X-ray structure of the curare alkaloid (+)-tubocurarine dibromide.

X-ray structure determination of the compound (C37H42N2O6)2+ .2Br-.4CH3OH, confirms that (+)-tubocurarine is a monoquaternary salt and has established that the molecule adopts different conformations in crystals of the dibromide and dichloride salts. The crystal structure is stabilised by a number of hydrogen bonds involving the two free hydroxyl groups and the tertiary nitrogen of the tubocurarine molecule, the bromide ions and the solvent molecules. The absolute configuration of the molecule, determined by X-ray anomalous scattering, confirms the configuration assigned earlier by chemical studies.

Ribonuclease A. Analysis of the hydrogen bond geometry, and spatial accessibility at the active site.

The hydrogen bonding of bovine ribonuclease A derived from the high resolution X-ray structure has been studied in detail. Correlations have been examined for main-chain-main-chain hydrogen bond angles, torsion angles and distances, respectively. Differences are found consistently for correlations associated with alpha-helix and beta-sheet, respectively. Ten of the 124 side-chains have four or more hydrogen bond contacts; two, including Glu-101, have five or more. Three potential C = O---H, three N---X and three potential side-chain H-bonds fail to form. A search for highly inaccessible buried residues resulted in nine outstanding examples, all of which are conserved across 38 known mammalian ribonuclease A sequences, indicating the importance of these residues for structural stability. Of the two histidines in the active site, His-12 has five hydrogen bonds and His-119 three. The conformational space accessible to these two catalytically important residues studied by means of simple non-bonded contact energy calculations confirms the existence of two alternative, interchangeable locations for His-119, while His-12 is locked in a local energy minimum.

Novel non-productively bound ribonuclease inhibitor complexes--high resolution X-ray refinement studies on the binding of RNase-A to cytidylyl-2',5'-guanosine (2',5'CpG) and deoxycytidylyl-3',5'-guanosine (3',5'dCpdG).

The X-ray structures of two complexes of bovine ribonuclease-A produced by soaking pre-grown crystals in solutions of the inhibitors cytidylyl-2',5'-guanosine (2',5' CpG) and deoxycytidylyl-3',5'-guanosine (3',5'dCpdG) have been determined at 1.5 A resolution and refined by restrained least squares to R = 21.0% for 17,855 reflections, and R = 19.1% for 16,347 reflections, respectively. Binding of the substrate analogs to the protein has taken place in a completely unexpected and previously unreported manner. In each case the guanine base occupies the well characterized B1 pyrimidine binding site adjacent to Thr-45 (described by Richards, F.M., Wyckoff, H.W., Carlson, W.D., Allewell, N.M., Lee, B. and Mitsui, Y. (1971) Cold Spring Harbor Symp. Quant. Biol. 36, 35-54, and others including Palmer, R.A., Moss, D.S., Haneef, I. and Borkakoti, N. (1984) Biochim. Biophys. Acta 785, 81-88) having entered through a secondary channel external to the active site itself. We designate this reversed non-productive mode as retro-binding. In this mode of binding the SO4(2-) anion bound in the active site of the native protein crystals (Borkakoti, N., Moss, D.S. and Palmer, R.A. (1982) Acta Crystallogr. B38 2210-2217) has not been displaced by the phosphate of the inhibitor molecule as originally anticipated and observed in other studies. Instead the CMP or dCMP moiety of the inhibitor molecule is held loosely in a channel running towards the surface of the protein molecule and is thus completely external to the active site. Consequently, although it has been possible to model them, no attempt has been made to refine either the disordered cytosine in the CpG complex or the deoxycytosine in the dCpdG complex. The traditional B2 purine binding site of RNase (Richards et al., 1971) is unoccupied by the soaked inhibitors. Important changes that have taken place in the protein structure include: stabilization of both Lys-41 and Gln-11 via H-bonding to SO4(2-); stabilization of His-119 in the A conformation (Borkakoti, N., Moss, D.S. and Palmer, R.A. (1982) Acta Crystallogr. B38 2210-2217); and stabilization of SO4(2-) by H-bonds formed with the retro-bound guanine base. Binding of the inhibitors and stabilization of the active site is accompanied by displacement and redistribution of solvent molecules.

Crystallization of mouse pancreatic ribonuclease.

Mouse pancreatic ribonuclease has been crystallized in a form suitable for X-ray structure determination. The crystals grown from solutions of 2-methyl-2,4-pentanediol diffract to high resolution and belong to the hexagonal space group P6(1) (P6(5)) with unit cells dimensions a = b = 64.44 A, c = 53.91 A, y = 120 degrees and V = 1.94 x 10(5) A3 (1 A = 0.1 nm). There are six molecules per unit cell (1 molecule/asymmetric unit), and Vm = 2.3 A3/dalton.

Crystallization of the ribosome inactivating protein ML1 from Viscum album (mistletoe) complexed with beta-D-galactose.

A ribosome inactivating protein (ML1) from the mistletoe plant (Viscum album) has been crystallized. The crystals, grown in the presence of beta-D-galactose, are hexagonal, space group P6(1)22 or P6(5)22, a = b = 111.0 A, c = 309.3 A with 24 molecules per unit cell (assuming 33% solvent by weight). The protein of molecular mass 63 kDa is a heterodimer consisting of two chains, A and B, joined by a disulfide bond. The A-chain, 29 kDa, inhibits protein synthesis by depurinating an adenine residue (A4324) in a highly conserved RNA loop of the 28 S ribosomal subunit. The toxicity of the protein is mediated by the B-chain, 34 kDa, which has lectin activity, interacting with sugar residues of glycoproteins and glycolipids on the surface of target cells.

X-ray refinement study on the binding of cytidylic acid (2'-CMP) to ribonuclease A.

The X-ray structure of the inhibitor complex of bovine ribonuclease A with cytidylic acid (2'-CMP) has been determined at 2.3 A (1 A = 0.1 nm) resolution and refined by restrained least-squares refinement to R = 0.132 for 5650 reflections. Incorporation of the inhibitor molecule has occurred with little disturbance of the protein main-chain atoms, although significant displacement of some side-chain atoms has occurred, particularly in the region of the active site. The binding of 2'-CMP to ribonuclease A is different from that of the related cytidine-N(3)-oxide 2'-phosphate, which has an extra oxygen on N(3) of the cytidine base. The PO4(2-) group is held by hydrogen bond interactions to the side-groups of His 12, Glu 11 and His119. Thr45 is involved in stabilizing the enzyme-ligand complex by forming hydrogen bond interactions between O(gamma) and the pyrimidine base N(3) atom and between the main-chain N(45) and O(2) of the base. Phe120 is much closer to the inhibitor than in the cytidine N(3)-oxide 2'-phosphate structure.

Specificity of pancreatic ribonuclease-A. An X-ray study of a protein-nucleotide complex.

The modified purine nucleotide 8-oxo-guanosine-2'-phosphate binds at the pyrimidine binding site of ribonuclease-A. The O8-2'GMP inhibitor is in a syn conformation, with an intramolecular hydrogen bond between the N-3 atom of the base and the O-5' atom of the ribose. The essential groups of the protein involved in base recognition are O gamma 45 and N-45, which form hydrogen bonds to the five-membered ring of the heterocyclic base. Mobility of enzyme side-chains (viz. Lys41, Lys66, His119) close to the catalytic cleft of the protein allows conformational flexibility in the substrate binding region of ribonuclease-A. Inhibitor binding alters the solvent structure of the protein but the overall shape of the enzyme is not effected.

Newly observed binding mode in pancreatic ribonuclease.

Dinucleotides containing guanine, when soaked into crystals of bovine pancreatic ribonuclease, have been found to bind in an unexpected manner, quite unlike interpretations of earlier X-ray diffraction studies. This finding has prompted a reexamination of three mononucleotide-RNase complexes from this laboratory resulting in a re-interpretation of the complex that involved a guanine mononucleotide.

Audit of the use of psoralen photochemotherapy (PUVA) and narrowband UVB phototherapy in the treatment of psoriasis.

BACKGROUND: Psoralen photochemotherapy (PUVA), the combined use of psoralen and long wave ultraviolet (UVA) irradiation, was introduced around 1974 and its beneficial effects were rapidly confirmed worldwide. In an attempt to minimize its recognized long-term photocarcinogenic risk after some 150-200 exposures while also maintaining efficacy, however, the narrowband (311-312 nm) ultraviolet B (UVB) lamp (TL-01) was introduced in 1984, and has moved towards replacing PUVA except for severe or resistant disease. AIMS: To discover whether our use of these therapies complied with established British Photodermatology Group guidelines for PUVA and guidelines formulated within our unit for narrowband UVB. METHODS: The study was retrospective over 6 months from November 2001 to April 2002, all relevant information being obtained from the patients' hospital notes. RESULTS: Thirty-one patients received PUVA (18 oral, 11 bath and two uncertain because of missing notes) and 20 narrowband UVB during this period. CONCLUSIONS: Our PUVA and narrowband UVB phototherapy guidelines were shown to have been followed relatively closely with the following exceptions: one PUVA patient received a high cumulative exposure by mutual agreement because there was no other suitable therapy; a failure to measure minimal phototoxic doses (MPDs) in some PUVA patients; and slightly prolonged referral delays, but generally by patient choice.

The role of structural domains in RIP II toxin model membrane binding.

The interaction of plant toxin ricin and MLI binding subunits to liposomes containing monosialoganglioside (GM1), bearing a terminal galactose residue, has been examined as a possible receptor model. For the first time we demonstrate that ricin B-chain but not ricin provokes liposome aggregation at 10 M% GM1 concentration, whereas in the presence of either ricin A-chain or galactose the aggregation is inhibited. The B-subunit of plant toxin MLI from Viscum album has similar lectin specificity and activity but cannot aggregate GM1 liposomes. The ability of the B-chain to aggregate liposomes adds a new crucial step in the toxin transmembrane penetration mechanism. We demonstrate here possible ricin B-chain interactions with membranes proceeding via two sites, namely (a) a galactose-binding domain and (b) a hydrophobic interchain domain. In close contact with two phospholipid bilayers, ricin B-chain may determine the geometry of the fusion site. These events can provoke A-chain translocation which follows membrane fusion.

Mistletoe lectin I forms a double trefoil structure.

The quaternary structure of mistletoe lectin I (MLI), a type II ribosome inactivating protein, has been determined by X-ray crystallography. A definitive molecular replacement solution was determined for MLI using the co-ordinates of the homologue ricin as a search model. MLI exists as an [AB]2 dimer with internal crystallographic two-fold symmetry. Domain I of the B chains is non-covalently associated through interactions involving three looped chains (alpha, beta, gamma) in each molecule of the dimer, forming a double trefoil structure. The ricin molecule which shares 52% sequence homology with MLI has a disulphide bridge between Cys20 and Cys39 in the alpha loop. An evolutionary mutation has replaced Cys39 with serine in MLI. This mutation appears to allow the alpha loop the flexibility required to take up its place at the dimer interface, and also suggests a rationale for why ricin does not form dimers. Measurement of retention times using FPLC gel filtration confirms that dimerisation also occurs in solution between MLI B chains with an association constant Ka = 10(6) M.

The antagonism of acetylcholine excitatory and inhibitory responses of Helix central neurones, by some neuromuscular blocking drugs.

Intracellular recordings were made from identified central neurones of the snail Helix aspersa. The antagonism of acetylcholine excitatory and inhibitory responses by d-tubocurarine, HS818 and HS626 was investigated. d-Tubocurarine was equipotent against both the inhibitory and excitatory responses, while HS626 and HS818 were of similar potency to d-tubocurarine in antagonising acetylcholine excitation but were approximately one order of magnitude less potent in antagonising the inhibitory response.

A double mesogen based on linked p-terphenyl units.

The structure of bis(4,4"-decyloxy-p-terphenyl-2'-ylmethyl) carbonate, C(79)H(110)O(7), (I), has been determined at 123 K. It is a new type of twin mesogen. No two adjacent aromatic rings are coplanar and the four decyloxy side chains are maximally extended. Molecules of the compound are packed along the crystallographic a axis. The molecular arrangement is a precursor of a smectic A phase.

The hospital library is crucial to quality healthcare.

In a 1988 report, an advisory committee of the Medical Library Association (MLA) wrote that "no one would argue against information as the foundation for efficient cost-effective business or against access to knowledge as a prerequisite for developing new knowledge." Yet the increasing number of threats to the hospital library--largely from within the industry--suggest that many hospitals do not value information in the same way their counterparts in other businesses do. In the first of two articles in this issue on hospital library and information services, the executive director of the MLA uses the MLA's experience and a variety of research findings to restate the case for the hospital library's vital role in quality care.