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PubMed1997

Structural characterization of the myoglobin active site using infrared crystallography.

We use polarized IR absorption on single crystals to determine the orientation of carbon monoxide bound at the active site of myoglobin, and conclude that the C-O bond lies approximately 7 degrees from the normal to the mean plane of the heme. This result disagrees with much larger angular displacements reported in structural models derived from X-ray and neutron diffraction measurements. The insensitivity of the IR-derived orientation to changes in pH or crystal packing contrasts with the wide variations in CO orientation among diffraction-based models and suggests that the latter are in error. The small energies required to displace the C-O bond 7 degrees from its energetically preferred upright geometry suggest that distortion of the surrounding protein, rather than the relatively undeformable Fe-C-O unit, is the main steric mechanism inhibiting CO binding to myoglobin.

Animals↗
PubMed1998

Dehydration of biological membranes by cooling: an investigation on the purple membrane.

The lamellar spacing dl of purple membrane (PM) multilayer systems was investigated with neutron diffraction as a function of temperature and of the level of hydration. The observed large T-dependent variations of dl indicate that PM is partially dehydrated when cooled below a "hydration water freezing point". This phenomenon is reversible, but a hysteresis is observed when PM is rehydrated upon reheating. The hydration water remaining bound to the membrane below about 240 K is non-freezing. Its amount was found to be hnf=0.24(+/-0.02) g 2H2O/g BR for all samples equilibrated at room temperature in the presence of 2H2O vapour at >/=84% r.h. It is evident, that the dehydration/rehydration behaviour of PM is strongly correlated with the temperature-dependent behaviour of the dynamical structure factor. Above the well-known "dynamical transition" announcing the onset of localized diffusive molecular motions between 190 K and 230 K, a second dynamical transition is caused by the temperature-induced rehydration of the PM starting near 255 K. This is also correlated with the deviation from a pure Arrhenius law of the rate-limiting process in the photocycle, known to occur upon cooling beyond the ice point into the same temperature region. Our results suggest that the phenomenon of dehydration and rehydration induced by cooling and reheating, respectively, is a general property of biological membranes.

Cell Membrane↗
PubMed1999

Deuterium REDOR: principles and applications for distance measurements

The application of short composite pulse schemes ( and ) to the rotational echo double-resonance (REDOR) spectroscopy of X-2H (X: spin 12, observed) systems with large deuterium quadrupolar interactions has been studied experimentally and theoretically and compared with simple 180 degrees pulse schemes. The basic properties of the composite pulses on the deuterium nuclei have been elucidated, using average Hamiltonian theory, and exact simulations of the experiments have been achieved by stepwise integration of the equation of motion of the density matrix. REDOR experiments were performed on 15N-2H in doubly labeled acetanilide and on 13C-2H in singly 2H-labeled acetanilide. The most efficient REDOR dephasing was observed when composite pulses were used. It is found that the dephasing due to simple 180 degrees deuterium pulses is about a factor of 2 less efficient than the dephasing due to the composite pulse sequences and thus the range of couplings observable by X-2H REDOR is enlarged toward weaker couplings, i.e., larger distances. From these experiments the 2H-15N dipolar coupling between the amino deuteron and the amino nitrogen and the 2H-13C dipolar couplings between the amino deuteron and the alpha and beta carbons have been elucidated and the corresponding distances have been determined. The distance data from REDOR are in good agreement with data from X-ray and neutron diffraction, showing the power of the method. Copyright 1999 Academic Press.

Journal Article↗
PubMed2001

Calculation of 13C chemical shifts in rna nucleosides: structure-13C chemical shift relationships.

Isotropic 13C chemical shifts of the ribose sugar in model RNA nucleosides are calculated using SCF and DFT-GIAO ab initio methods for different combinations of ribose sugar pucker, exocyclic torsion angle, and glycosidic torsion angle. Idealized conformations were obtained using structures that were fully optimized by ab initio DFT methods starting with averaged parameters from a collection of crystallographic data. Solid-state coordinates of accurate crystal or neutron diffraction structures were also examined directly without optimization. The resulting 13C chemical shifts for the two sets of calculations are then compared. The GIAO-DFT method overestimates the shifts by an average of 5 ppm while the GIAO-SCF underestimates the shifts by the same amount. However, in the majority of cases the errors appear to be systematic, as the slope of a plot of calculated vs experimental shifts is very close to unity, with minimal scatter. The values of the 13C NMR shifts of the ribose sugar are therefore sufficiently precise to allow for statistical separation of sugar puckering modes and exocyclic torsion angle conformers, based on the canonical equation model formulated in a previous paper.

Algorithms↗
PubMed2002

Obtaining molecular and structural information from 13C-14N systems with 13C FIREMAT experiments.

The effect of dipolar coupling to 14N on 13C FIREMAT (five pi replicated magic angle turning) experiments is investigated. A method is developed for fitting the 13C FIREMAT FID employing the full theory to extract the 13C-14N dipolar and 13C chemical shift tensor information. The analysis requires prior knowledge of the electric field gradient (EFG) tensor at the 14N nucleus. In order to validate the method the analysis is done for the amino acids alpha-glycine, gamma-glycine, l-alanine, l-asparagine, and l-histidine on FIREMAT FIDs recorded at 13C frequencies of 50 and 100 MHz. The dipolar and chemical shift data obtained with this analysis are in very good agreement with the previous single-crystal 13C NMR results and neutron diffraction data on alpha-glycine, l-alanine, and l-asparagine. The values for gamma-glycine and l-histidine obtained with this new method are reported for the first time. The uncertainties in the EFG tensor on the resultant 13C chemical shift and dipolar tensor values are assessed.

Alanine↗
PubMed1995

Structural comparison of the plant satellite viruses.

Detailed structures are now available for three plant satellite viruses, satellite tobacco necrosis virus (STNV), satellite tobacco mosaic virus (STMV), and satellite panicum mosaic virus (SPMV). It is, therefore, possible to compare the tertiary structure of viral protein subunits, their quaternary interactions, and the interactions of protein subunits with the RNA genome. This analysis indicates that, in spite of common function and preservation of a "jelly-roll" motif in the protein monomer, the three viruses are remarkably different. The differences include the arrangement of secondary structural elements, interactions of adjacent subunits, and the disposition of subunits relative to icosahedral symmetry axes. In each of the three viruses, however, the narrow end of the jelly roll forms fivefold contacts. The fivefold protein interactions are organized about a Ca2+ ion for STNV, an anion for STMV, and, apparently, neither of these for SPMV. Low-resolution neutron diffraction studies using H2O/D2O solvent contrast variation revealed the general location of the RNA genome within the STNV. In the case of SPMV, regions of electron density on the interior of the capsid could be assigned to RNA, although it was not possible to model the nucleic acid. Only for STMV was nucleic acid visible in election density maps, and this was manifested as double-helical RNA segments associated with each coat protein dimer. The observations presented here provide no support for any common evolutionary relationship.

Amino Acid Sequence↗
PubMed1987

A molecular dynamics simulation of crystalline alpha-cyclodextrin hexahydrate.

The structure of crystalline alpha-cyclodextrin (alpha-CD) hexahydrate, form I (C36H60O30.6H2O, space group P2(1)2(1)2(1)) is experimentally so well determined by X-ray and by neutron diffraction analyses that the positions of all the hydrogen atoms are available. This provides an opportunity for testing an empirical force field that is currently used in simulations of protein and nucleic acid structures by performing molecular dynamics studies employing the GROMOS program package on a system of 4 unit cells containing 16 alpha-CD molecules and 96 water molecules. A detailed comparison of the simulated and experimentally determined crystal structures shows that the experimental positions of the alpha-CD atoms are reproduced within 0.025 nm, well within the overall experimental accuracy of 0.036 nm; that the water molecules are on average within 0.072 nm from their experimental sites, with two thirds reproduced within experimental accuracy by the calculations; that high correlation is produced between the occurrence of simulated and experimentally observed hydrogen bonds. The good agreement between simulated and experimental results suggests that the tested force field is reliable.

Carbohydrate Conformation↗
PubMed1987

Molecular dynamics simulation of crystalline beta-cyclodextrin dodecahydrate at 293 K and 120 K.

Molecular dynamics (MD) simulations for crystalline beta-cyclodextrin dodecahydrate (beta-CD) at two different temperatures, 293 K and 120 K, have been performed using the GROMOS program package. The calculated structural properties are compared to those obtained from neutron diffraction studies of this system at the quoted temperatures. The simulation was carried out over a period of 20 ps on four unit cells containing 8 beta-CD molecules and 96 water molecules, whereby all atoms were allowed to move. At room temperature, the experimental positions of the (non-hydrogen) glucose atoms are reproduced within 0.034 nm, a value which is smaller than the experimental (0.041 nm) or simulated (0.049 nm) overall root mean square (rms) positional fluctuation. The corresponding numbers for the low temperature study are 0.046 nm, 0.019 nm and 0.022 nm. At both temperatures the experimentally observed degree of anisotropy of the atomic motions is also found in the simulations. The comparison of a variety of structural properties leads to the conclusion that the molecular model and force field used are able to simulate the cyclodextrin system very well. Experimentally observed differences in properties as a function of number of glucose units in the CD molecule (alpha-CD, 6 versus beta-CD, 7) and as a function of temperature are qualitatively reproduced by the simulations.

Carbohydrate Conformation↗
PubMed2002

Speciation of nitrogen - [N(3-)] and [N(2)(2-)] - in binary compounds.

The nitrogen content of the binary compounds SrN = Sr(4)[N](2)[N(2)], Sr[N(2)], and Ba[N(2)] (prepared by high-pressure syntheses) was determined analytically by using the carrier gas hot extraction method. For handling of the air- and moisture-sensitive samples, a transfer chamber was constructed to protect the compounds against decomposition before being analyzed. Additionally, it was necessary to develop a method allowing controlled and variable heating of the electrode furnace to get analytical results with high precision and accuracy. By means of a suitable temperature program it was possible not only to verify the existence but also to quantify the two different nitrogen species ([N(3-)] and [N(2)(2-)]), and thus confirm the results of recent neutron diffraction studies.

Journal Article↗
PubMed2003

Size and shape of mineralites in young bovine bone measured by atomic force microscopy.

Atomic force microscopy (AFM) was used to obtain three-dimensional images of isolated mineralites extracted from young postnatal bovine bone. The mean mineralite size is 9 nm x 6 nm x 2.0 nm, significantly shorter and thicker than the mineralites of mature bovine bone measured by the same technique. Mineralites of the young postnatal bone can be accommodated within the hole zone regions of a quasi-hexagonally packed collagen fibril in the fashion described by Hodge [9] in which laterally adjacent hole zone regions form continuous "channels" across the diameter of a fibril for a distance of at least 10 nm. Deposition of mineralites of the size noted above in this void volume of the fibrils would result in little or no distortion of the collagen molecules or supramolecular structure of the collagen fibril. The new AFM data supporting this claim is consistent with findings obtained by electron microscopy and low-angle x-ray and neutron diffraction that mineralites formed within collagen fibrils during initial stages of calcification occur within the hole zone region. However, the deposition of additional mineralites in the intermolecular spaces between collagen molecules in the overlap region of the fibrils would significantly distort the fibrils since the space available between adjacent molecules is considerably less than even the smallest dimension of the mineralites.

Animals↗
PubMed2006

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.

Aldose-Ketose Isomerases↗
PubMed2006

Conditioning action of the environment on the protein dynamics studied through elastic neutron scattering.

The dynamics of lysozyme in the picosecond timescale has been studied when it is in dry and hydrated powder form and when it is embedded in glycerol, glycerol-water, glucose and glucose-water matrices. The investigation has been undertaken through elastic neutron scattering technique on the backscattering spectrometer IN13. The dynamics of dry powder and embedded-in-glucose lysozyme can be considered purely vibrational up to 100 K, where the onset of an anharmonic contribution takes place. This contribution can be attributed to the activation of methyl group reorientations and is described with an Arrhenius trend. An additional source of anharmonic dynamics appears at higher temperatures for lysozyme in hydrated powders and embedded in glycerol, glycerol-water and glucose-water matrices. This second process, also represented with an Arrhenius trend, corresponds to the so-called protein dynamical transition. Both the temperature where such a transition takes place and the magnitude of the protein mean square displacements depend on the environment. The dynamical response of the protein to temperature is put in relationship with its thermal stability.

Animals↗
PubMed2006

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.

Crystallography↗
PubMed1977

Orientation and flexibility of the choline head group in phosphatidylcholine bilayers.

The average orientation and flexibility of the phosphorylcholine group are deduced from deuterium and phosphorus-31 nuclear magnetic resonance measurements of unsonicated phosphatidylcholine bilayers in the liquid crystalline state. The experimental data are consistent with a model in which the polar head group exhibits a restricted flexibility characterized by rapid transitions between two enantiomeric conformations. A completely flexible or a completely rigid head group structure can be excluded. The phosphorylcholine residue is found to be bent at the position of the phosphate group, due to a gauche-gauche conformation of the phosphodiester linkage. The choline dipole is aligned parallel to the plane of the membrane, which is in agreement with X-ray and neutron diffraction studies. The average orientation of the phosphorylcholine group is therfore the same as that of the phosphorylethanolamine head group.

Choline↗
PubMed1977

The 30 S subunit of the Escherichia coli ribosome. Topographical model of its component proteins.

This topographical model of the proteins of the 30 S subunit of the Escherichia coli ribosome was built to be consistent with the 37 published spectroscopic and chemical experiments that indicate proximity and with the two neutron diffraction experiments that indicate S3 and S7 as well as S2 and S5 to be separated by 110 A. The model is quite consistent with the protein arrangement suggested by assembly pathways, the various RNA binding sites, and the streptomycin-associated proteins, This consistency is encouraging since none of these data were considered during the construction of the model. The model differs significantly from those proposed by Traut et at. ((1974) Ribosomes 271-308) and by Tischendorf et al. ((1975) Proc. Natl. Acad. Sei. U.S. 72, 4820-4824).

Binding Sites↗
PubMed1987

Ultrasonic studies of proton-transfer reactions at the catalytic site of alpha-chymotrypsin.

Ultrasonic relaxation measurements for alpha-chymotrypsin in phosphate, sulfite and arsenate buffers exhibit a high peak of absorption at neutral pH. The analysis is based on: comparison of the relaxation measurements for the enzyme and for the zymogen and inhibited enzyme; X-ray and neutron diffraction data, and high-resolution NMR data. The ultrasonic relaxation is shown to result mainly from a proton-transfer reaction that involves the histidine at the catalytic site (His-57). The question is raised of whether the enhanced ultrasonic effect observed in the enzyme is indicative of a property that plays a part in the catalytic activity.

Binding Sites↗
PubMed1987

Correlation between calculated local stability and hydrogen exchange rates in proteins.

The attempt is made to find new correlations between local structural characteristics of proteins and the hydrogen exchange rates of their individual main-chain amides, and to relate such correlations to possible mechanisms of hydrogen exchange. It is found that in bovine pancreatic trypsin inhibitor (BPTI) the surface area buried by a particular residue and its neighbors correlates with the exchange rate of the main-chain amide of that residue. As the area buried by a particular fragment can be associated with the stabilization of the protein structure by this fragment, the correlation suggests a role for the energetics of the local unfolding in the mechanism of hydrogen exchange. Calculations based on the assumption that the exchange mechanism involves local unfolding lead to quantitative agreement between the calculated and experimentally measured exchange rates for 80% of the amides of BPTI that are buried or hydrogen bonded to the main-chain or to internal water molecules. The same degree of correlation is found between the calculated exchange rates and partial exchange data for ribonuclease S, hen lysozyme and cytochrome c. A similarly strong correlation is found between calculated exchange rates and the exchange rates of ribonuclease A determined by neutron diffraction in the crystal. The criteria of correlation are, however, less stringent in this case because of the experimental errors, which are larger than for solution data. It is suggested that the observed correlation be used for predictions of hydrogen exchange rates in proteins.

Animals↗
PubMed1987

Assignment of resonances in the 1H nuclear magnetic resonance spectrum of the carbon monoxide complex of sperm whale myoglobin by phase-sensitive two-dimensional techniques.

Phase-sensitive two-dimensional nuclear magnetic resonance (n.m.r.) experiments have been used to obtain extensive proton resonance assignments for the carbon monoxide complex of sperm whale myoglobin. Multiple quantum experiments were particularly important in the assignment procedure. The assignments are the most complete yet reported for a protein of such high molecular weight (approximately 18,000) and make possible new and comprehensive studies of the structure and dynamics of carbonmonoxymyoglobin in solution. Assignments for seven of the histidine residues are reported, including the critical proximal and distal histidines. Most of these are at variance with the assignments already in the literature. The present n.m.r. data indicate that histidines 24 (B5) and 119 (GH1) are hydrogen bonded to each other and, in contrast to neutron diffraction data, show that His24 does not protonate at pH greater than 5. The aromatic rings of all the phenylalanine and tyrosine residues undergo rapid flips about the ring axis. The side-chains of Leu89 (F4) and Phe138 (H15), which border a large hydrophobic cavity, are particularly mobile.

Amino Acid Sequence↗