Atomic and residue hydrophilicity in the context of folded protein structures

Water-protein interactions drive protein folding, stabilize the folded structure, and influence molecular recognition and catalysis. We analyzed the closest protein contacts of 10,837 water molecules in crystallographic structures to define a specific hydrophilicity scale reflecting specific rather than bulk solvent interactions. The tendencies of different atom and residue types to be the nearest protein neighbors of bound water molecules correlated with other hydrophobicity scales, verified the relevance of crystallographically determined water positions, and provided a direct experimental measure of water affinity in the context of the folded protein. This specific hydrophilicity was highly correlated with hydrogen-bonding capacity, and correlated better with experimental than computationally derived measures of partitioning between aqueous and organic phases. Atoms with related chemistry clustered with respect to the number of bound water molecules. Neutral and negatively charged oxygen atoms were the most hydrophilic, followed by positively-charged then neutral nitrogen atoms, followed by carbon and sulfur atoms. Agreement between observed side-chain specific hydrophilicity values and values derived from the atomic hydrophilicity scale showed that hydrophilicity values can be synthesized for different functional groups, such as unusual side or main chains, discontinuous epitopes, and drug molecules. Two methods of atomic hydrophilicity analysis provided a measure of complementarity in the interfaces of trypsin:pancreatic trypsin inhibitor and HIV protease:U-75875 inhibitor complexes. © 1995 Wiley-Liss, Inc.     

Fermenter production of an artificial fab fragment,rationally designed for the antigen cystatin,and its optimized crystallization through constant domain shuffling

The synthetic antibody model “M41” was rationally designed with a binding site complementary to chicken egg white cystatin as the prescribed antigen. In order to permit comparison between the computer model and an experimental three-dimensional structure of the artificial protein, its X-ray crystallographic analysis was pursued. For this purpose, M41 was expressed as a recombinant Fab fragment in E. coli by medium cell density fermentation employing the tightly regulated tetracycline promoter. The Fab fragment was efficiently purified via a His-6 tail fused to its heavy chain and immobilized metal affinity chromatography. To raise the chances for the productive formation of crystal packing contacts, three versions of the Fab fragment were generated with differing constant domains. One of these, the variant with murine C_K and C_H 1_γ1 domains, was successfully crystallized by microseeding in a sitting drop. The orthorhombic crystals exhibited symmetry of the space group P2₁2₁2₁ with unit cell dimensions a = 104.7 Å, b = 113.9 Å, c = 98.8 Å and diffracted X-rays to a nominal resolution of 2.5 Å. © 1995 Wiley-Liss, Inc.     

A multivariate approach to the selection of biological reserves

Multivariate analysis provides an effective context for the examination of some significant aspects of biodiversity and conservation. The framework is a multidimensional space that integrates sample sites, taxa and environments. This approach enables terms such as representativeness, complementarity and irreplaceability to be integrated within an intuitive and practical framework for reserve design. Cluster analysis is proposed to determine what is there by defining a set of complementary clusters. These clusters are sampled in a representative manner; from the core outward. The degree of irreplaceability of a site is defined as the multivariate distance of each potential reserve site to its nearest neighbour.     

The Alacoil: A very tight,antiparallel coiled-coil of helices

The Alacoil is an antiparallel (rather than the usual parallel) coiled-coil of α-helices with Ala or another small residue in every seventh position, allowing a very close spacing of the helices (7.5–8.5 Å between local helix axes), often over four or five helical turns. It occurs in two distinct types that differ by which position of the heptad repeat is occupied by Ala and by whether the closest points on the backbone of the two helices are aligned or are offset by half a turn. The aligned, or ROP, type has Ala in position “d” of the heptad repeat, which occupies the “tip-to-tip” side of the helix contact where the Cα–Cβ bonds point toward each other. The more common offset, or ferritin, type of Alacoil has Ala in position “a” of the heptad repeat (where the Cα-Cβ bonds lie back-to-back, on the “knuckle-touch” side of the helix contact), and the backbones of the two helices are offset vertically by half a turn. In both forms, successive layers of contact have the Ala first on one and then on the other helix. The Alacoil structure has much in common with the coiled-coils of fibrous proteins or leucine zippers: both are α-helical coiled-coils, with a critical amino acid repeated every seven residues (the Leu or the Ala) and a secondary contact position in between. However, Leu zippers are between aligned, parallel helices (often identical, in dimers), whereas Alacoils are between antiparallel helices, usually offset, and much closer together. The Alacoil, then, could be considered as an “Ala anti-zipper.” Leu zippers have a classic “knobs-into-holes” packing of the Leu side chain into a diamond of four residues on the opposite helix; for Alacoils, the helices are so close together that the Ala methyl group must choose one side of the diamond and pack inside a triangle of residues on the other helix. We have used the ferritin-type Alacoil as the basis for the de novo design of a 66-residue, coiled helix hairpin called “Alacoilin.” Its sequence is: cmSP DQWDKE A AQYDAHA QE FEKKS HRNng TPEA DQYRHM A SQY QAMA QK LKAIA NQLKK Gseter (with “a” heptad positions underlined and nonhelical parts in lowercase), which we will produce and test for both stability and uniqueness of structure.     

Degradation of the endosperm cell walls of Lactuca sativa L., cv. grand rapids in relation to the mobilisation of proteins and the production of hydrolytic enzymes in the axis,cotyledons and endosperm

The timing of changes in total nitrogen and soluble amino nitrogen content, and in the activities of proteinase (pH 7.0), isocitrate lyase, catalase, phytase, phosphatase (pH 5.0), -galactosidase and -mannosidase were studied in extracts from the cotyledons, axis and endosperms of germinating and germinated light-promoted lettuce seeds. The largest amount of total nitrogen (2.7% seed dry weight) occurs within the cotyledons, as storage protein. As this decreases the total nitrogen content of the axis increases and the soluble amino nitrogen in the cotyledons and axis increases. Proteinase activity in the cotyledons increases coincidentally with the depletion of total nitrogen therein. Enzymes for phytate mobilisation and for gluconeogenesis of hydrolysed lipids increase in activity in the cotyledons as the appropriate stored reserves decline. Beta-mannosidase, an enzyme involved in the hydrolysis of oligo-mannans released by the action of endo--mannase on mannan reserves in the endosperm, arises within the cotyledons. This indicates that complete hydrolysis of mannans to the monomer does not occur within the endosperm. Mobilisation of all cotyledon reserves occurs after the endosperm has been degraded, providing further evidence that the endosperm is an early source of food reserves for the growing embryo.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid - EDTA ethylenediamine tetraacetic acid disodium salt - TCA trichloroacetic acid Part 2 of a series, of which the first was published in Planta 139, 1–8 (1978)     

Kinetics of the cooperativity of the Ca2+-transporting adenosine triphosphatase of sarcoplasmic reticulum and the mechanism of the ATP interaction.

The extent of the negative cooperativity with MgATP of the Ca²⁺-stimulated ATPase activity of sarcoplasmic reticulum has been studied with various membrane preparations and under various conditions. Preparations studied were fragmented sarcoplasmic reticulum vesicles, deoxycholate-solubilized and fractionated ATPase, triton extracted reticulum, vesicles reconstituted from either detergent, and limited trypsin digests of the reticulum. Conditions studied were suboptimal, optimal, and inhibitory Ca²⁺ concentrations; temperatures from 13 to 46 °C; 1 or 5 mm MgCl₂; 0.1 m KCl, 0.1 m NaCl, or no added salt; and Triton or deoxycholate present in the assay. With preparations in which vesicles could accumulate Ca²⁺ ion, the ionophore A23187 was added to prevent inhibition by internal Ca²⁺ ions. Under all circumstances, the negative cooperativity of MgATP was present (Hill coefficient of 0.2 to 0.8), indicating the persistence of the properties of the enzyme molecule and its lipid environment giving rise to kinetic negative cooperativity. Attempts to measure the number of ATP sites by protection against N-ethylmaleimide inactivation and by binding of an analog suggested, but did not prove, that there was only one specific, active ATP binding site below 0.5 mm. These results are interpreted to be consistent with either of two mechanisms for ATP cooperativity of the Ca²⁺-stimulated ATPase activity of sarcoplasmic reticulum: (a) a single, high affinity ATP active site and a second, lower affinity “allosteric” activator site; or (b) a single ATP site which demonstrates two affinities through some kinetic mechanism such as a substrate-induced, slow transition.     

经济开发区生态规划与建设研究

生态规划和建设是协调经济发展和生态环境关系,创造和谐的人工化环境的调控对策。木文研究了沈阳开发区生态规划和建设的理论、方法,着重探讨了开发区经济、环境协调发展的途径:1)选择理想的协调发展优化方案,2)控制发展热电生产,发展集中供热和低能耗高技术产业,3)节约水资源,提高水循环利用率,4)在生态学原理指导下进行生态管理。     

Application of empirical design methodologies to the study of the influence of reaction conditions and N-alpha-protecting group structure on the enzymatic X-Phe-Leu-NH(2) dipeptide synthesis in buffer/dimethylformamide solvents systems

The influence of five different N-terminal protecting groups (For, Ac, Boc, Z, and Fmoc) and reaction conditions (temperature and dimethylformamide content) on the alpha-chymotrypsin-catalyzed synthesis of the dipeptide derivative X-Phe-Leu-NH(2) was studied. Groups such as For, Ac, Boc, and Z always rendered good peptide yields (82% to 85%) at low reaction temperatures and DMF concentrations, which depended on the N-alpha protection choice. Boc and Z were the most reactive N-alpha groups and, in addition, the most suitable for peptide synthesis. On the other hand, the use of empirical design methodologies allowed, with minimal experimentation and by multiple regression, to deduce an equation, which correlates the logarithm of the first order kinetic constant (log k') with reaction temperature, DMF concentration, and hydrophobicity (log P values) of the different protecting groups. The predictive value of the equation was tested by comparing the performance of another protective group, such as Aloc, with the performance predicted by said equation. Experimental and calculated k' values were found to be in good agreement.     

Utilization of the tricarboxylic acid cycle, a reactor design criterion for the microaerobic production of 2,3-butanediol

A new parameter, the relative utilization of tricarboxylic acid (TCA) cycle beta, is introduced to quantitatively account for the involvement of fermentation pathways and TCA cycle in the utilization of oxygen under oxygen-limiting (microaerobic) conditions. With the facultative anaerobe Enterobacter aerogenes, which produces 2,3-butanediol, a method is proposed to calculate beta from measurement of metabolites and exhaust gas. In continuous culture beta was found to be small under oxygen limitation, indicating that the fermentation pathways were preferred over the TCA cycle and oxygen was almost entirely consumed through oxidation of reduced nicotinamide adenine dinucleotide (NADH(2)) released by fermentation under these conditions. The increase of beta at high oxygen supply revealed a saturation of oxygen utilization through fermentation pathways. It could be concluded that, for the optimal performance of a microaerobic culture, oxygen uptake rate must be kept at such a level that as much NADH(2) as possible from fermentation pathways is oxidized by oxygen, and at the same time the utilization of TCA cycle is kept at a minimum. As the dynamics of the microaerobic culture can be fast, a significant effect of reactor hydrodynamics, i.e., mixing, on the overall performance can be expected. This was confirmed experimentally, and the parameter beta proved to be a useful reactor design criterium for the microaerobic cultivation. (c) 1992 John Wiley & Sons, Inc.