Crystal quality and differential crystal-growth behaviour of three proteins crystallized in gel at high hydrostatic pressure.

Pressure is a non-invasive physical parameter that can be used to control and influence protein crystallization. It is also found that protein crystals of superior quality can be produced in gel. Here, a novel crystallization strategy combining hydrostatic pressure and agarose gel is described. Comparative experiments were conducted on hen and turkey egg-white lysozymes and the plant protein thaumatin. Crystals could be produced under up to 75-100 MPa (lysozymes) and 250 MPa (thaumatin). Several pressure-dependent parameters were determined, which included solubility and supersaturation of the proteins, number, size and morphology of the crystals, and the crystallization volume. Exploration of three-dimensional phase diagrams in which pH and pressure varied identified growth conditions where crystals had largest size and best morphology. As a general trend, nucleation and crystal-growth kinetics are altered and nucleation is always enhanced under pressure. Further, solubility of the lysozymes increases with pressure while that of thaumatin decreases. Likewise, changes in crystallization volumes at high and atmospheric pressure are opposite, being positive for the lysozymes and negative for thaumatin. Crystal quality was estimated by analysis of Bragg reflection profiles and X-ray topographs. While the quality of lysozyme crystals deteriorates as pressure increases, that of thaumatin crystals improves, with more homogeneous crystal morphology suggesting that pressure selectively dissociates ill-formed nuclei. Analysis of the thaumatin structure reveals a less hydrated solvent shell around the protein when pressure increases, with approximately 20% less ordered water molecules in crystals grown at 150 MPa when compared with those grown at atmospheric pressure (0.1 MPa). Noticeably, the altered water distribution is seen in depressurized crystals, indicating that pressure triggers a stable structural alteration on the protein surface while its polypeptide backbone remains essentially unaltered.

Origin of the opalescence at the alpha-beta transition of quartz: role of the incommensurate phase studied by synchrotron radiation.

The origin of the light scattering observed at the alpha-beta transition of quartz is still a subject of controversy. We present structural studies performed during the coexistence of the alpha and the intermediate incommensurate (inc) phases using simultaneously synchrotron x-ray diffraction and optical techniques. The small and large angle light scatterings are due, respectively, to the orientation domains of the 3q inc phase and to the alpha phase twins revealed by diffuse x-ray scattering. In the vicinity of the interphase boundary, the two light scattering regions, both with perturbed properties, form a complex multiscale structure.

Crystallization within agarose gel in microgravity improves the quality of thaumatin crystals.

To prevent crystals from moving in orbit and sedimenting upon their return to earth, the model protein thaumatin was crystallized in agarose gel in the Advanced Protein Crystallization Facility during the eight-day Space Shuttle mission STS-95 (November 1998). The quality of tetragonal crystals grown in microgravity was compared with that of controls prepared in parallel in the laboratory. On the basis of their diffraction properties, microgravity crystals were more ordered than crystals grown in gel on earth (the latter being, on average, better than reference crystals obtained in solution on earth). It is concluded that protein crystallization within a gel in microgravity may yield crystals of superior quality by combining the advantages of both environments. A possible explanation for the positive effect of microgravity on protein crystallization in gels involving the better quality of the nucleus is discussed.

Mosaic spread characterization of microgravity-grown tetragonal lysozyme single crystals.

Mosaic spread values for crystals grown in microgravity were measured using synchrotron radiation. Full width at half maximum (FWHM) values for diffraction line profiles in the range 10-20" (arc seconds, 1" = 1 degrees /3600) were observed. These values are similar to those measured for crystals grown on earth using the gel-acupuncture method. The crystals analysed are composed of from two to five domains producing peaks having widths from 5 to 15". The distribution of these domains is neither homogeneous (with domains of lower quality concentrated in the centre of the crystal) nor isotropic (producing peaks whose width changes depending on the observation direction). Methodological aspects are also discussed, with special consideration of the effects of mosaic spread on the data-collection procedures for high-resolution (low-intensity) reflections.

In-situ measurement of rocking curves during lysozyme crystal growth.

The rocking curve of protein crystals contains a lot of useful information concerning crystal quality, most of which is lost owing to the superimposition of spurious features appearing in these fragile materials after growth, during handling and mounting. To minimize such data spoiling, an experimental setup to perform in situ X-ray diffraction experiments during crystal growth has been designed. The setup, which includes video observation to allow the correlation of crystal shape, size and growth rate with X-ray data, has been used to assess the mosaicity of tetragonal lysozyme crystals during crystal growth. The full width at half maximum (FWHM) of diffraction peaks collected from these crystals changes during the growth process as a (directly proportional) response to the growth rates and the different development of different domain blocks. These changes in the domain distribution and FWHM with time involve a 'zonation' of the crystals, which show very different rocking curves in different parts of their volume. The rocking curves recorded in situ from growing crystals are easier to understand than those from crystals that have suffered even minor handling.

The Perfection of Protein Crystals Probed by Direct Recording of Bragg Reflection Profiles with a Quasi-Planar X-ray Wave.

Profiles of Bragg reflections from earth-grown crystals of lysozyme from hen egg-white and collagenase from Hypoderma lineatum were directly recorded with a quasi-planar X-ray wave. One crystal of each protein was chosen for a detailed investigation. Each sample is shown to consist of only a few (three and two, respectively) highly ordered domains, misoriented with respect to each other by a few arc s. The smallest rocking widths were observed for the large domain of the collagenase sample (FWHM corrected for instrumental broadening: 0.0016 degrees for a strong reflection at 3 A resolution). With appropriate improvements, this method might become a quantitative tool for characterizing the perfection of crystals from biological macromolecules.

Thickness-shear mode shapes and mass-frequency influence surface of a circular and electroded AT-cut quartz resonator.

Finite-element solutions for the fundamental thickness shear mode and the second-anharmonic overtone of a circular, 1.87-MHz AT-cut quartz plate with no electrodes are presented and compared with previously obtained results for a rectangular plate of similar properties. The edge flexural mode in circular plates, a vibration mode not seen in the rectangular plate is also presented. A 5-MHz circular and electroded AT-cut quartz plate is studied. A portion of the frequency spectrum is constructed in the neighborhood of the fundamental thickness-shear mode. A convergence study is also presented for the electroded 5-MHz plate. A new two-dimensional (2-D) technique for visualizing the vibration mode solutions is presented. This method departs substantially from the three-dimensional (3-D) ;wire-frame' plots presented in the previous analysis. The 2-D images can be manipulated to produce nodal line diagrams and can be color coded to illustrate mode shapes and energy trapping phenomenon. A contour plot of the mass-frequency influence surface for the plated 5-MHz resonator is presented. The mass-frequency influence surface is defined as a surface giving the frequency change due to a small localized mass applied to the resonator surface.