Evolution of methionine initiator and phenylalanine transfer RNAs

Summary Sequence data from methionine initiator and phenylalanine transfer RNAs were used to construct phylogenetic trees by the maximum parsimony method. Although eukaryotes, prokaryotes and chloroplasts appear related to a common ancestor, no firm conclusion can be drawn at this time about mitochondrial-coded transfer RNAs. tRNA evolution is not appropriately described by random hit models, since the various regions of the molecule differ sharply in their mutational fixation rates. Hot mutational spots are identified in the TC, the amino acceptor and the upper anticodon stems; the D arm and the loop areas on the other hand are highly conserved. Crucial tertiary interactions are thus essentially preserved while most of the double helical domain undergoes base pair interchange. Transitions are about half as costly as transversions, suggesting that base pair interchanges proceed mostly through G-U and A -C intermediates. There is a preponderance of replacements starting from G and C but this bias appears to follow the high G + C content of the easily mutated base paired regions.     

付里叶变换红外光谱法研究水在稳定多肽构象中的作用

在０—１００％相对湿度（ＲＨ）范围内用竹里叶变换红外光谱研究了水合对聚赖氨酸溴化氢结合物构象的影响。随相对湿度ＲＨ增加,水合分为：无规卷曲、β－折叠和α－螺旋三个稳定的阶段．多肽的水合行为远比报导的复杂,在三个阶段中都不是纯单一结构组分,在任何一个ＲＨ下都存在３－４个吸收组分。在ＲＨ＞４４％以上β特征吸收发生复杂变化,表明β－结构的类型可能发生变化。实验表明,水是肽链柔性的增塑剂,运动性和构象的催化剂。     

Conformational properties of l-fucose and the tetrasaccharide building block of the sulfated l-fucan from Lytechinus variegatus

Although the 3D structure of carbohydrates is known to contribute to their biological roles, conformational studies of sugars are challenging because their chains are flexible in solution and consequently the number of 3D structural restraints is limited. Here, we investigate the conformational properties of the tetrasaccharide building block of the Lytechinus variegatus sulfated fucan composed of the following structure [l-Fucp4(SO₃⁻)-α(1-3)-l-Fucp2,4(SO₃⁻)-α(1-3)-l-Fucp2(SO₃⁻)-α(1-3)-l-Fucp2(SO₃⁻)] and the composing monosaccharide unit Fucp, primarily by nuclear magnetic resonance (NMR) experiments performed at very low temperatures and using H₂O as the solvent for the sugars rather than using the conventional deuterium oxide. By slowing down the fast chemical exchange rates and forcing the protonation of labile sites, we increased the number of through-space ¹H–¹H distances that could be measured by NMR spectroscopy. Following this strategy, additional conformational details of the tetrasaccharide and l-Fucp in solution were obtained. Computational molecular dynamics was performed to complement and validate the NMR-based measurements. A model of the NMR-restrained 3D structure is offered for the tetrasaccharide.     

Solid-state NMR Study Reveals Collagen I Structural Modifications of Amino Acid Side Chains upon Fibrillogenesis

In vivo, collagen I, the major structural protein in human body, is found assembled into fibrils. In the present work, we study a high concentrated collagen sample in its soluble, fibrillar, and denatured states using one and two dimensional {¹H}-¹³C solid-state NMR spectroscopy. We interpret ¹³C chemical shift variations in terms of dihedral angle conformation changes. Our data show that fibrillogenesis increases the side chain and backbone structural complexity. Nevertheless, only three to five rotameric equilibria are found for each amino acid residue, indicating a relatively low structural heterogeneity of collagen upon fibrillogenesis. Using side chain statistical data, we calculate equilibrium constants for a great number of amino acid residues. Moreover, based on a ¹³C quantitative spectrum, we estimate the percentage of residues implicated in each equilibrium. Our data indicate that fibril formation greatly affects hydroxyproline and proline prolyl pucker ring conformation. Finally, we discuss the implication of these structural data and propose a model in which the attractive force of fibrillogenesis comes from a structural reorganization of 10 to 15% of the amino acids. These results allow us to further understand the self-assembling process and fibrillar structure of collagen.     

Cdc37 (Cell Division Cycle 37) Restricts Hsp90 (Heat Shock Protein 90) Motility by Interaction with N-terminal and Middle Domain Binding Sites

The ATPase-driven dimeric molecular Hsp90 (heat shock protein 90) and its cofactor Cdc37 (cell division cycle 37 protein) are crucial to prevent the cellular depletion of many protein kinases. In complex with Hsp90, Cdc37 is thought to bind an important lid structure in the ATPase domain of Hsp90 and inhibit ATP turnover by Hsp90. As different interaction modes have been reported, we were interested in the interaction mechanism of Hsp90 and Cdc37. We find that Cdc37 can bind to one subunit of the Hsp90 dimer. The inhibition of the ATPase activity is caused by a reduction in the closing rate of Hsp90 without obviously bridging the two subunits or affecting nucleotide accessibility to the binding site. Although human Cdc37 binds to the N-terminal domain of Hsp90, nematodal Cdc37 preferentially interacts with the middle domain of CeHsp90 and hHsp90, exposing two Cdc37 interaction sites. A previously unreported site in CeCdc37 is utilized for the middle domain interaction. Dephosphorylation of CeCdc37 by the Hsp90-associated phosphatase PPH-5, a step required during the kinase activation process, proceeds normally, even if only the new interaction site is used. This shows that the second interaction site is also functionally relevant and highlights that Cdc37, similar to the Hsp90 cofactors Sti1 and Aha1, may utilize two different attachment sites to restrict the conformational freedom and the ATP turnover of Hsp90.     

Stabilization,Characterization, and Selective Removal of Cystatin C Amyloid Oligomers

The pathophysiological process in amyloid disorders usually involves the transformation of a functional monomeric protein via potentially toxic oligomers into amyloid fibrils. The structure and properties of the intermediary oligomers have been difficult to study due to their instability and dynamic equilibrium with smaller and larger species. In hereditary cystatin C amyloid angiopathy, a cystatin C variant is deposited in arterial walls and cause brain hemorrhage in young adults. In the present investigation, we use redox experiments of monomeric cystatin C, stabilized against domain swapping by an intramolecular disulfide bond, to generate stable oligomers (dimers, trimers, tetramers, decamers, and high molecular weight oligomers). These oligomers were characterized concerning size by gel filtration, polyacrylamide gel electrophoresis, and mass spectrometry, shape by electron and atomic force microscopy, and, function by assays of their capacity to inhibit proteases. The results showed the oligomers to be highly ordered, domain-swapped assemblies of cystatin C and that the oligomers could not build larger oligomers, or fibrils, without domain swapping. The stabilized oligomers were used to induce antibody formation in rabbits. After immunosorption, using immobilized monomeric cystatin C, and elution from columns with immobilized cystatin C oligomers, oligomer-specific antibodies were obtained. These could be used to selectively remove cystatin C dimers from biological fluids containing both dimers and monomers.     

Ion-specific Effects on Prion Nucleation and Strain Formation

Ordered, fibrous, self-seeding aggregates of misfolded proteins known as amyloids are associated with important diseases in mammals and control phenotypic traits in fungi. A given protein may adopt multiple amyloid conformations, known as variants or strains, each of which leads to a distinct disease pattern or phenotype. Here, we study the effect of Hofmeister ions on amyloid nucleation and strain generation by the prion domain-containing fragment (Sup35NM) of a yeast protein Sup35p. Strongly hydrated anions (kosmotropes) initiate nucleation quickly and cause rapid fiber elongation, whereas poorly hydrated anions (chaotropes) delay nucleation and mildly affect the elongation rate. For the first time, we demonstrate that kosmotropes favor formation of amyloid strains that are characterized by lower thermostability and higher frangibility in vitro and stronger phenotypic and proliferation patterns effectively in vivo as compared with amyloids formed in chaotropes. These phenomena point to inherent differences in the biochemistry of Hofmeister ions. Our work shows that the ionic composition of a solution not only influences the kinetics of amyloid nucleation but also determines the amyloid strain that is preferentially formed.     

B3LYP/6-311++G* * study of structure and spin-spin coupling constant in heparin disaccharide

Structures of heparin disaccharide have been analyzed by DFT using the B3LYP/6-311++G( * *) method. The optimized geometries of two forms of this disaccharide, differing in the conformation ((1)C(4) and (2)S(0)) of the IdoA2S residue, confirmed considerable influences of the sulfate and the carboxylate groups upon the pyranose ring geometries. The computed energies showed that disaccharide having the (1)C(4) form of the IdoA2S residue is more stable than that with the (2)S(0) form. Interatomic distances, bond and torsion angles showed that interconversion of the IdoA2S residue results in geometry changes in the GlcN,6S residue as well. Three-bond proton-proton and proton-carbon spin-spin coupling constants computed for both forms agree with the experimental data and indicate that only two chair forms contribute to the conformational equilibrium in disaccharide. Influences of the charged groups upon the magnitudes of spin-spin coupling constants are also discussed.     

Carbanilation of cereal beta-glucans for molecular weight determination and conformational studies

Cereal beta-glucans can form aggregates in aqueous solution. The presence of aggregates in cereal beta-glucan solutions led to inaccurate determination of molecular weights and it was believed that intermolecular hydrogen bonding caused the aggregation. To eliminate aggregates, a carbanilation method for molecular weight determination of cereal beta-glucans was developed. Wheat beta-glucan samples were selected for investigation. The carbanilation method can prevent intermolecular hydrogen bonding by blocking hydroxyl groups with phenyl carbamate groups. The carbanilates of cereal beta-glucans were prepared by the reaction of cereal beta-glucans with phenylisocyanate catalyzed by DMSO and pyridine. To avoid degradation during the carbanilation reaction, relatively mild conditions were used, which led to incomplete substitution (DS: approximately 2). However, after the carbanilation reaction, the carbanilates dissolved completely in 1,4-dioxane solution without any detectable aggregates, which allowed accurate molecular weight determination. The degree of substitution (DS) of carbanilates was determined by both a nitrogen content method and an FT-IR method. The FT-IR method proved to be the more effective for DS estimation. Using this method, the converted molecular weights of cereal beta-glucans were in good agreement with the results measured in 0.5M NaOH solution, which previously was shown to be a good solvent for cereal beta-glucans. After the carbanilation reaction, conformational changes of carbanilates were studied by static and dynamic light scattering techniques. The fractal dimension (d(f)=2.27) and the structure sensitive parameters (rho >2) suggested a porous globular structure for partially carbanilated beta-glucans.     

有机物料对土壤镉形态及其生物有效性的影响

采用盆栽试验,研究了淹水种稻条件下添加猪粪和泥炭对红壤和潮土中内源和外源Cd形态及其生物有效性的影响。结果表明,土壤中内源Cd在各形态之间的分布比较均匀;添加外源Cd时Cd主要分布于交换态,从分蘖期到成熟期,内源Cd交换态普遍升高,添加外源Cd时交换态普遍降低。有机物料对内源Cd交换态的影响不显著,但当添加外源Cd时则对交换态有显著影响,在不添加外源Cd的条件下,有机物料普遍促进水稻根系对Cd的吸收。在添加外源Cd的条件下,有机物料普遍抑制水稻根系对Cd的吸收,猪粪的抑制效果强于泥炭,水稻根系对Cd与Fe的累积呈显著抑制作用。