A model for water motion in crystals of lysozyme based on an incoherent quasielastic neutron-scattering study.

This paper reports an incoherent quasielastic neutron scattering study of the single particle, diffusive motions of water molecules surrounding a globular protein, the hen egg-white lysozyme. For the first time such an analysis has been done on protein crystals. It can thus be directly related and compared with a recent structural study of the same sample. The measurement temperature ranged from 100 to 300 K, but focus was on the room temperature analysis. The very good agreement between the structural and dynamical studies suggested a model for the dynamics of water in triclinic crystals of lysozyme in the time range approximately 330 ps and at 300 K. Herein, the dynamics of all water molecules is affected by the presence of the protein, and the water molecules can be divided into two populations. The first mainly corresponds to the first hydration shell, in which water molecules reorient themselves fivefold to 10-fold slower than in bulk solvent, and diffuse by jumps from hydration site to hydration site. The long-range diffusion coefficient is five to sixfold less than for bulk solvent. The second group corresponds to water molecules further away from the surface of the protein, in a second incomplete hydration layer, confined between hydrated macromolecules. Within the time scale probed they undergo a translational diffusion with a self-diffusion coefficient reduced approximately 50-fold compared with bulk solvent. As protein crystals have a highly crowded arrangement close to the packing of macromolecules in cells, our conclusion can be discussed with respect to solvent behavior in intracellular media: as the mobility is highest next to the surface, it suggests that under some crowding conditions, a two-dimensional motion for the transport of metabolites can be dominant.

Rapid neutron-diffraction data collection for hydrogen-bonding studies: application of the Laue diffractometer (LADI) to the case study zinc (tris)thiourea sulfate.

The successful application of the newly developed image-plate neutron Laue diffractometer (LADI) at the Institut Laue-Langevin (ILL), Grenoble, France, for rapid hydrogen-bonding characterization is reported. The case study concerns the promising non-linear optical material zinc (tris)thiourea sulfate (ZTS), which contains 30 atoms in the asymmetric unit and crystallizes in the orthorhombic space group, Pca2(1), a = 11.0616 (9), b = 7.7264 (6), c = 15.558 (1) A [T = 100.0 (1) K]. The results from a 12 h data collection from ZTS on LADI are compared with those obtained over 135 h using the monochromatic four-circle diffractometer D9 at the same reactor source with a crystal 13 times larger in volume. Both studies reveal the extensive hydrogen bonding and other close non-bonded contacts within the material. As expected, the results from D9 are more precise than those obtained from LADI; however, the bond geometry determined from the two experiments is the same within the larger estimated standard deviations. Furthermore, the conclusions drawn from the two studies separately regarding the nature of all supramolecular features are identical. This illustrates that LADI is eminently suitable for rapid characterization of hydrogen-bonded structures by neutron diffraction, with the gain in speed compared with traditional instrumentation being several orders of magnitude.

Quasi-Laue neutron-diffraction study of the water arrangement in crystals of triclinic hen egg-white lysozyme.

Triclinic crystals of lysozyme, hydrogen-deuterium exchanged in deuterated solvent, have been studied using neutron quasi-Laue techniques and a newly developed cylinder image-plate detector. The wavelength range employed was from 2.7 to 3.5 A, which gave 9426 significant reflections [F >/= 2sigma(F)] to a resolution limit of 1. 7 A. The deuteration states of the H atoms in the protein molecule were identified, followed by an extensive analysis of the water structure surrounding the protein. The final R factor was 20.4% (Rfree = 22.1%). In total, the 244 observed water molecules form approximately one layer of water around the protein with far fewer water molecules located further away. Water molecules covering the apolar patches make tangential layers at 4-5 A from the surface or form C-H...O contacts, and several water-molecule sites can be identified in the apolar cavities. Many of the water molecules are apparently orientationally disordered, and only 115 out of the 244 water molecules sit in mean single orientations. Comparison of these results with quasi-elastic neutron scattering observations of the water dynamics leads to a picture of the water molecules forming an extended constantly fluctuating network covering the protein surface.

Neutron Laue diffractometry with an imaging plate provides an effective data collection regime for neutron protein crystallography.

Neutron quasi-Laue diffraction data (2 A resolution) from tetragonal hen egg-white lysozyme were collected in ten days with neutron imaging plates. The data processing Laue software, LAUEGEN, developed for X-ray Laue diffractometry, was adapted for neutron diffractometry with a cylindrical detector. The data analysis software, X-PLOR, was modified and used for the refinement of hydrogen atoms, and the positions of 960 hydrogen atoms in the protein and 157 bound water molecules, were determined. Several examples are given of the methods used to identify hydrogen atoms and water molecules.

Anomalous Dispersion with Edges in the Soft X-ray Region: First Results of Diffraction from Single Crystals of Trypsin Near the K-Absorption Edge of Sulfur.

Anomalous dispersion of X-ray diffraction at wavelengths near the X-ray K-absorption edge of sulfur at wavelengths around 5 A has been applied to single crystals of trypsin obtained from an ammonium sulfate solution. The multiwavelength anomalous-dispersion method based on 775 unique reflections (+183 Bijvoet mates) measured at three wavelengths near the K-absorption edge of sulfur in trypsin (two methionines and disulfide bridges of six cystines) reproduces the known features of the trypsin structure of a resolution of 4 A. It appears that there is anisotropic anomalous scattering from the disulfide bridges of cystine. The multiwavelength anomalous solvent contrast shows up at wavelengths near the K-absorption edge of the sulfate ions, which is shifted by 10 eV to higher energies with respect to that of sulfur in trypsin. The influence of the complex contrast of trypsin in 2.5 M ammonium sulfate on the dispersion of a low-order reflection is analyzed. The measurement of anomalous dispersion of X-ray diffraction at long wavelengths beyond 5 A requires a special diffractometer, the features of which are presented. An outstanding one is a detector system consisting of four multiwire proportional counters. Its efficiency is compared with that of imaging plates. The influence of radiation damage with soft X-ray diffraction from single crystals of trypsin is presented and possible remedies are discussed.

A neutron image plate quasi-Laue diffractometer for protein crystallography.

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 200 microm x 200 microm 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.

Structural basis of cell-cell adhesion by cadherins.

Crystal structures of the amino-terminal domain of N-cadherin provide a picture at the atomic level of a specific adhesive contact between cells. A repeated set of dimer interfaces is common to the structure in three lattices. These interactions combine to form a linear zipper of molecules that mirrors the linear structure of the intracellular filaments with which cadherins associate. This cell-adhesion zipper may provide a mechanism to marshal individual molecular adhesive interactions into strong bonds between cells.

Image plate detectors for macromolecular neutron diffractometry.

Neutron diffraction studies of macromolecules require large position sensitive detectors. It is proposed that such a device can be based on image plate technology, which relies on re-usable photostimuable phosphors, combined with a neutron to gamma-ray converter. Design parameters such as the best wavelength for the neutron radiation and the optimum sample to detector distance are discussed, and a design for a cylindrical detector is outlined. Presently a prototype of such a detector is being built, and the very first test-recording of an X-ray diffraction pattern from a protein crystal is presented.

Crystal structure of hydrophobic protein from soybean; a member of a new cysteine-rich family.

X-ray diffraction methods have been used to determine the structure of the 8.3 kDa hydrophobic protein from soybean and to refine the atomic co-ordinates to a crystallographic R-factor of 18.7% at 1.8 A resolution. The molecule is a four-helix bundle, which together with the connecting loops and a twisted beta-strand form a spiral. The surface contains 70% apolar atoms, and the crystal packing is dominated by hydrophobic interactions, producing a two-dimensional sheet of protein molecules. Most of the 59 water molecules located are involved in hydrophilic contacts and their structural organization does not seem to be affected by the high hydrophobicity of the molecule. From the protein fold it appears that three of the four disulphide bridges are important for keeping the amino and carboxyl-terminal segments in place in the native form, while the central part of the molecule is stabilized by many hydrophobic interactions. Although the protein function is not known, a number of possibilities can be excluded on experimental grounds and by comparison with other members of the family.

Protein single-crystal diffraction with 5 A synchrotron X-rays at the sulfur K-absorption edge.

. Sulfur atoms, an integral part of many proteins, are possible candidates for anomalous scattering in phase determination by multiple-wavelength methods. The main difficulty encountered is that a wavelength of about 5 A is required to obtain a large anomalous signal from these atoms, leading to very large absorption effects. Initial experiments have been carried out using a synchrotron X-ray source, evacuated beam tubes, a diffractometer inside a vacuum chamber, a special sample holder and a suitable scattering geometry. The results are encouraging, showing that Bragg reflections can be measured, and that changes in their intensities around the absorption edge are observable.

Crystallographic data for the 9000 dalton wheat non-specific phospholipid transfer protein.

The wheat non-specific phospholipid transfer protein belongs to a family of small proteins sharing a common pattern of four disulphide bridges. Its function in vivo is not known, but it has a high affinity to phospholipids and is involved in phospholipid transfer in vitro. The molecular weight is 9607, and it crystallizes in the space group P2(1) with a = 40.73 A, b = 112.11 A, c = 50.44 A and beta = 106.80 degrees. The crystals diffract to 3 A resolution.

Crystallographic data for soybean hydrophobic protein.

The soybean hydrophobic protein belongs to a family of proteins that contains a number of storage and phospholipid binding proteins. Its function is not known, but its overall hydrophobic nature is typical of many membrane proteins of similar size. The molecular weight is 8.3 x 10(3), and it crystallizes in the space group P2(1)2(1)2(1), with a = 52.01 A, b = 43.50 A and c = 28.80 A. The crystals diffract to 1.8 A resolution, and are thus suitable for X-ray structural studies.

Binding of dimethyl sulfoxide to lysozyme in crystals, studied with neutron diffraction.

Crystals of hen egg white lysozyme soaked in 15% (v/v) dimethyl sulfoxide have been studied with single-crystal neutron diffraction to determine the effect of the solvent molecules on the protein configuration. A total of 9423 statistically significant Bragg reflections to a resolution of approximately 1.8 A were used to locate 6 dimethyl sulfoxide molecules, and structure refinements including a model for the flat solvent lead to a final crystallographic agreement factor of 0.130. The mode of location of the dimethyl sulfoxide molecules was compared with that in previous studies employing ethanol. This showed that hydrophobic interactions can be an essential factor in fixing the probe molecules on the protein surface. There was, however, no sign of any significant change in the protein configuration; so although possibly at higher concentrations of dimethyl sulfoxide the protein will unfold, there was no indication of any precursor effect.

Study of ethanol-lysozyme interactions using neutron diffraction.

Single-crystal neutron diffraction has been used to observe the interactions between deuterated ethanol (CD3CD2OH) and lysozyme in triclinic crystals of hen egg white lysozyme soaked in 25% (v/v) ethanol solutions. A total of 6047 observed reflections to a resolution of 2 A were used, and 13 possible ethanol sites were identified. The three highest occupied sites are close to locations for bromoethanol found in an earlier study by Yonath et al. [Yonath, A., Podjarny, A., Honig, B., Traub, W., Sielecki, A., Herzberg, O., & Moult, J. (1978) Biophys. Struct. Mech. 4, 27-36]. Structure refinements including a model for the flat solvent lead to a final crystallographic agreement factor of 0.097. Comparison with earlier neutron studies on triclinic lysozyme showed that neither the molecular structure nor the thermal motions were affected significantly by the ethanol. A detailed analysis of the ethanol-lysozyme contacts showed 61% of these to be with hydrophobic sites, in agreement with the dominant hydrophobic nature of ethanol. This, together with the fact that the molecular structure of lysozyme is not perturbed, suggests a model for denaturation of lysozyme by alcohol, which proceeds via a dehydration of the protein at high alcohol concentration.

Neutron small-angle scattering studies of ribonuclease in mixed aqueous solutions and determination of the preferentially bound water.

Neutron small-angle measurements of ribonuclease A in mixed solutions of ethanol-water and glycerol-water have been used to estimate the region near the molecule that only contains water. When glycerol is used as probe, the region corresponds to an interaction parameter xi of 0.23 +/- 0.05 g of water/g of protein. For ethanol xi is -0.07 +/- 0.05, corresponding to the macromolecule being equally accessible to ethanol and water. The observations of the radius of gyration in the mixed solutions are used to show that the volume excluding glycerol is found on the exterior of the protein.