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 delta-endotoxin CytB from Bacillus thuringiensis and the ligand-binding domain of the human nuclear retinoid-X receptor RXR-alpha. 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.

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)6CI3. 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 GNU 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 GOT pair found in DNA fragments. The CU mismatch involves one hydrogen-bonded contact between the bases and a bridging water molecule which ensures a good fit ofthe 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.Tertiary structure is essential to the biological function of many single-stranded RNAs. Base pairing between complementary segments in a sequence generates a series of doublehelical stems that connect unpaired regions or loops. These can fold further into compact assemblies stabilized by additional hydrogen bonds and by hydrophobic and electrostatic interactions (1). The various levels of structural order are most completely characterized in tRNAs, for which a number of crystal structures have been determined (2-4). In the case of larger and more complex RNA species, including ribosomal components and RNase P, present understanding of secondary structure relies heavily on assumptions about the relative stabilities of possible base-pairing arrangements (5). These follow the specificities of adenine for uracil and guanine for cytosine observed in both ribonucleotides and deoxyribonucleotides during replication and transcription and are also mirrored in the experimentally measured stabilities of short oligonucleotide duplexes. Although the double helix is a central feature of these systems, none is a conspicuously direct model of the environment within a globular RNA structure.The present analysis of an RNA double helix is the second to be reported for an oligonucleotide containing a "tetraloop" sequence. Tetraloops are common in natural singlestranded RNA and, although the stems of such structural elements are variable, the unpaired regions are found to be tetranucleotides of mainly two types: a group with the general sequence r(GNRA) and the specific r(UUCG). Examples of both classes have been examined extensively in solution, where they exist as monomeric, thermally stable, looped species which parallel the behavior of the parent sequences in RNA of higher molecular weight (6,7).In crystals of both the present sequence and a dodecamer studied by Holbrook et al. (8), where r(UUCG) is embedded in se...

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The crystal structure of the deoxyoctamer d(G-G-Br U-A-BrU-A-C-C) was refined to a resolution of 1.7 A using combined diffractometer and synchrotron data. The analysis was carried out independently in two laboratories using different procedures. Although the final results are identical the comparison of the two approaches highlights potential problems in the refinement of oligonucleotides when only limited data are available. As part of the analysis the positions of 84 solvent molecules in the asymmetric unit were established. The DNA molecule is highly solvated, particularly the phosphate-sugar back-bone and the functional groups of the bases. The major groove contains, in the central BrU-A-BrU-A region, a ribbon of water molecules forming closed pentagons with shared edges. These water molecules are linked to the base O and N atoms and to the solvent chains connecting the O-1 phosphate oxygen atoms on each strand. The minor groove is also extensively hydrated with a continuous network in the central region and other networks at each end. The pattern of hydration is briefly compared with that observed in the structure of a B-dodecamer.

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 structure of marcfortine A, a novel alkaloid isolated from Penicilliurn roqueforti, has been established by X-ray analysis; two minor alkaloids, marcfortine B and C, as well as the previously known roquefortine have also been isolated.

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