Showing papers in "Basic life sciences in 1996"

A neutron image plate quasi-Laue diffractometer for protein crystallography

Abstract: An instrument which is based on image plate technology has been constructed to perform cold neutron Laue crystallography on protein structures. The crystal is mounted at the center of a cylindrical detector which is 400mm long and has a circumference of 1000mm, with gadolinium oxide-containing image plates mounted on its exterior surface. Laue images registered on the plate are read out by rotating the drum and translating a laser read head parallel to the cylinder axis, giving a pixel size of 200µm × 200µm and a total read time of 5 minutes. Preliminary results indicate that it should be possible to obtain a complete data set from a protein crystal to atomic resolution in about two weeks.

The determination of the in situ structure by nuclear spin contrast variation.

Abstract: Polarized neutron scattering from polarized nuclear spins in hydrogenous substances opens a new way of contrast variation. The enhanced contrast due to proton spin polarization was used for the in situ structure determination of tRNA of the functional complex of the E.coli ribosome.

DNA hydration studied by neutron fiber diffraction.

Abstract: The development of neutron high angle fiber diffraction to investigate the location of water around the deoxyribonucleic acid (DNA) double-helix is described. The power of the technique is illustrated by its application to the D and A conformations of DNA using the single crystal diffractometer, D19, at the Institut Laue-Langevin, Grenoble and the time of flight diffractometer, SXD, at the Rutherford Appleton ISIS Spallation Neutron Source. These studies show the existence of bound water closely associated with the DNA. The patterns of hydration in these two DNA conformations are quite distinct and are compared to those observed in X-ray single crystal studies of two-stranded oligodeoxynucleotides. Information on the location of water around the DNA double-helix from the neutron fiber diffraction studies is combined with that on the location of alkali metal cations from complementary X-ray high angle fiber diffraction studies at the Daresbury Laboratory SRS using synchrotron radiation. These analyses emphasize the importance of viewing DNA, water and ions as a single system with specific interactions between the three components and provide a basis for understanding the effect of changes in the concentration of water and ions in inducing conformational transitions in the DNA double-helix.

Neutron reflectivity studies of single lipid bilayers supported on planar substrates.

Abstract: Neutron reflectivity was used to probe the structure of single phosphatidylcholine (PC) lipid bilayers adsorbed onto a planar silicon surface in an aqueous environment. Fluctuations in the neutron scattering length density profiles perpendicular to the silicon/water interface were determined for different lipids as a function of the hydrocarbon chain length. The lipids were studied in both the gel and liquid crystalline phases by monitoring changes in the specularly-reflected neutron intensity as a function of temperature. Contrast variation of the neutron scattering length density was applied to both the lipid and the solvent. Scattering length density profiles were determined using both model-independent and model-dependent fitting methods. During the reflectivity measurements, a novel experimental set-up was implemented to decrease the incoherent background scattering due to the solvent. Thus, the reflectivity was measured to Q ~ 0.3A-1, covering up to seven orders of magnitude in reflected intensity, for PC bilayers in D2O and silicon-matched (38% D2O/62% H2O) water. The kinetics of lipid adsorption at the silicon/water interface were also explored by observing changes in the reflectivity at low Q values under silicon-matched water conditions.

The structure of the muscle protein complex 4Ca2+.troponin C.troponin I. Monte Carlo modeling analysis of small-angle X-ray data.

Abstract: Analysis of scattering data based on a Monte Carlo integration method was used to obtain a low resolution model of the 4Ca2+.troponin C.troponin I complex. This modeling method allows rapid testing of plausible structures where the best fit model can be ascertained by a comparison between model structure scattering profiles and measured scattering data. In the best fit model, troponin I appears as a spiral structure that wraps around 4Ca2+.troponin C which adopts an extended dumbbell conformation similar to that observed in the crystal structures of troponin C. The Monte Carlo modeling method can be applied to other biological systems in which detailed structural information is lacking.

Neutron Diffraction Studies of Amphipathic Helices in Phospholipid Bilayers

Abstract: The structural feature which is thought to facilitate the interaction of many peptides with phospholipid bilayers is the ability to fold into an amphipathic helix. In most cases the exact location and orientation of this helix with respect to the membrane is not known, and may vary with factors such as pH and phospholipid content of the bilayer. The growing interest in this area is stimulated by indications that similar interactions can contribute to the binding of certain hormones to their cell-surface receptors. We have been using the techniques of neutron diffraction from stacked phospholipid bilayers in an attempt to investigate this phenomenon with a number of membrane-active peptides. Here we report some of our findings with three of these: the bee venom melittin; the hormone calcitonin; and a synthetic peptide representing the ion channel fragment of influenza A M2 protein.

Structural Model of the 50S Subunit of E. Coli Ribosomes from Solution Scattering

Abstract: The application of new methods of small-angle scattering data interpretation to a contrast variation study of the 50S ribosomal subunit of Escherichia coli in solution is described. The X-ray data from contrast variation with sucrose are analyzed in terms of the basic scattering curves from the volume inaccessible to sucrose and from the regions inside this volume occupied mainly by RNA and by proteins. From these curves models of the shape of the 50S and its RNA-rich core are evaluated and positioned so that their difference produces a scattering curve which is in good agreement with the scattering from the protein moiety. Based on this preliminary model, the X-ray and neutron contrast variation data of the 50S subunit in aqueous solutions are interpreted in the frame of the advanced two-phase model described by the shapes of the 50S subunit and its RNA-rich core taking into account density fluctuations inside the RNA and the protein moiety. The shape of the envelope of the 50S subunit and of the RNA-rich core are evaluated with a resolution of about 40A. The shape of the envelope is in good agreement with the models of the 50S subunit obtained from electron microscopy on isolated particles. The shape of the RNA-rich core correlates well with the model of the entire particle determined by the image reconstruction from ordered sheets indicating that the latter model which is based on the subjective contouring of density maps is heavily biased towards the RNA.

Myoglobin solvent structure at different temperatures

Abstract: The structure of the solvent surrounding myoglobin crystals has been analyzed using neutron diffraction data, and the results indicate that the water around the protein is not disordered, but rather lies in well-defined hydration shells. We have analyzed the structure of the solvent surrounding the protein by collecting neutron diffraction data at four different temperatures, namely, 80, 130, 180, and 240K. Relative Wilson Statistics applied to low resolution data showed evidence of a phase transition in the region of 180K. A plot of the liquidity factor, Bsn, versus distance from the protein surface begins with a high plateau near the surface of the protein and drops to two minima at distances from the protein surface of about 2.35A and 3.85A. Two distinct hydration shells are observed. Both hydration shells are observed to expand as the temperature is increased.

Theoretical Description of Biomolecular Hydration

Abstract: The local density of water molecules around a biomolecule is constructed from calculated two- and three-points correlation functions of polar solvents in water using a Potential-of-Mean-Force (PMF) expansion. As a simple approximation, the hydration of all polar (including charged) groups in a biomolecule is represented by the hydration of water oxygen in bulk water, and the effect of non-polar groups on hydration are neglected, except for excluded volume effects. Pair and triplet correlation functions are calculated by molecular dynamics simulations. We present calculations of the structural hydration for ideal A-DNA molecules with sequences [d(CG)5]2 and [d(C5G5)]2. We find that this method can accurately reproduce the hydration patterns of A-DNA observed in neutron diffraction experiments on oriented DNA fibers (P. Langan et al. J. Biomol. Struct. Dyn., 10, 489(1992)).

Understanding water: molecular dynamics simulations of myoglobin.

Abstract: Molecular dynamics simulations were performed on CO myoglobin to evaluate the stability of the bound water molecules as determined in a neutron diffraction analysis. The myoglobin structure derived from the neutron analysis provided the starting coordinate set used in the simulation. The simulations show that only a few water molecules are tightly bound to protein atoms, while most solvent molecules are labile, breaking and reforming hydrogen bonds. Comparison between myoglobin in solution and in a single crystal highlighted some of the packing effects on the solvent structure and shows that water solvent plays an indispensable role in protein dynamics and structural stability. The described observations explain some of the differences in the experimental results of protein hydration as observed in NMR, neutron and X-ray diffraction studies.

High-level expression and deuteration of sperm whale myoglobin. A study of its solvent structure by X-ray and neutron diffraction methods.

Abstract: Neutron diffraction has become one of the best ways to study light atoms, such as hydrogens. Hydrogen however has a negative coherent scattering factor, and a large incoherent scattering factor, while deuterium has virtually no incoherent scattering, but a large positive coherent scattering factor. Beside causing high background due to its incoherent scattering, the negative coherent scattering of hydrogen tends to cancel out the positive contribution from other atoms in a neutron density map. Therefore a fully deuterated sample will yield better diffraction data with stronger density in the hydrogen position. On this basis, a sperm whale myoglobin gene modified to include part of the A c11 protein gene has been cloned into the T7 expression system. Milligram amounts of fully deuterated holo-myoglobin have been obtained and used for crystallization. The synthetic sperm whale myoglobin crystallized in P2(1) space group isomorphous with the native protein crystal. A complete X-ray diffraction dataset at 1.5A has been collected. This X-ray dataset, and a neutron data set collected previously on a protonated carbon-monoxymyoglobin crystal have been used for solvent structure studies. Both X-ray and neutron data have shown that there are ordered hydration layers around the protein surface. Solvent shell analysis on the neutron data further has shown that the first hydration layer behaves differently around polar and apolar regions of the protein surface. Finally, the structure of per-deuterated myoglobin has been refined using all reflections to a R factor of 17%.

Intercalation of Small Hydrophobic Molecules in Lipid Bilayers Containing Cholesterol

Abstract: Partitioning of small hydrophobic molecules into lipid bilayers containing cholesterol has been studied using the 2XC diffractometer at the University of Missouri Research Reactor. Locations of the compounds were determined by Fourier difference methods with data from both deuterated and undeuterated compounds introduced into the bilayers from the vapor phase. Data fitting procedures were developed for determining how well the compounds were localized. The compounds were found to be localized in a narrow region at the center of the hydrophobic layer, between the two halves of the bilayer. The structures are therefore intercalated structures with the long axis of the molecules in the plane of the bilayer.

Neutron scattering studies on chromatin higher-order structure.

Abstract: We have been engaged in studies of the structure and condensation of chromatin into the 30nm filament using small-angle neutron scattering. We have also used deuterated histone H1 to determine its location in the chromatin 30nm filament. Our studies indicate that chromatin condenses with increasing ionic strength to a limiting structure that has a mass per unit length of 6–7 nucleosomes/11nm. They also show that the linker histone H1/H5 is located in the interior of the chromatin filament, in a position compatible with its binding to the inner face of the nucleosome. Analysis of the mass per unit length as a function of H5 stoichiometry suggests that 5–7 contiguous nucleosomes need to have H5 bound before a stable higher order structure can exist.

Probing Self Assembly in Biological Mixed Colloids by Sans, Deuteration, and Molecular Manipulation

Abstract: Small-angle neutron scattering was used to obtain information on the form and molecular arrangement of particles in mixed colloids of bile salts with phosphatidylcholine, and bile salts with monoolein. Both types of systems showed the same general characteristics. The particle form was highly dependent on total lipid concentration. At the highest concentrations the particles were globular mixed micelles with an overall size of 50A. As the concentration was reduced the mixed micelles elongated, becoming rodlike with diameter about 50A. The rods had a radial core-shell structure in which the phosphatidylcholine or monoolein fatty tails were arranged radially to form the core with the headgroups pointing outward to form the shell. The bile salts were at the interface between the shell and core with the hydrophilic parts facing outward as part of the shell. The lengths of the rods increased and became more polydispersed with dilution. At sufficiently low concentrations the mixed micelles transformed into single bilayer vesicles. These results give insight on the physiological function of bile and on the rules governing the self assembly of bile particles in the hepatic duct and the small intestine.

Time-of-flight Laue fiber diffraction studies of perdeuterated DNA.

Abstract: The diffractometer SXD at the Rutherford Appleton Laboratory ISIS pulsed neutron source has been used to record high resolution time-of-flight Laue fiber diffraction data from DNA These experiments, which are the first of their kind, were undertaken using fibers of DNA in the A conformation and prepared using deuterated DNA in order to minimise incoherent background scattering These studies complement previous experiments on instrument D19 at the Institut Laue Langevin using monochromatic neutrons Sample preparation involved drawing large numbers of these deuterated DNA fibers and mounting them in a parallel array The strategy of data collection is discussed in terms of camera design, sample environment and data collection The methods used to correct the recorded time-of-flight data and map it into the final reciprocal space fiber diffraction dataset are also discussed Difference Fourier maps showing the distribution of water around A-DNA calculated on the basis of these data are compared with results obtained using data recorded from hydrogenated A-DNA on D19 Since the methods used for sample preparation, data collection and data processing are fundamentally different for the monochromatic and Laue techniques, the results of these experiments also afford a valuable opportunity to independently test the data reduction and analysis techniques used in the two methods

Protein-Detergent Interactions in Single Crystals of Membrane Proteins Studied by Neutron Crystallography

Abstract: The detergent micelles surrounding membrane protein molecules in single crystals can be investigated using neutron crystallography combined with H2O/D2O contrast variation. If the protein structure is known then the contrast variation method allows phases to be determined at a contrast where the detergent dominates the scattering. The application of various constraints allows the resulting scattering length density map to be realistically modeled. The method has been applied to two different forms of the membrane protein porin. In one case both hydrogenated and partially deuterated protein were used, allowing the head group and tail to be distinguished.

Small-Angle Neutron Scattering Instrument of Institute for Solid State Physics, the University of Tokyo (SANS-U) and its Application to Biology

Abstract: A small-angle neutron spectrometer (SANS-U) suitable for the study of mesoscopic structure in the field of polymer chemistry and biology, has been constructed at the guide hall of JRR-3M reactor at the Japan Atomic Energy Research Institute. The instrument is 32m long and utilizes a mechanical velocity selector and pinhole collimation to provide a continuous beam with variable wavelength in the range from 5 to 10A. The neutron detector is a 65 × 65cm2 2D position sensitive proportional counter. The practical Q range of SANS-U is 0.0008 to 0.45A-1. The design, characteristics and performance of SANS-U are described with some biological studies using SANS-U.

Determination of Protein and Solvent Volumes in Protein Crystals from Contrast Variation Data

Abstract: By varying the relative values of protein and solvent scattering densities in a crystal, it is possible to obtain information on the shape and dimensions of protein molecular envelopes. Neutron diffraction methods are ideally suited to these contrast variation experiments because H/D exchange leads to large differential changes in the protein and solvent scattering densities and is structurally non-perturbing. Low resolution structure factors have been measured from cubic insulin crystals with differing H/D contents. Structure factors calculated from a simple binary density model, in which uniform scattering densities represent the protein and solvent volumes in the crystals, were compared with these data. The contrast variation differences in the sets of measured structure factors were found to be accurately fitted by this simple model. Trial applications to two problems in crystal structure determination illustrate how this fact may be exploited, (i) A translation function that employs contrast variation data gave a sharp minimum within 1–9A of the correctly positioned insulin molecule and is relatively insensitive to errors in the atomic model, (ii) An ab initio phasing method for the contrast variation data, based on analyzing histograms of the density distributions in trial maps, was found to recover the correct molecular envelope.

In Situ Shape and Distance Measurements in Neutron Scattering and Diffraction

Abstract: Neutron scattering combined with selective isotopic labeling and contrast matching is useful for obtaining in situ structural information about a selected particle, or particles, in a macromolecular complex. The observed intensities, however, may be distorted by inter-complex interference and by scattering-length-density fluctuations of the (otherwise) contrast-matched portions. Methods have been proposed to cancel out such distortions (Hoppe’s method, the Statistical Labeling Method, and the Triple Isotopic Substitution Method). With these methods as well as related unmixed-sample methods, structural information about the selected particle(s) can be obtained without these distortions. We have generalized these methods so that, in addition to globular particles in solution, they can be applied to in situ structures of systems having underlying symmetry and/or net orientation as well. The information obtainable from such experiments is discussed.

The Chemical Reactivity and Structure of Collagen Studied by Neutron Diffraction

Abstract: The chemical reactivity of collagen can be studied using neutron diffraction (a nondestructive technique), for certain reaction types. Collagen contains a number of lysine and hydroxylysine side chains that can react with aldehydes and ketones, or these side chains can themselves be converted to aldehydes by lysyl oxidase. The reactivity of these groups not only has an important role in the maintenance of mechanical strength in collagen fibrils, but can also manifest pathologically in the cases of aging, diabetes (reactivity with a variety of sugars) and alcoholism (reactivity with acetaldehyde). The reactivity of reducing groups with collagen can be studied by neutron diffraction, since the crosslink formed in the adduction process is initially of a Schiff base or keto-imine nature. The nature of this crosslink allows it to be deuterated, and the position of this relatively heavy scattering atom can be used in a process of phase determination by multiple isomorphous replacement. This process was used to study the following: the position of natural crosslinks in collagen; the position of adducts in tendon from diabetic rats in vivo and the in vitro position of acetaldehyde adducts in tendon.

Hydroxyl and Water Molecule Orientations in Trypsin

Abstract: A comparison is presented of experimentally observed hydroxyl and water hydrogens in trypsin determined from neutron density maps with the results of a 140ps molecular dynamics (MD) simulation. Experimental determination of hydrogen and deuterium atom positions in molecules as large as proteins is a unique capability of neutron diffraction. The comparison addresses the degree to which a standard force-field approach can adequately describe the local electrostatic and van der Waals forces that determine the orientations of these hydrogens. The molecular dynamics simulation, based on the all-atom AMBER force-field, allowed free rotation of all hydroxyl groups and movement of water molecules making up a bath surrounding the protein. The neutron densities, derived from 2.1A D2O-H2O difference Fourier maps, provide a database of 27 well-ordered hydroxyl hydrogens. Virtually all of the simulated hydroxyl orientations are within a standard deviation of the experimentally-observed positions, including several examples in which both the simulation and the neutron density indicate that a hydroxyl group is shifted from a ‘standard’ rotamer. For the most highly ordered water molecules, the hydrogen distributions calculated from the trajectory were in good agreement with neutron density; simulated water molecules that displayed multiple hydrogen bonding networks had correspondingly broadened neutron density profiles. This comparison was facilitated by development of a method to construct a pseudo 2A density map based on the hydrogen atom distributions from the simulation. The degree of disorder of internal water molecules is shown to result primarily from the electrostatic environment surrounding that water molecule as opposed to the cavity size available to the molecule. A method is presented for comparing the discrete observations sampled in a dynamics trajectory with the time-averaged data obtained from X-ray or neutron diffraction studies. This method is particularly useful for statically-disordered water molecules, in which the average location assigned from a trajectory may represent a site of relatively low occupancy.