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Noble gases are chemically inert, and it was therefore thought they would have little effect on biology. Paradoxically, it was found that they do exhibit a wide range of biological effects, many of which are target-specific and potentially useful and some of which have been demonstrated in vivo. The underlying mechanisms by which useful pharmacology, such as tissue and neuroprotection, anti-addiction effects, and analgesia, is elicited are relatively unexplored. Experiments to probe the interactions of noble gases with specific proteins are more difficult with gases than those with other chemicals. It is clearly impractical to conduct the large number of gas–protein experiments required to gain a complete picture of noble gas biology. Given the simplicity of atoms as ligands, in silico methods provide an opportunity to gain insight into which noble gas–protein interactions are worthy of further experimental or advanced computational investigation. Our previous validation studies showed that in silico methods can accurately predict experimentally determined noble gas binding sites in X-ray structures of proteins. Here, we summarize the largest reported in silico reverse docking study involving 127 854 protein structures and the five nonradioactive noble gases. We describe how these computational screening methods are implemented, summarize the main types of interactions that occur between noble gases and target proteins, describe how the massive data set that this study generated can be analyzed (freely available at group18.csiro.au), and provide the NDMA receptor as an example of how these data can be used to understand the molecular pharmacology underlying the biology of the noble gases. We encourage chemical biologists to access the data and use them to expand the knowledge base of noble gas pharmacology, and to use this information, together with more efficient delivery systems, to develop “atomic drugs” that can fully exploit their considerable and relatively unexplored potential in medicine.

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 Rsymvalues. 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.

Cryptogein is a proteinaceous elicitor of plant defense reactions which also exhibits sterol carrier properties. In this study, we report that this protein binds fatty acids. The stoichiometry of the fatty acid–cryptogein complex is 1:1. Linoleic acid and dehydroergosterol compete for the same site, but elicitin affinity is 27 times lower for fatty acid than for sterol. We show that C7 to C12 saturated and C16 to C22 unsaturated fatty acids are the best ligands. The presence of double bonds markedly increases the affinity of cryptogein for fatty acids. A comparison between elicitins and known lipid transfer proteins is discussed.

Cryptogein is a proteinaceous elicitor of plant defense reactions which also exhibits sterol carrier properties. In this study, we report that this protein binds fatty acids. The stoichiometry of the fatty acid–cryptogein complex is 1:1. Linoleic acid and dehydroergosterol compete for the same site, but elicitin affinity is 27 times lower for fatty acid than for sterol. We show that C7 to C12 saturated and C16 to C22 unsaturated fatty acids are the best ligands. The presence of double bonds markedly increases the affinity of cryptogein for fatty acids. A comparison between elicitins and known lipid transfer proteins is discussed.

This paper presents a survey of developments in de novophasing methods and instrumentation in protein crystallography that have been carried out over the past 20 years at the French synchrotron radiation facility LURE. This includes progress in detector technology, particularly with multiwire proportional chambers, contributions to the development of the MAD and MASC methods for experimental phase‐determination viaanomalous dispersion, the exploration of the use of xenon and krypton as heavy atoms and anomalous scatterers, as well as the substantialization of parts of the `Bayesian programme' for structure determination in highly efficient and user‐friendly software. It is shown how the conjunction of high‐quality data collection with novel phasing methods and with optimized data‐processing schemes can bring about major improvements, even when the signal is very weak, in the accuracy of structural determinations.

In order to define the active site(s) of human tumour necrosis factor (hTNF), we mutagenized its gene at random and directly screened the resulting population for loss of cytotoxic activity on L929 cells. Four biologically inactive mutant proteins (Arg32‐‐‐‐Trp, Leu36‐‐‐‐Phe, Ser86‐‐‐‐Phe and Ala84‐‐‐‐Val) behaved similar to the wild‐type in various physico‐chemical assays. The residues were positioned on a 3D structural model and were found to cluster together at the base of the molecule at each side of the groove that separates two monomers in the trimeric structure. A very conservative mutation at one of these sites (Ala84‐‐‐‐Val) almost completely abolished cytotoxic activity. Amino acid alterations in three other residues in close proximity to this receptor binding site were introduced: replacements at positions 29 and 146 clearly reduced cytotoxicity only when non‐conservative alterations were introduced (Leu29‐‐‐‐Ser and Glu146‐‐‐‐Lys), suggesting an indirect influence on the active site. However, a conservative mutation at position 91 (Val‐‐‐‐Ala) caused a significant drop (500‐fold) in bioactivity which suggests that Val91 may also play a direct role in receptor recognition. Our results favor a model in which each TNF molecule has three receptor‐interaction sites (between the three subunits), thus allowing signal transmission by receptor clustering.

The noble gas krypton is shown to bind to crystallized proteins in a similar way to xenon [Schiltz, Prangé & Fourme (1994). J. Appl. Cryst.27, 950–960]. Preliminary tests show that the major krypton binding sites are essentially identical to those of xenon. Noticeable substitution is achieved only at substantially higher pressures (above 50 × 105Pa). As is the case for xenon, the protein complexes with krypton are highly isomorphous with the native structure so that these complexes can be used for phase determination in protein crystallography. Krypton is not as heavy as xenon, but its K‐absorption edge is situated at a wavelength (0.86 Å) that is readily accessible on synchrotron radiation sources. As a test case, X‐ray diffraction data at the high‐energy side of the Kedge were collected on a crystal of porcine pancreatic elastase (molecular weight of 25.9 kDa) put under a krypton gas pressure of 56 × 105Pa. The occupancy of the single Kr atom is approximately 0.5, giving isomorphous and anomalous scattering strengths of 15.2 and 1.9 e, respectively. This derivative could be used successfully for phase determination with the SIRASmethod (single isomorphous replacement with anomalous scattering). After phase improvement by solvent flattening, the resulting electron‐density map is of exceptionally high quality, and has a correlation coefficient of 0.85 with a map calculated from the refined native structure. Careful data collection and processing, as well as the correct statistical treatment of isomorphous and anomalous signals have proven to be crucial in the determination of this electron‐density map. Heavy‐atom refinement and phasing were carried out with the program SHARP, which is a fully fledged implementation of the maximum‐likelihood theory for heavy‐atom refinement [Bricogne (1991). Crystallographic Computing5, edited by D. Moras, A. D. Podjarny & J. C. Thierry, pp. 257–297. Oxford: Clarendon Press]. It is concluded that the use of xenon and krypton derivatives, when they can be obtained, associated with statistical heavy‐atom refinement will allow one to overcome the two major limitations of the isomorphous replacement method i.e.non‐isomorphism and the problem of optimal estimation of heavy‐atom parameters.

Structure of beticolin O has been established through examination of its spectroscopic data including X-Ray diffraction and through its chemical transformation into the previously identified beticolin 2.

In order to define the active site(s) of human tumour necrosis factor (hTNF), we mutagenized its gene at random and directly screened the resulting population for loss of cytotoxic activity on L929 cells. Four biologically inactive mutant proteins (Arg32‐‐‐‐Trp, Leu36‐‐‐‐Phe, Ser86‐‐‐‐Phe and Ala84‐‐‐‐Val) behaved similar to the wild‐type in various physico‐chemical assays. The residues were positioned on a 3D structural model and were found to cluster together at the base of the molecule at each side of the groove that separates two monomers in the trimeric structure. A very conservative mutation at one of these sites (Ala84‐‐‐‐Val) almost completely abolished cytotoxic activity. Amino acid alterations in three other residues in close proximity to this receptor binding site were introduced: replacements at positions 29 and 146 clearly reduced cytotoxicity only when non‐conservative alterations were introduced (Leu29‐‐‐‐Ser and Glu146‐‐‐‐Lys), suggesting an indirect influence on the active site. However, a conservative mutation at position 91 (Val‐‐‐‐Ala) caused a significant drop (500‐fold) in bioactivity which suggests that Val91 may also play a direct role in receptor recognition. Our results favor a model in which each TNF molecule has three receptor‐interaction sites (between the three subunits), thus allowing signal transmission by receptor clustering.

The antibiotic nogalamycin, a drug with high specificity for TG and CG steps in double-stranded DNA, has been crystallized as a 1:1 complex with the hexamer d(CCCGGG). The antibiotic is inserted at the central CG step of the duplex, with the two sugars oriented in the same direction and with strong interactions with the DNA within the grooves. The amino-glucose residue makes an integral part of a well defined major groove hydration network with van der Waals contacts and several strong hydrogen bonds to the duplex. The nogalose residue resides in the minor groove, making primarily van der Waals contacts. The single site allows an accurate molecular description of the intercalation, without perturbations from end effects observed previously. The local unwinding induced by nogalamycin is completely relaxed 2 base pairs away from the intercalation site. The two strands of the DNA show a continuous deformation from the A to the B form: 1) the cytosines toward the 5' end of the nogalomycin site in each strand have c3'-endo conformations while 5 guanosines toward the 3' ends have c2'-endo conformations; 2) within each strand, the phosphate-phosphate distances increase in a continuous manner from 5.7 A (A-form) to 7.1 A (B-form).

A protein crystallography experiment at the xenon K‐edge (λ= 0.358 Å) has been successfully carried out at the materials science beamline (BL2/ID11) of the ESRF. The samples used in this methodological study were crystals of porcine pancreatic elastase, a 26 kDa protein of known structure. The diffraction data are of excellent quality. The combination of isomorphous replacement and anomalous dispersion of a single xenon heavy‐atom derivative allowed accurate phase determination and the computation of a high‐quality electron density map of the protein molecule. This is the first fully documented report on a complete protein crystallography experiment, from data collection up to phase determination and calculation of an electron density map, carried out with data obtained at ultra‐short wavelengths. Experimental considerations as well as possible advantages and drawbacks of protein crystallography at very short and ultra‐short wavelengths are discussed.

4-Cyclohexyl-3-(1',3'-dithianyl)-4-oxobutanoic acid methyl ester 2, prepared by alkylation of cyclohexanoyl-1,3-dithiane 1, undergoes a basepromoted rearrangement affording the 1,4-dithiaepino-furan-2-one system.

Structural study using X-ray cristallography of the complexes of the title has been done. Alendronate is already known to be a very good chelator of divalent cations and supramolecular species obtained with alendronate and these cations are very stable and make them able to be used in biological systems. Our study points out that the size of the cation strongly modifies the structure of the complex. With Cu(II) and Zn(II) which are two cations with proximate ionic radii one cation is put in the centre of the coordination system and structures are isomorphous. On the other hand, with Cd(II), a binuclear complex is obtained. Results are discussed on the basis of these structures.

The nonamer r(GCUUCGGC)dBrU, where dBrU is 5-bromo-2'-deoxyuridine, contains the tetraloop sequence UUCG. It crystallizes in the presence of Rh(NH3)6Cl3. In solution the oligomer is expected to form a hairpin loop but the x-ray structure analysis, to a resolution of 1.6 A, indicates an eight-base-pair A-RNA duplex containing a central block of two G.U and two C.U pairs. Self-pairs which approximate to Watson-Crick geometry are also formed in the extended crystal structure between symmetry-related BrU residues and are part of infinite double-helical stacks. The G.U pair is a wobble base pair analogous to the G.T pair found in DNA fragments. The C.U mismatch involves one hydrogen-bonded contact between the bases and a bridging water molecule which ensures a good fit of the base pair in the RNA helix. The BrU.BrU pair is held by two hydrogen bonds in an orientation which is compatible with duplex geometry. The structure observed within the crystal has some parallels with the structure of globular RNAs, and the presence of stable, noncanonical base pairs has implications for the prediction of RNA secondary structure.