Roger Fourme

Exploring hydrophobic sites in proteins with xenon or krypton.

X‐ray diffraction is used to study the binding of xenon and krypton to a variety of crystallised proteins: porcine pancreatic elastase; subtilisin Carlsberg from Bacillus licheniformis; cutinase from Fusarium solani; collagenase from Hypoderma lineatum; hen egg lysozyme, the lipoamide dehydrogenase domain from the outer membrane protein P64k from Neisseria meningitidis; urate‐oxidase from Aspergillus flavus, mosquitocidal δ‐endotoxin CytB from Bacillus thuringiensis and the ligand‐binding domain of the human nuclear retinoid‐X receptor RXR‐α. Under gas pressures ranging from 8 to 20 bar, xenon is able to bind to discrete sites in hydrophobic cavities, ligand and substrate binding pockets, and into the pore of channel‐like structures. These xenon complexes can be used to map hydrophobic sites in proteins, or as heavy‐atom derivatives in the isomorphous replacement method of structure determination. Proteins 30:61–73, 1998.

High-pressure protein crystallography (HPPX): instrumentation, methodology and results on lysozyme crystals

A new set-up and associated methodology for the collection of angle-dispersive diffraction data from protein crystals submitted to high hydrostastic pressure have been developed on beamline ID30 at the ESRF. The instrument makes use of intense X-rays of ultra-short wavelength emitted by two collinear undulators, and combines a membrane-driven diamond-anvil cell mounted on a two-axis goniometer and an imaging-plate scanner. Sharp and clean diffraction pictures from tetragonal crystals of hen egg-white lysozyme (tHEWL) and orthorhombic crystals of bovine erythrocyte Cu, Zn superoxide dismutase (SOD) were recorded at room temperature and pressures up to 0.915 and 1.00 GPa, respectively. The compressibility of tHEWL was determined from unit-cell parameters determined at 24 different pressures up to 0.915 GPa. High-pressure diffraction data sets from several crystals of tHEWL were collected and analyzed. Merging of data recorded on different crystals at 0.30 and 0.58 GPa produced two sets of structure amplitudes with good resolution, completeness, redundancy and R(sym) values. A third set at 0.69 GPa was of a similar quality except a lower completeness. The three structures have been refined. The pressure-induced loss of crystalline order in a tHEWL crystal beyond 0.82 GPa was captured through a series of diffraction pictures.

On the preparation and X-ray data collection of isomorphous xenon derivatives

A simple method for the preparation of isomorphous xenon derivatives is presented. A device has been designed that allows diffraction studies on protein crystals under xenon gas pressures up to 50 × 105 Pa. Crystal mounting and X-ray data collection do not significantly differ from standard techniques. Tests carried out on crystals of the protein porcine pancreatic elastase reveal a single xenon binding site with high occupancy at a pressure of 8 × 105 Pa. Xenon binding to several other crystallized proteins has also been investigated and results indicate that the method is generally applicable. Time-resolved studies show that, at 297 K, xenon binding is essentially completed within a few minutes. At pressures above 106 Pa, successful data collection is hampered by X-ray absorption and by the formation of xenon hydrate. Absorption can be reduced by using short-wavelength radiation and by mounting crystals in small capillaries. To circumvent xenon hydrate formation, higher working temperatures and the use of cryoprotective mother liquors are advocated.

Macromolecular crystallography with synchrotron radiation: photographic data collection and polarization correction

The design and operation of a focusing camera for high-resolution macromolecular crystallography with synchrotron radiation (SR) are described. The performance of this service-oriented instrument is evaluated on the basis of five years of use. Standard procedures for data collection, data processing and data reduction have been modified to take unusual features of the SR source into account; the effect of polarization is thoroughly discussed.

Bent crystal, bent multilayer optics on a multipole wiggler line for an x‐ray diffractometer with an imaging plate detector

A setup for diffraction and diffuse scattering studies on biological crystals, in the wavelength range 1.5–0.9 A, is operating on the superconducting wiggler line of the storage ring DCI at LURE. Double focusing and rejection of harmonic contribution are achieved by combining two Bragg reflectors with elliptical curvature, respectively, a Si or Ge single crystal and a large W/Si interferential reflector (layered synthetic microstructure). Area detectors include photographic films and an imaging plate scanner device.

Crystal structure study of Opsanus tau parvalbumin by multiwavelength anomalous diffraction

The crystal structure of a small calcium‐binding protein, the parvalbumin IIIf from Opsanus tau in which Tb was substituted for Ca, has been analysed by multiwavelength anomalous diffraction. Data at a resolution of 2.3 Å were collected at three wavelengths near the L3 absorption edge of Tb (1.645–1.650 Å), using the synchrotron radiation emitted by a storage ring and a multiwire proportional counter. The phases of the reflections were determined from this single derivative, without native data. Prior to any refinement, the resulting electron density map shows a good agreement with the model of the homologous carp parvalbumin in regions of identical amino‐acid sequence.

The catalytic site of serine proteinases as a specific binding cavity for xenon

BACKGROUND Under moderate pressure, xenon can bind to proteins and form weak but specific interactions. Such protein-xenon complexes can be used as isomorphous derivatives for phase determination in X-ray crystallography. RESULTS Investigation of the serine proteinase class of enzymes shows that the catalytic triad, the common hydrolytic motif of these enzymes, is a specific binding site for one xenon atom and shows high occupancy at pressures below 12 bar. Complexes of xenon with two different serine proteinases, elastase and collagenase, were analyzed and refined to 2.2 A and 2.5 A resolution, respectively. In both cases, a single xenon atom with a low temperature factor is located in the active site at identical positions. Weak interactions exist with several side chains of conserved amino acids at the active site. Xenon binding does not induce any major changes in the protein structure and, as a consequence, crystals of the xenon complexes are highly isomorphous with the native protein structures. Xenon is also found to bind to the active site of subtilisin Carlsberg, a bacterial serine proteinase, that also has a catalytic triad motif. CONCLUSIONS As the region around the active site shows conserved structural homology in all serine proteinases, it is anticipated that xenon binding will prove to be a general feature of this class of proteins.

Structure-Function Perturbation and Dissociation of Tetrameric Urate Oxidase by High Hydrostatic Pressure

Structure-function relationships in the tetrameric enzyme urate oxidase were investigated using pressure perturbation. As the active sites are located at the interfaces between monomers, enzyme activity is directly related to the integrity of the tetramer. The effect of hydrostatic pressure on the enzyme was investigated by x-ray crystallography, small-angle x-ray scattering, and fluorescence spectroscopy. Enzymatic activity was also measured under pressure and after decompression. A global model, consistent with all measurements, discloses structural and functional details of the pressure-induced dissociation of the tetramer. Before dissociating, the pressurized protein adopts a conformational substate characterized by an expansion of its substrate binding pocket at the expense of a large neighboring hydrophobic cavity. This substate should be adopted by the enzyme during its catalytic mechanism, where the active site has to accommodate larger intermediates and product. The approach, combining several high-pressure techniques, offers a new (to our knowledge) means of exploring structural and functional properties of transient states relevant to protein mechanisms.

Opening the high-pressure domain beyond 2 kbar to protein and virus crystallography--technical advance.

The combined use of a diamond anvil cell and ultrashort-wavelength undulator radiation has allowed the collection of high-resolution diffraction data from protein and virus crystals submitted to hydrostatic pressures beyond 2 kbar. Crystals of cubic cowpea mosaic virus (CPMV) can be compressed to at least 3.5 kbar. Diffraction from CPMV crystals displaying an unusual disorder at atmospheric pressure was considerably enhanced by application of pressure. These experiments suggest that pressure may be used in some cases to improve order in crystals.

Use of noble gases xenon and krypton as heavy atoms in protein structure determination.

Publisher Summary This chapter presents various aspects of the preparation of xenon and krypton derivatives and their use as heavy atoms or anomalous scatterers in protein crystallography. The noble gas atoms are able to diffuse rapidly toward potential interaction sites in proteins via the solvent channels that are always present in crystals of macromolecules. Xenon and krypton derivatives of proteins can be obtained by subjecting a native protein crystal to a xenon or krypton gas atmosphere pressurized in the range of 1–100 bar. The number and occupancies of xenon/krypton-binding sites vary with the applied pressure. The interaction of noble gas atoms with proteins is the result of noncovalent weak-energy van der Waals forces, and therefore the process of xenon/krypton binding is completely reversible. With proteins, however, the interactions of xenon and krypton are of noncovalent origin and are therefore similar in nature to the forces that give rise to the formation of other well-known complexes of xenon and krypton with small molecules. The key physical parameter in xenon/krypton–protein interactions is the electronic polarizability of the noble gas atoms. The usual repulsive forces between atoms and molecules that are in close contact with each other also play an important role because they determine the minimum size that a cavity must have for xenon or krypton to bind into it. With the current emphasis on high-throughput macromolecular crystallography, the use of xenon and krypton derivatives should feature prominently in the list of available tools to solve protein structures.