Comparison of the intermediate complexes of human growth hormone bound to the human growth hormone and prolactin receptors.

The crystal structures of complexes of human growth hormone (hGH) with the growth hormone and prolactin receptors (hGHR and hPRLR, respectively), together with the mutational data available for these systems, suggest that an extraordinary combination of conformational adaptability, together with finely tuned specificity, governs the molecular recognition processes operative in these systems. On the one hand, in the active 1:2 ligand-receptor complexes, 2 copies of the same receptor use the identical set of binding determinants to recognize topographically different surfaces on the hormone. On the other hand, comparing the 1:1 hGH-hGHR and hGH-hPRLR complexes, 2 distinct receptors use this same set of binding determinants to interact with the identical binding site on the ligand, even though few residues among the binding determinants are conserved. The structural evidence demonstrates that this versatility is accomplished by local conformational flexibility of the binding loops, allowing adaptation to different binding environments, together with rigid-body movements of the receptor domains, necessary for the creation of specific interactions with the same binding site.     

Crystal structure of cholera toxin B-pentamer bound to receptor GM1 pentasaccharide.

Cholera toxin (CT) is an AB5 hexameric protein responsible for the symptoms produced by Vibrio cholerae infection. In the first step of cell intoxication, the B-pentamer of the toxin binds specifically to the branched pentasaccharide moiety of ganglioside GM1 on the surface of target human intestinal epithelial cells. We present here the crystal structure of the cholera toxin B-pentamer complexed with the GM1 pentasaccharide. Each receptor binding site on the toxin is found to lie primarily within a single B-subunit, with a single solvent-mediated hydrogen bond from residue Gly 33 of an adjacent subunit. The large majority of interactions between the receptor and the toxin involve the 2 terminal sugars of GM1, galactose and sialic acid, with a smaller contribution from the N-acetyl galactosamine residue. The binding of GM1 to cholera toxin thus resembles a 2-fingered grip: the Gal(beta 1-3)GalNAc moiety representing the "forefinger" and the sialic acid representing the "thumb." The residues forming the binding site are conserved between cholera toxin and the homologous heat-labile enterotoxin from Escherichia coli, with the sole exception of His 13. Some reported differences in the binding affinity of the 2 toxins for gangliosides other than GM1 may be rationalized by sequence differences at this residue. The CTB5:GM1 pentasaccharide complex described here provides a detailed view of a protein:ganglioside specific binding interaction, and as such is of interest not only for understanding cholera pathogenesis and for the design of drugs and development of vaccines but also for modeling other protein:ganglioside interactions such as those involved in GM1-mediated signal transduction.     

A structural basis for the interaction of urea with lysozyme.

The effect of urea on the crystal structure of hen egg-white lysozyme has been investigated using X-ray crystallography. High resolution structures have been determined from crystals grown in the presence of 0, 0.7, 2, 3, 4, and 5 M urea and from crystals soaked in 9 M urea. All the forms are essentially isomorphous with the native type II crystals, and the derived structures exhibit excellent geometry and RMS differences from ideality in bond distances and angles. Comparison of the urea complex structures with the native enzyme (type II form, at 1.5 A resolution) indicates that the effect of urea is minimal over the concentration range studied. The mean difference in backbone conformation between the native enzyme and its urea complexes varies from 0.18 to 0.49 A. Conformational changes are limited to flexible surface loops (Thr 69-Asn 74, Ser 100-Asn 103), the active site loop (Asn 59-Cys 80), and the C-terminus (Cys 127-Leu 129). Urea molecules are bound to distinct sites on the surface of the protein. One molecule is bound to the active site cleft's C subsite, at all concentrations, in a fashion analogous to that of the N-acetyl substituent of substrate and inhibitor sugars normally bound to this site. Occupation of this subsite by urea alone does not appear to induce the conformational changes associated with inhibitor binding.     

两种钙藻热解产出的气态和液态烃类

在２００℃至４００℃高温和还原条件下分别对仙掌藻和乳节藻两种钙质藻类进行热模拟降解实验,分离获得气态和液态烃类化合物。随着热解温度升高,两种钙藻产烃气量明显上升,其中甲烷与乙烷气的增加最多,同时两种钙藻产出的乙烷与乙烯比值都有规律地增加,但仙掌藻产烃气量高于乳节藻。这表明钙质藻类可能是天然气的一种重要母质来源。虽然这两种钙藻随温度增加热解产出的可溶有机质及族组分的变化规律不明显,但它们的正烷烃分布特征具有相类似的变化。未经热解时它们都以Ｃ１７为主峰的低碳数正烷烃占优势;当热解温度从２００℃增加到４００℃时,它们都又表现出以Ｃ２５或Ｃ２３为主峰的较高碳数正烷烃占优势的分布特征。这与富含钙藻化石的沉积岩样品中正烷烃的分布特征相一致,说明Ｃ２５或Ｃ２３为主峰的较高碳数正烷烃占优势的分布特征可能是钙藻热解有机质的一种判识标志．     

Cold adaption of enzymes: Structural comparison between salmon and bovine trypsins

The crystal structure of an anionic form of salmon trypsin has been determined at 1.82 Å resolution. We report the first structure of a trypsin from a phoikilothermic organism in a detailed comparison to mammalian trypsins in order to look for structural rationalizations for the cold-adaption features of salmon trypsin. This form of salmon trypsin (T II) comprises 222 residues, and is homologous to bovine trypsin (BT) in about 65% of the primary structure. The tertiary structures are similar, with an overall displacement in main chain atomic positions between salmon trypsin and various crystal structures of bovine trypsin of about 0.8 Å. Intramolecular hydrogen bonds and hydrophobic interactions are compared and discussed in order to estimate possible differences in molecular flexibility which might explain the higher catalytic efficiency and lower thermostability of salmon trypsin compared to bovine trypsin. No overall differences in intramolecular interactions are detected between the two structures, but there are differences in certain regions of the structures which may explain some of the observed differences in physical properties. The distribution of charged residues is different in the two trypsins, and the impact this might have on substrate affinity has been discussed. © 1994 Wiley-Liss, Inc.     

Direct resolution of ergot alkaloid enantiomers on a novel chiral silica-based stationary phase

A new covalently-bonded, silica-based stationary phase, using as the chiral selector the 1-(3-aminopropyl) derivative of (+)-(5R,8S,10R)-terguride, has been developed to resolve optically active isomers by HPLC. Good resolution of structurally related racemic ergot alkaloids were obtained using water-methanol mixtures as the eluent. Analysis of the influence of the type and concentration of the organic modifier, and the pH of the buffer in the mobile phase allowed the enantioseparation of these compounds to be optimized. Determination of the optical purity of a lisuride-containig drug is reported. © 1994 Wiley-Liss, Inc.     

Exothermic responses of dormant Salix stems during exposure to subzero temperatures

Differential thermal analysis (DTA) was used to determine the exothermic responses in dormant stems and excised lengths of stem of Salix dasyclados Wimmer subjected to artificial freezing treatments.
The presence of ice on the surfaces of intact stems restricted the mechanism of freezing avoidance to temperatures above –4°C. In contrast, excised lengths of stem started to freeze as soon as the ambient temperature fell below –2°C, demonstrating that extracellular ice formation takes place earlier if cut surfaces are present. Exposure of dormant excised lengths of stem to subfreezing temperatures for more than 8 weeks did not alter their nucleation temperature not their exothermic differential responses. Early extracellular crystallisation of freezable cellular water provides conditions that allow dormant Salix dasyclados stems or excised lengths of stem to survive extreme freezing stress.
Crystallisation of extracellular and cellular water took place in the cortex, and did not result in visual damage or reduced survival. This nucleation of extracellular water took place over the same temperature range whether the excised dormant lengths of stem were partly (bark only) or completely thawed. Exposure of dormant tissue to 20°C for up to 24 h did not alter the level of freezing tolerance, nor did it increase the susceptibility of excised lengths of stem to damage by extreme temperature fluctuations.     

The influence of temperature on the dynamics of protein precrystallization clusters,studied by photon correlation spectroscopy

Hen-egg white lysozyme was used for studying the influence of temperature on crystallization. The reaction was initiated at variable temperatures, covering the range between 5–50 °C, and was monitored with photon correlation spectroscopy. When aggregation was induced by addition of NaCl, the clusters formed exhibited diffusion limited aggregation behavior and crystals appeared in less than two days. In contrast, (NH₄)₂SO₄ induced aggregation took place mostly in the cross-over regime. In this case, solutions either remained transparent and void of crystals or formed gels within a few weeks. In both cases the kinetics could be dynamically scaled into master curves indicating that the precrystallization formed aggregates are fractals resulting from different collision processes.     

Myosin light chain kinase expression during smooth muscle development

The expression of smooth muscle myosin light chain kinase (MLCK) was investigated during chicken gizzard development. The molecular weight and the antigenic properties of MLCK did not change during development. The use of anion exchange high performance liquid chromatography (HPLC) enabled us to distinguish between MLCKs from post-hatched and adult chickens. A partial amino acid sequence determination of 4-day-old gizzard MLCK failed to disclose differences in the primary sequences of the two proteins. The results suggest that MLCK has the same primary sequence in all sequences of the two proteins. The results suggest that MLCK has the same primary sequence in all stages of gizzard development, although charge variants due to post-translational modifications may exist.