Detection and characterization of an ovine placental lactogen stable intermediate in the urea-induced unfolding process

The urea-induced equilibrium unfolding of ovine placental lactogen, purified from ovine placenta, was followed by size-exclusion chromatography, far-UV CD, and intrinsic tryptophan fluorescence. The data obtained by each of these methods showed a poor fit to a two-state model involving only a native and an unfolded form. A satisfactory fit required, instead, a model that involved a stable, partially folded form in addition to the native and unfolded ones. The results obtained from the best-fitting theoretical curves for the three-state model indicated that this intermediate state, which is the predominant species in solution at 3.6 M of urea activity, is compact, largely alpha-helical, and changes considerably the native-like tertiary packing around its tryptophan residues. These findings suggest that this stable intermediate exhibits properties similar to those that characterize the molten globule state.     

Chimeras of human lysozyme and alpha-lactalbumin: an interesting tool for studying partially folded states during protein folding

Protein folding is an extremely active field of research where biology, chemistry, computer science and physics meet. Although the study of protein-folding intermediates in general and equilibrium intermediates in particular has grown considerably in recent years, many questions regarding the conformational state and the structural features of the various partially folded intermediate states remain unanswered. Performing kinetic measurements on proteins that have had their structures modified by site-directed mutagenesis, the so-called protein-engineering method, is an obvious way to gain fine structural information. In the present review, this method has been applied to a variety of proteins belonging to the lysozyme/alpha-lactalbumin family. Besides recombinants obtained by point mutations of individual critical residues, chimeric proteins in which whole structural elements (10-25 residues) from alpha-lactalbumin were inserted into a human lysozyme matrix are examined. The conformational properties of the equilibrium intermediate states are discussed together with the structural characterization of the partially unfolded states encountered in the kinetic folding pathway.     

Touring the landscapes: partially folded proteins examined by hydrogen exchange

Recent studies on Escherichia coli ribonuclease H and several other proteins reveal a specific region in each protein that remains structured in partially folded conformations. These regions play a dominant role in determining the fold and stability of the protein.     

Kinetic and spectroscopic characterization of an intermediate peroxynitrite complex in the nitrogen monoxide induced oxidation of oxyhemoglobin

A rare case of absence of the azygos vein associated with double superior vena cava is presented. Imaging findings on plain chest film and on contrast-enhanced computed tomography are described, and the embryology of azygos and hemiazygos veins is reviewed.     

Kinetic and spectroscopic characterization of an intermediate peroxynitrite complex in the nitrogen monoxide induced oxidation of oxyhemoglobin

Two forms of the gamma subunit of G protein were purified from bovine lung, and were identified as gamma10 and gamma11 by analyses of partial amino acid sequences and reactivity with specific antibodies. The N-terminal amino acid residue of gamma11 was an unmodified Pro2, and the purified gamma11 was freed from beta even under non-denaturing conditions. Western blots with specific antibodies against gamma10 and gamma11 showed that both gamma subunits are present in a variety of tissues in the rat, with a particular abundance of gamma11 in the platelets.     

Characterization of folded, intermediate, and unfolded states of recombinant human interstitial collagenase

Recombinant interstitial collagenase (rMMP-1) forms insoluble inclusion bodies when over-expressed in Escherichia coli. We surveyed conditions for renaturation of purified rMMP-1 in 6 M guandine hydrochloride (GdnHCl) and found that optimal folding occurred when the denatured protein was diluted at 4 degrees C in approximately 2 M guanidine HCl, 20% glycerol, 2.5 mM reduced and oxidized glutathione, and 5 mM CaCl2, followed by buffer exchange to remove denaturant and thiols. The circular dichroism spectrum and catalytic constants of the refolded enzyme were similar to those of native MMP-1. The propeptide, which comprises approximately 20% of the mass of proMMP-1, was not required for folding to a functional enzyme. Size exclusion chromatography and spectroscopic measurements at intermediate [GdnHCl] revealed two intermediate folding states. The first, observed at 1 M GdnHCl, had a slightly larger Stokes' radius than the folded protein. CD and fluorescence analysis showed that it contained ordered tryptophan residues with a higher quantum yield than the fully folded state. The second intermediate, which appeared between 2 and 4 M GdnHCl, exhibited properties consistent with the molten globule, including secondary structure, lack of ordered tryptophan, exposed hydrophobic binding sites, and a Stokes' radius between that of the folded and unfolded states.     

X-ray diffraction analysis of scrapie prion: intermediate and folded structures in a peptide containing two putative alpha-helices

Small proteinaceous infectious particles called prions cause certain neurodegenerative diseases in human and animals. Limited proteolysis of infectious scrapie prions PrP(Sc) yields an N-truncated polypeptide termed PrP 27-30, which encompasses residues 90 to 231 of PrP(Sc) and which assembles into 100 to 200 A wide amyloid rods. It has been hypothesized that the infectious prion is converted from its non-infectious cellular form (PrP(C)) by means of an alpha-helical to beta-sheet conformational change. Secondary structure analysis, computer modeling, and structural biophysics methods support this hypothesis. Residues 90 to 145 of PrP, which contain two putative alpha-helical domains H1 and H2, may be of particular relevance to the disease pathogenesis, as C-terminal truncation at residue 145 was found in a patient with an inherited prion disease. Moreover, our recent X-ray diffraction analysis suggests that the peptide consisting of these residues (designated SHa 90-145) closely models the amyloidogenic beta-sheet core of PrP. In the current study, we have analyzed in detail the X-ray diffraction patterns of SHa 90-145. Two samples were examined: one that was dehydrated under ambient conditions whilst in an external magnetic field (to induce fibril orientation), and another that was sealed after partial drying. The dried, magnetically oriented sample showed a cross-beta diffraction pattern in which the fiber axis (rotation axis) was parallel to the H-bonding direction of the beta-sheets. The major wide-angle peaks indicate the presence of approximately 40 A wide beta-crystallites, which constitute the protofilament. Each crystallite is composed of several orthogonal unit cells, normal to the fiber (a-axis) direction, having lattice constants a = 9.69 A, b = 6.54 A, and c = 18.06 A. Electron density maps were calculated by iterative Fourier synthesis using beta-silk as an initial phase model. The distribution of density indicated that there were two types of beta-sheet, suggesting that larger and smaller side-chains localized to different sheets. This would arise from folding of the polypeptide in which there are turns in the middle of both the H1 and H2 domains. A monoclinic macrolattice, with a = 9.61 A, b = c = 52.99 A and alpha = 114.6 degrees, was found to index all the reflections, including those in the low-angle region. This suggests that the beta-crystallites are nearly hexagonally packed. To account for the approximately 100 A wide fibers visualized by negative staining in the electron microscope, the beta-crystallites would be arranged in 4-mers. The partially dried sample showed a sharp 4.7 A reflection (from H-bonding) and five broad peaks superimposed on monotonically decreasing diffuse scattering. This solution-like scattering was modeled by an anisometric rectangle with a thickness comparable to a singe beta-chain. The structure, which occurred during dehydration, could be a transient in the alpha-helical to beta-sheet conversion, suggesting that formation of hydrogen bonding precedes the inter-sheet interaction and assembly into the amyloid of scrapie prion.     

Interaction of GroEL with a highly structured folding intermediate: iterative binding cycles do not involve unfolding

The GroE proteins are molecular chaperones involved in protein folding. The general mechanism by which they facilitate folding is still enigmatic. One of the central open questions is the conformation of the GroEL-bound nonnative protein. Several suggestions have been made concerning the folding stage at which a protein can interact with GroEL. Furthermore, the possibility exists that binding of the nonnative protein to GroEL results in its unfolding. We have addressed these issues that are basic for understanding the GroE-mediated folding cycle by using folding intermediates of an Fab antibody fragment as molecular probes to define the binding properties of GroEL. We show that, in addition to binding to an early folding intermediate, GroEL is able to recognize and interact with a late quaternary-structured folding intermediate (Dc) without measurably unfolding it. Thus, the prerequisite for binding is not a certain folding stage of a nonnative protein. In contrast, general surface properties of nonnative proteins seem to be crucial for binding. Furthermore, unfolding of a highly structured intermediate does not necessarily occur upon binding to GroEL. Folding of Dc in the presence of GroEL and ATP involves cycles of binding and release. Because in this system no off-pathway reactions or kinetic traps are involved, a quantitative analysis of the reactivation kinetics observed is possible. Our results indicate that the association reaction of Dc and GroEL in the presence of ATP is rather slow, whereas in the absence of ATP association is several orders of magnitude more efficient. Therefore, it seems that ATP functions by inhibiting reassociation rather than promoting release of the bound substrate.     

Components of the psychotherapy relationship: Their interaction and unfolding during treatment.

C. J. Gelso and J. A. Carter (see record 1986-09708-001) suggested that all counseling and psychotherapy relationships, regardless of theoretical orientation, consist of 3 components: a working alliance, a transference configuration (including therapist countertransference), and a real relationship. Drawing on theoretical and research literature and using clinical examples, this article offers 19 propositions about how these 3 relationship components interact with one another, how each operates across the course of psychotherapy, and how they affect the treatment in both brief and longer term therapies. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

In vitro enzyme activation and folded stability of Pseudomonas aeruginosa exotoxin A and its C-terminal peptide

Pseudomonas aeruginosa exotoxin A(ETA) and its C-terminal, enzymatically active fragment (PE40, 375 residues) were studied by high-performance size-exclusion chromatography, steady-state and stopped-flow fluorescence spectroscopy, and circular dichroism spectroscopy. Both proteins have been overexpressed and purified by high-performance liquid chromatography. The effect of various activation conditions (pH, urea, and DTT) on enzymatic activity was studied. Upon enzymatic activation, structural changes induced within both proteins' structures were monitored, and these changes were correlated with concomitant alterations in the catalytic activity of the proteins. The pH optimum of enzymatic activity for both ETA and PE40 was between 7.0 and 8.0, decreasing to nearly zero at acidic (pH 5.0) and basic (pH 11-12) values. Analysis of the pH titration data revealed the presence of two distinct pKa values which implicate a His residue(s) (likely His-440 and -426) and a Tyr or Lys residue (possibly Tyr-481). The identity and possible role of an active site Lys residue is not known. Additionally, a significant increase in the Stokes radii of both proteins was detected when the pH was lowered from 8.0 to 6.0. The enzymatic activity of PE40 was not affected by urea or DTT, and its Stokes radius decreased monotonically with increasing urea concentration in the presence of DTT. In contrast, the enzymatic activity of ETA peaked when the protein was preincubated with 4.0 M urea, and this coincided with a large transition (increase) in the protein's Stokes radius between 3 and 5 M urea. Furthermore, loss of helical secondary structure of both PE40 and ETA commenced at approximately 2 M urea and progressively diminished at higher denaturant concentrations. The unfolding of both proteins in urea (and DTT) was reversible, and the free energies of unfolding were determined by both circular dichroism and fluorescence spectroscopy and were found to be 13.7 +/- 2.9 and 9.8 +/- 3.4 kJ/mol, respectively, for ETA and were 17.8 +/- 6.8 and 7.5 +/- 3.6 kJ/mol, respectively, for PE40. The refolding rate of PE40 was relatively rapid [t 1/2(1) = 27 s, t 1/2(2) = 624 s], which was in stark contrast to the refolding rate of ETA (t 1/2 = several hours). The relative refolding rates of PE40 and ETA help to explain the mechanism of in vitro enzyme activation and assay.     

中厚板中间坯冷却过程中晶粒长大及控制方法

为控制中厚板中间坯长时间待温导致的晶粒长大,研究了中间强制水冷却对奥氏体组织的影响.通过对Q345B钢和含Nb-Ti钢采用1050℃变形后快冷至1050~950℃预定温度保温的热模拟方法,确定了中间坯冷却过程中的晶粒尺寸变化规律,提出了中厚板冷却过程中晶粒长大的控制方法,建立了Q345B钢和含Nb-Ti钢在中间冷却过程中的晶粒长大模型.在中间冷却过程中,Q345B钢晶粒稳定性较差,而含Nb-Ti钢晶粒稳定性良好,归因于以铌为主的析出相对奥氏体晶界的钉扎作用.中间坯的强制冷却可控制奥氏体晶粒长大,63mm厚中间坯强制冷却可有效减小平均晶粒尺寸约20μm.在实际生产中,经中间强制冷却后16 mm厚度Q345B钢板的冲击韧性提高25%~70%.     

IR spectroscopic characterization of 2-thiohydantoins and 2-thiobarbiturates

A characterization of 2-thiohydantoins and 2-thiobarbiturates by IR spectra is proposed, using three characteristic group frequencies: the "thioureide band" around 1500 cm-1 and the antisymmetric-symmetric stretching modes of NCS bonds around 1400 and 1200 cm-1. The general characteristic absorption areas are found by comparison with N-phenylthioureas and thioanilides.     

Dissecting the structure of a partially folded protein. Circular dichroism and nuclear magnetic resonance studies of peptides from ubiquitin

The nature and interaction of structural elements in a partially ordered form of ubiquitin, the A-state, which is populated at low pH in 40 to 60% aqueous methanol, have been investigated. Two synthetic peptides have been studied under the same conditions: U(1-21), corresponding to the N-terminal beta-hairpin in the native (N) and A-states of ubiquitin and U(1-35), which includes this hairpin plus an alpha-helix. Circular dichroism studies indicate that, although these peptides are largely unfolded in water, their structural content in 30 and 60% methanol is comparable with the corresponding native secondary structure. Sequence-specific assignments of the 1H n.m.r. spectra of U(1-35) in aqueous methanol and subsequent secondary structure determination confirm the conservation in detail of native-like secondary structure. Corresponding resonances in spectra of U(1-35), U(1-21) and the A-state itself were found to have closely similar chemical shifts, suggesting that the beta-hairpin exists independently in the partially folded protein, with little or no influence from the rest of the molecule. This is confirmed by the virtual absence in nuclear Overhauser enhancement spectroscopy and rotating frame nuclear Overhauser enhancement spectroscopy spectra of nuclear Overhauser enhancement effects between structural elements. c.d. and n.m.r. evidence suggests that structure in the C-terminal half of the molecule in the A-state is largely non-native. Thus, although methanol is necessary to assure its stability in the absence of wider native interactions, the structure of the beta-hairpin, including the register of its hydrogen bonding, appears to be determined entirely by its own sequence. This intrinsic structural preference in the first part of the ubiquitin sequence is much stronger than in the C-terminal half, a conclusion reflected in the results from a variety of secondary structure prediction algorithms.     

Role of the colchicine ring A and its methoxy groups in the binding to tubulin and microtubule inhibition

The roles of the methoxy substituents on ring A of two ring colchicine (COL) analogues were probed by the synthesis of a number of drugs and the examination of their effect on binding to tubulin, inhibition of microtubule assembly, and induction of GTPase activity. Selective elimination of ring A methoxy groups at positions 2, 3, and 4 weakened all three processes. The effects on binding and inhibition were independent of the nature of ring C (or C'). Specifically, excision of the 2- or 3-methoxy groups weakened binding by ca. 0.4 kcal mol-1, while that of the 4-methoxy group of ring A was weakened by 1.36 +/- 0.15 kcal mol-1. The effect on the inhibition of microtubule assembly, expressed as the equilibrium constant for the binding of the tubulin-drug complex to the end of a microtubule, was more complex and strongly dependent on the nature of ring C (or C'). This was attributed to the abilities of various groups on ring C' to overcome the wobbling in the tubulin-drug complex introduced by the weakening of the anchoring provided by ring A. It is concluded that ring A of COL is not germane to the mechanism of the inhibition of tubulin self-assembly. It serves only as a complex-stabilizing anchor. The control of this process resides in the interactions that key oxygen atoms of ring C of COL or C' of structural analogues establish with the protein. It is proposed that the 4-methoxy group of ring A serves as a key attachment point for immobilization of the drugs on the protein.     

Presenilins: detection and characterization of Alzheimer''s disease genes]

Melatonin's timekeeping function is undoubtedly related to the fact that it is primarily produced during nighttime darkness; that is, melatonin and light occur at opposite times. The human phase response curve (PRC) to melatonin appears to be about 12h out of phase with the PRC to light. These striking complementarities, together with light's acute suppressant effect on melatonin production, suggest that a function for endogenous melatonin is to augment entrainment of the circadian pacemaker by the light-dark cycle. The melatonin PRC also indicates correct administration times for using exogenous melatonin to treat circadian phase disorders.     

X-ray analysis and spectroscopic characterization of M121Q azurin. A copper site model for stellacyanin

The dependence of the properties of the azurin blue copper site on the nature of the axial ligand at position 121 was tested by site-directed mutagenesis. This residue was substituted for a glutamine, the purported fourth copper ligand in the related protein stellacyanin. M121Q azurin was isolated and purified from Escherichia coli and characterized by spectroscopic methods. The mutant copper site has the ultra-violet-vis and electron paramagnetic resonance (EPR) characteristics of a type I site, but the spectroscopic details differ significantly from wild-type (wt) azurin. The X and S-band EPR spectra of M121Q azurin can be well stimulated with the parameters for stellacyanin, indicating that the copper sites of both proteins in the oxidized state are similar. The midpoint potential of M121Q is 263 mV, 25 mV lower than for wt azurin. The reactivity of the mutant was probed by measuring the electron self exchange rate by nuclear magnetic resonance spectroscopy. The rate was 8 x 10(3) mol-1 s-1, almost two orders of magnitude lower than the value for wt azurin (5 x 10(5) mol-1 s-1). Detailed structural information on the M121Q Cu(II)-site was obtained by X-ray analysis of M121Q azurin crystals at 1.9 A resolution. The histidine and cysteine copper ligand distances and angles in the equatorial plane around the copper are very similar to the wt protein. Gln121 is co-ordinated in a monodentate fashion via its side-chain oxygen atom at a distance of 2.26 A. The distance between copper and the carbonyl group of Gly45 is increased from 3.13 A (wt) to 3.37 A resulting in a distorted tetrahedral N2SO copper co-ordination. The possible significance of these results for the structure of the copper site of stellacyanin, the only small blue copper protein lacking a methionine ligand, is discussed. Conformational changes with respect to the wt azurin are seen in some of the connecting loops between secondary structure elements, in the mutation site and in the beta-strand 2a. The side-chains involved in the hydrophobic patch surrounding His117 are subject to large changes in their conformations. In contrast to wt azurin, the copper site in M121Q azurin undergoes significant structural changes on reduction.(ABSTRACT TRUNCATED AT 400 WORDS)     

钙处理中间产物的形成及其对氧化铝夹杂的改性

通过观测钙处理前后夹杂物形貌和成分的变化,对钙处理效果、中间产物的形成及不同中间产物对氧化铝夹杂的改性路径进行了研究.结果表明,钙处理可将钢液中不规则固态夹杂改性为球形液态夹杂,并且各炉次夹杂物的改性程度不同.热力学分析表明,Als和S含量越高,氧化铝夹杂改性为液态的难度越大.钙处理后的短时间内,Ca S和CaO作为中间产物存在.通过建立中间产物生成的动力学模型,确定了生成不同中间产物的临界硫质量分数为11.1×10^-6（钢液中溶解氧质量分数为4×10^-6）.由该模型结合结果分析,推断出不同中间产物对氧化铝的改性路径.     

选矿过程自动检测与自动化综述

介绍了碎矿、磨矿、浮选等选矿过程中自动控制的研究现状,以及选矿自动化目前存在的主要问题和发展趋势.     

Expression of prolactin gene in mouse placenta during late pregnancy: detection of mRNA and its translation product

Threshold photoelectron spectra of N2+ were measured between 23.4 and 27.6 eV with high resolution and high intensity by using the penetrating field technique and synchrotron radiation. Five vibrational progressions were observed. The first of these progressions was the C2Sigmau+ state. The second progression was identified as the transition to the second state of 2Pig symmetry found by P. Baltzer, M. Larsson, L. Karlsson, B. Wannberg, and M. Carlsson (1992. Phys. Rev. A 46, 5545). The third progression, which was discovered by F. Merkt and P. M. Guyon (1993. J. Chem. Phys. 99, 3400), can be designated as the 2Sigmau- state by comparison with previous theoretical study (E. W. Thulstrup and A. Andersen, 1975. J. Phys. B 8, 965). The fourth and fifth progressions were designated as the 2Deltau and 2(2) Piu states by similar comparison with previous theories. Copyright 1997 Academic Press. Copyright 1997Academic Press     

Identification and characterization of the unfolding transition state of chymotrypsin inhibitor 2 by molecular dynamics simulations

Temperature-induced unfolding of chymotrypsin inhibitor 2 (CI2) in water has been investigated using molecular dynamics simulations. One simulation (2.2 ns) has been analyzed in detail and three additional simulations (each > or = 1 ns) were performed to check the generality of the results. Concurrent loss of secondary and tertiary structure during unfolding was observed in all the simulations. For each simulation, the major transition state of unfolding was identified based on conformational analysis of protein structures along the unfolding trajectory. The transition state has a considerably weakened hydrophobic core and disrupted secondary structure. Nevertheless, the overall structure of the transition state is closer to the native state than to the unfolded state. The disruption of the hydrophobic core appears to be rate limiting. However, other energy barriers have to be overcome before reaching the major transition state. A method is described to quantitatively compare the structure of the simulated transition state with that characterized by protein engineering experiments. Good agreement with the experimental data is obtained for all four transition state models (the correlation coefficient R = 0.80 to 0.93) and the average over all four models gives the best correlation (R = 0.94). These simulations provide the first comprehensive atomic-level view of what the unfolding transition state of C12 may look like.