

Lysozyme hydrolyzes the glycosidic bonds between N-acetylmuramic acid and N-acetylglucosamine in peptidoglycans located in the bacterial cell wall. The mechanism of the hydrolysis reaction of lysozyme was first studied more than 50 years ago; however, it has not yet been fully elucidated and various mechanisms are still being investigated. One reaction system that has commonly been proposed is that the lysozyme intermediate undergoes covalent ligand binding during hydrolysis. However, these findings resulted from experiments performed under laboratory conditions using fluorine-based ligands, which facilitate the formation of covalent bonds between the ligands and the catalytic side chain of lysozyme. More recently, high-resolution X-ray structural analysis was used to study the complex of lysozyme with an N-acetylglucosamine tetramer. As a result, the carboxyl group of Asp52 was found to form a relatively strong hydrogen-bond network and had difficulty binding covalently to C1 of the carbohydrate ring. To confirm this hydrogen-bond network, neutron test measurements were successfully performed to a resolution of better than 1.9 Å.

Current status and near future plan of neutron protein crystallography at J-PARC.

The IBARAKI Biological Crystal Diffractometer (iBIX) has been available for use at MLF (Material and Life Science Facility) in J-PARC (Japan Proton Accelerator Research Complex) since 2008. The development in state-of-the-art detector systems could enable iBIX to become one of the highest-performance neutron single-crystal diffractometers in the world. Here, together with other various developments, such as data reduction software, crystal growth, and new techniques in measurement coupled analysis, we provided new hydrogen and water structural data of several proteins and macromolecules. Although the proton power at MLF has not yet reached its planned maximum (1MW), a more powerful neutron source will be soon needed for neutron protein crystallography. A future idea is also proposed and discussed in this article.

Cryoprotectant-free high-pressure cooling and dynamic nuclear polarization for more sensitive detection of hydrogen in neutron protein crystallography.

To improve the sensitivity of hydrogen detection using neutrons, a proton-polarization technique together with a high-pressure cooling method is necessary. The highest pressure (200 MPa) used in the experiment described here enabled relatively large protein crystals to be cooled without any cryoprotectants while retaining the protein structure, and it was confirmed that high-pressure-cooled crystals diffracted to nearly the same resolution as flash-cooled small crystals soaked with cryoprotectants. Dynamic nuclear polarization was used as a proton-polarization technique for protein crystals, and ∼300 mg polycrystalline protein doped with TEMPOL gave a maximum proton polarization of 22.3% at a temperature of 0.5 K in a 2.5 T magnetic field.

Ligation-Dependent Picosecond Dynamics in Human Hemoglobin As Revealed by Quasielastic Neutron Scattering.

Hemoglobin, the vital O2 carrier in red blood cells, has long served as a classic example of an allosteric protein. Although high-resolution X-ray structural models are currently available for both the deoxy tense (T) and fully liganded relaxed (R) states of hemoglobin, much less is known about their dynamics, especially on the picosecond to subnanosecond time scales. Here, we investigate the picosecond dynamics of the deoxy and CO forms of human hemoglobin using quasielastic neutron scattering under near physiological conditions in order to extract the dynamics changes upon ligation. From the analysis of the global motions, we found that whereas the apparent diffusion coefficients of the deoxy form can be described by assuming translational and rotational diffusion of a rigid body, those of the CO form need to involve an additional contribution of internal large-scale motions. We also found that the local dynamics in the deoxy and CO forms are very similar in amplitude but are slightly lower in frequency in the former than in the latter. Our results reveal the presence of rapid large-scale motions in hemoglobin and further demonstrate that this internal mobility is governed allosterically by the ligation state of the heme group.

DNA conformational transitions inferred from re-evaluation of m|Fo| - D|Fc| electron-density maps.

Conformational flexibility of DNA plays important roles in biological processes such as transcriptional regulation and DNA packaging etc. To understand the mechanisms of these processes, it is important to analyse when, where and how DNA shows conformational variations. Recent analyses have indicated that conventional refinement methods do not always provide accurate models of crystallographic heterogeneities and that some information on polymorphism has been overlooked in previous crystallographic studies. In the present study, the m|Fo| - D|Fc| electron-density maps of double-helical DNA crystal structures were calculated at a resolution equal to or better than 1.5 Šand potential conformational transitions were found in 27% of DNA phosphates. Detailed analyses of the m|Fo| - D|Fc| peaks indicated that some of these unassigned densities correspond to ZI ↔ ZII or A/B → BI conformational transitions. A relationship was also found between ZI/ZII transitions and metal coordination in Z-DNA from the detected peaks. The present study highlights that frequent transitions of phosphate backbones occur even in crystals and that some of these transitions are affected by the local molecular environment.

Structural Insight Into Protein Binding of Boron Tracedrug UTX-97 Revealed by the Co-Crystal Structure With Lysozyme at 1.26 Å Resolution.

Boron neutron capture therapy (BNCT) is one of the numbers of radiotherapies for treatment of certain cancers. The ability of low-dose irradiation with neutrons or radioactive beams to provide an acceptable quality of life is an objective which has not yet been achieved; therefore it will be necessary to increase the efficiency of the neutron capture reaction by lower dose irradiation and by achieving higher drug concentrations in living cells. Drug selectivity in targeting the affected cellular compartment is most important. Molecular design and synthesis of drugs should be based on high resolution structures and analysis of specific compounds and host molecules; however, it is necessary to obtain crystals for X-ray structural analysis. Because compounds containing bulky functional groups are difficult to crystalize due to their flexibility, the method described here makes it possible to stabilize these compounds by complexing them with protein molecules. We have first solved the three-dimensional structure of a BNCT drug-protein molecule combination at 1.26 Å resolution, and discuss the nature of the interaction between a BNCT drug and the protein molecule residues.

A technique for determining the deuterium/hydrogen contrast map in neutron macromolecular crystallography.

A difference in the neutron scattering length between hydrogen and deuterium leads to a high density contrast in neutron Fourier maps. In this study, a technique for determining the deuterium/hydrogen (D/H) contrast map in neutron macromolecular crystallography is developed and evaluated using ribonuclease A. The contrast map between the D2O-solvent and H2O-solvent crystals is calculated in real space, rather than in reciprocal space as performed in previous neutron D/H contrast crystallography. The present technique can thus utilize all of the amplitudes of the neutron structure factors for both D2O-solvent and H2O-solvent crystals. The neutron D/H contrast maps clearly demonstrate the powerful detectability of H/D exchange in proteins. In fact, alternative protonation states and alternative conformations of hydroxyl groups are observed at medium resolution (1.8 Å). Moreover, water molecules can be categorized into three types according to their tendency towards rotational disorder. These results directly indicate improvement in the neutron crystal structure analysis. This technique is suitable for incorporation into the standard structure-determination process used in neutron protein crystallography; consequently, more precise and efficient determination of the D-atom positions is possible using a combination of this D/H contrast technique and standard neutron structure-determination protocols.

Neutron Nucleic Acid Crystallography.

The hydration shells surrounding nucleic acids and hydrogen-bonding networks involving water molecules and nucleic acids are essential interactions for the structural stability and function of nucleic acids. Water molecules in the hydration shells influence various conformations of DNA and RNA by specific hydrogen-bonding networks, which often contribute to the chemical reactivity and molecular recognition of nucleic acids. However, X-ray crystallography could not provide a complete description of structural information with respect to hydrogen bonds. Indeed, X-ray crystallography is a powerful tool for determining the locations of water molecules, i.e., the location of the oxygen atom of H2O; however, it is very difficult to determine the orientation of the water molecules, i.e., the orientation of the two hydrogen atoms of H2O, because X-ray scattering from the hydrogen atom is very small.Neutron crystallography is a specialized tool for determining the positions of hydrogen atoms. Neutrons are not diffracted by electrons, but are diffracted by atomic nuclei; accordingly, neutron scattering lengths of hydrogen and its isotopes are comparable to those of non-hydrogen atoms. Therefore, neutron crystallography can determine both of the locations and orientations of water molecules. This chapter describes the current status of neutron nucleic acid crystallographic research as well as the basic principles of neutron diffraction experiments performed on nucleic acid crystals: materials, crystallization, diffraction experiments, and structure determination.

Site specific oxidation of amino acid residues in rat lens γ-crystallin induced by low-dose γ-irradiation.

Although cataracts are a well-known age-related disease, the mechanism of their formation is not well understood. It is currently thought that eye lens proteins become abnormally aggregated, initially causing clumping that scatters the light and interferes with focusing on the retina, and ultimately resulting in a cataract. The abnormal aggregation of lens proteins is considered to be triggered by various post-translational modifications, such as oxidation, deamidation, truncation and isomerization, that occur during the aging process. Such modifications, which are also generated by free radical and reactive oxygen species derived from γ-irradiation, decrease crystallin solubility and lens transparency, and ultimately lead to the development of a cataract. In this study, we irradiated young rat lenses with low-dose γ-rays and extracted the water-soluble and insoluble protein fractions. The water-soluble and water-insoluble lens proteins were digested with trypsin, and the resulting peptides were analyzed by LC-MS. Specific oxidation sites of methionine, cysteine and tryptophan in rat water-soluble and -insoluble γE and γF-crystallin were determined by one-shot analysis. The oxidation sites in rat γE and γF-crystallin resemble those previously identified in γC and γD-crystallin from human age-related cataracts. Our study on modifications of crystallins induced by ionizing irradiation may provide useful information relevant to human senile cataract formation.

Structural fluctuation observed in Z-DNA d(CGCGCG)2 in the absence of divalent metal cations and polyamines.

In the present study, Z-DNA d(CGCGCG)2 was crystallized from a DNA solution in the absence of divalent metal cations and polyamines, and its X-ray structure was determined at 0.98 Å resolution. Comparison of this structure and previously reported Z-DNA structures, containing Mg(2+) cations and/or polyamines, demonstrated that Z-DNA can have structural fluctuations with respect to phosphate groups and hydration in the minor groove. At the GpC steps, a two-state structural equilibrium between the ZI and ZII conformations was frequently observed. In contrast, at the CpG steps, the phosphate groups exhibited rotational fluctuation, which could induce distortion of sugar puckering. In addition, alternative positions of water molecules were found in the middle of the minor groove of the Z-DNA. These structural fluctuations were likely observable because of the absence of Mg(2+) cations and polyamines. The results related to these phenomena were supported by those of other experimental methods, suggesting the possibility of these fluctuations occurring in biological conditions.

X-ray structure determination and deuteration of nattokinase.

Nattokinase (NK) is a strong fibrinolytic enzyme, which is produced in abundance by Bacillus subtilis natto. Although NK is a member of the subtilisin family, it displays different substrate specificity when compared with other subtilisins. The results of molecular simulations predict that hydrogen arrangements around Ser221 at the active site probably account for the substrate specificity of NK. Therefore, neutron crystallographic analysis should provide valuable information that reveals the enzymatic mechanism of NK. In this report, the X-ray structure of the non-hydrogen form of undeuterated NK was determined, and the preparation of deuterated NK was successfully achieved. The non-hydrogen NK structure was determined at 1.74 Å resolution. The three-dimensional structures of NK and subtilisin E from Bacillus subtilis DB104 are near identical. Deuteration of NK was carried out by cultivating Bacillus subtilis natto in deuterated medium. The D2O resistant strain of Bacillus subtilis natto was obtained by successive cultivation rounds, in which the concentration of D2O in the medium was gradually increased. NK was purified from the culture medium and its activity was confirmed by the fibrin plate method. The results lay the framework for neutron protein crystallography analysis.

Fundamental studies for the proton polarization technique in neutron protein crystallography.

The isotope effect in conventional neutron protein crystallography (NPC) can be eliminated by the proton polarization technique (ppt). Furthermore, the ppt can improve detection sensitivity of hydrogen (relative neutron scattering length of hydrogen) by approximately eight times in comparison with conventional NPC. Several technical difficulties, however, should be overcome in order to perform the ppt. In this paper, two fundamental studies to realise ppt are presented: preliminary trials using high-pressure flash freezing has shown the advantage of making bulk water amorphous without destroying the single crystal; and X-ray diffraction and liquid-chromatography/mass-spectrometry analyses of standard proteins after introducing radical molecules into protein crystals have shown that radical molecules could be distributed non-specifically around proteins, which is essential for better proton polarization.

Direct interactions between Z-DNA and alkaline earth cations, discovered in the presence of high concentrations of MgCl2 and CaCl2.

In this study, crystals of Z-DNA hexamer d(CGCGCG) complexed with MgCl2 and CaCl2 were obtained in the presence of high concentrations of alkaline earth salts (500mM) using a temperature control technique, and their crystal structures were determined at 1.3Å resolution. Mg(2+) and Ca(2+) cations in these structures tend to interact directly with phosphate groups of Z-DNA duplexes; however, they tend to form water-mediated interactions with Z-DNA in the presence of lower concentrations of alkaline earth salts. In these crystals, a DNA duplex was laid along its c-axis and interacted with its 6 neighboring DNA duplexes through coordination bonds of PO…(Mg(2+) or Ca(2+))…OP. A symmetrical hexagonal Z-DNA duplex assembly model may explain DNA condensation caused by alkaline earth salts. These structures offer insights into the functions of alkaline earth cations essential to the structures and assembly of Z-DNA duplexes.

High-resolution X-ray study of the effects of deuteration on crystal growth and the crystal structure of proteinase K.

Deuteration of macromolecules is an important technique in neutron protein crystallography. Solvent deuteration of protein crystals is carried out by replacing water (H(2)O) with heavy water (D(2)O) prior to neutron diffraction experiments in order to diminish background noise. The effects of solvent deuteration on the crystallization of proteinase K (PK) with polyethylene glycol as a precipitant were investigated using high-resolution X-ray crystallography. In previous studies, eight NO(3)(-) anions were included in the PK crystal unit cell grown in NaNO(3) solution. In this study, however, the PK crystal structure did not contain NO(3)(-) anions; consequently, distortions of amino acids arising from the presence of NO(3)(-) anions were avoided in the present crystal structures. High-resolution (1.1 Å) X-ray diffraction studies showed that the degradation of PK crystals induced by solvent deuteration was so small that this degradation would be negligible for the purpose of neutron protein crystallography experiments at medium resolution. Comparison of the nonhydrogen structures of nondeuterated and deuterated crystal structures demonstrated very small structural differences. Moreover, a positive correlation between the root-mean-squared differences and B factors indicated that no systematic difference existed.

Purification, crystallization and preliminary X-ray diffraction experiment of nattokinase from Bacillus subtilis natto.

Nattokinase is a single polypeptide chain composed of 275 amino acids (molecular weight 27,724) which displays strong fibrinolytic activity. Moreover, it can activate other fibrinolytic enzymes such as pro-urokinase and tissue plasminogen activator. In the present study, native nattokinase from Bacillus subtilis natto was purified using gel-filtration chromatography and crystallized to give needle-like crystals which could be used for X-ray diffraction experiments. The crystals belonged to space group C2, with unit-cell parameters a=74.3, b=49.9, c=56.3 Å, ß=95.2°. Diffraction images were processed to a resolution of 1.74 Šwith an Rmerge of 5.2% (15.3% in the highest resolution shell) and a completeness of 69.8% (30.0% in the highest resolution shell). This study reports the first X-ray diffraction analysis of nattokinase.

Protonation states of histidine and other key residues in deoxy normal human adult hemoglobin by neutron protein crystallography.

The protonation states of the histidine residues key to the function of deoxy (T-state) human hemoglobin have been investigated using neutron protein crystallography. These residues can reversibly bind protons, thereby regulating the oxygen affinity of hemoglobin. By examining the OMIT F(o)-F(c) and 2F(o)-F(c) neutron scattering maps, the protonation states of 35 of the 38 His residues were directly determined. The remaining three residues were found to be disordered. Surprisingly, seven pairs of His residues from equivalent α or ß chains, αHis20, αHis50, αHis58, αHis89, ßHis63, ßHis143 and ßHis146, have different protonation states. The protonation of distal His residues in the α(1)ß(1) heterodimer and the protonation of αHis103 in both subunits demonstrates that these residues may participate in buffering hydrogen ions and may influence the oxygen binding. The observed protonation states of His residues are compared with their ΔpK(a) between the deoxy and oxy states. Examination of inter-subunit interfaces provided evidence for interactions that are essential for the stability of the deoxy tertiary structure.

Direct determination of protonation states of histidine residues in a 2 A neutron structure of deoxy-human normal adult hemoglobin and implications for the Bohr effect.

We have investigated the protonation states of histidine residues (potential Bohr groups) in the deoxy form (T state) of human hemoglobin by direct determination of hydrogen (deuterium) positions with the neutron protein crystallography technique. The reversible binding of protons is key to the allosteric regulation of human hemoglobin. The protonation states of 35 of the 38 His residues were directly determined from neutron scattering omit maps, with 3 of the remaining residues being disordered. Protonation states of 5 equivalent His residues--alpha His20, alpha His50, alpha His89, beta His143, and beta His146--differ between the symmetry-related globin subunits. The distal His residues, alpha His58 and beta His63, are protonated in the alpha 1 beta 1 heterodimer and are neutral in alpha 2 beta 2. Buried residue alpha His103 is found to be protonated in both subunits. These distal and buried residues have the potential to act as Bohr groups. The observed protonation states of His residues are compared to changes in their pK(a) values during the transition from the T to the R state and the results provide some new insights into our understanding of the molecular mechanism of the Bohr effect.