MgATP binding to the nucleotide-binding domains of the eukaryotic cytoplasmic chaperonin induces conformational changes in the putative substrate-binding domains

The eukaryotic cytosolic chaperonins are large heterooligomeric complexes with a cylindrical shape, resembling that of the homooligomeric bacterial counterpart, GroEL. In analogy to GroEL, changes in shape of the cytosolic chaperonin have been detected in the presence of MgATP using electron microscopy but, in contrast to the nucleotide-induced conformational changes in GroEL, no details are available about the specific nature of these changes. The present study identifies the structural regions of the cytosolic chaperonin that undergo conformational changes when MgATP binds to the nucleotide binding domains. It is shown that limited proteolysis with trypsin in the absence of MgATP cleaves each of the eight subunits approximately in half, generating two fragments of approximately 30 kDa. Using mass spectrometry (MS) and N-terminal sequence analysis, the cleavage is found to occur in a narrow span of the amino acid sequence, corresponding to the peptide binding regions of GroEL and to the helical protrusion, recently identified in the structure of the substrate binding domain of the archeal group II chaperonin. This proteolytic cleavage is prevented by MgATP but not by ATP in the absence of magnesium, ATP analogs (MgATPyS and MgAMP-PNP) or MgADP. These results suggest that, in analogy to GroEL, binding of MgATP to the nucleotide binding domains of the cytosolic chaperonin induces long range conformational changes in the polypeptide binding domains. It is postulated that despite their different subunit composition and substrate specificity, group I and group II chaperonins may share similar, functionally-important, conformational changes. Additional conformational changes are likely to involve a flexible helix-loop-helix motif, which is characteristic for all group II chaperonins.     

Cloning and characterization of the polyhydroxybutyrate depolymerase gene of Pseudomonas stutzeri and analysis of the function of substrate-binding domains

BACKGROUND: Overexpression of the 70 kDa heat shock protein (Hsp70) protects myocytes and neural cells from hypoxic injury. In contrast, Hsp70 induction in the kidney after ischemic or thermal preconditioning does not correlate well with protection from hypoxic injury. Herein, we directly tested if Hsp70 overexpression protects LLC-PK1 porcine tubular epithelial cells from hypoxic or thermal injury. METHODS: LLC-PK1 cells were either cotransfected with an Hsp70 and a luciferase expression vector or singly transfected with the luciferase expression vector. Loss of intracellular luciferase activity was used to assess injury after exposure to hypoxia or hyperthermia and after recovery under normal growth conditions. RESULTS: Overexpression of Hsp70 decreased loss of and improved restoration of intracellular luciferase activity in LLC-PK1 cells exposed to hyperthermia. In contrast, Hsp70 overexpression did not decrease the loss of or improve restoration of luciferase activity in cells exposed to hypoxia. CONCLUSIONS: These results suggest that Hsp70 overexpression is sufficient to protect LLC-PK1 proximal tubular cells from hyperthermia but is not sufficient for protection from hypoxia.     

The substrate-binding site in the lactose permease of Escherichia coli

Site-directed N-ethylmaleimide labeling was studied with Glu-126 and/or Arg-144 mutants in lactose permease containing a single, native Cys residue at position 148 in the substrate-binding site. Replacement of either Glu-126 or Arg-144 with Ala markedly decreases Cys-148 reactivity, whereas interchanging the residues, double-Ala replacement, or replacement of Arg-144 with Lys or His does not alter reactivity, indicating that Glu-126 and Arg-144 are charge-paired. Importantly, although alkylation of Cys-148 is blocked by ligand in wild-type permease, no protection whatsoever is observed with any of the Glu-126 or Arg-144 mutants. Site-directed fluorescence with 2-(4-maleimidoanilino)-naphthalene-6-sulfonic acid (MIANS) in mutant Val-331 --> Cys was also studied. In marked contrast to Val-331 --> Cys permease, ligand does not alter MIANS reactivity in mutant Glu-126 --> Ala/Val-331 --> Cys, Arg-144 --> Ala/Val-331 --> Cys, or Arg-144 --> Lys/Val-331 --> Cys and does not cause either quenching or a shift in the emission maximum of the MIANS-labeled mutants. However, mutation Glu-126 --> Ala or Arg-144 --> Ala and, to a lesser extent, Arg-144 --> Lys cause a red-shift in the emission spectrum and render the fluorophore more accessible to I-. The results demonstrate that Glu-126 and Arg-144 are irreplaceable for substrate binding and suggest a model for the substrate-binding site in the permease. In addition, the findings are consistent with the notion that alterations in the substrate translocation pathway at the interface between helices IV and V are transmitted conformationally to the H+ translocation pathway at the interface between helices IX and X.     

Pig heart fumarase contains two distinct substrate-binding sites differing in affinity

A eukaryotic fumarase is for the first time unequivocally shown to contain two distinct substrate-binding sites. Pig heart fumarase is a tetrameric enzyme consisting of four identical subunits of 50 kDa each. Besides the true substrates L-malate and fumarate, the active sites (sites A) also bind their analogs D-malate and oxaloacetate, as well as the competitive inhibitor glycine. The additional binding sites (sites B) on the other hand also bind the substrates and their analogs D-malate and oxaloacetate, as well as L-aspartate which is not an inhibitor. Depending on the pH, the affinity of sites B for ligands (Kd being in the millimolar range) is 1-2 orders of magnitude lower than the affinity of sites A (of which Kd is in the micromolar range). However, saturating sites B results in an increase in the overall activity of the enzyme. The benzenetetracarboxyl compound pyromellitic acid displays very special properties. One molecule of this ligand is indeed able to bind into a site A and a site B at the same time. Four molecules of pyromellitic acid were found to bind per molecule fumarase, and the affinity of the enzyme for this ligand is very high (Kd = 0.6 to 2.2 microM, depending on the pH). Experiments with this ligand turned out to be crucial in order to explain the results obtained. An essential tyrosine residue is found to be located in site A, whereas an essential methionine residue resides in or near site B. Upon limited proteolysis, a peptide of about 4 kDa is initially removed, probably at the C-terminal side; this degradation results in inactivation of the enzyme. Small local conformational changes in the enzyme are picked up by circular dichroism measurements in the near-UV region. This spectrum is built up of two tryptophanyl triplets, the first one of which is modified upon saturating the active sites (A), and the second one upon saturating the low affinity binding sites (B).     

Further characterization and purification of the flavin-dependent S-benzyl-L-cysteine S-oxidase activities of rat liver and kidney microsomes

Previously, we provided evidence that cysteine conjugate S-oxidase (S-oxidase) activities of rat liver and kidney microsomes may be associated with flavin-containing monooxygenases (FMOs). In this study, the biochemical properties of these activities were further investigated. When NADPH was replaced by NADH, the S-oxidase activities were reduced significantly. Removal of the flavin moiety from microsomes significantly reduced the S-oxidase activities; however, addition of exogenous FAD or FMN restored the activities of the flavin-depleted microsomes. Solubilization of hepatic or renal microsomes with Emulgen 911, Nonidet P-40, Triton X-100, or 3-[(3-cholamidopropyl)dimethyl-ammonio]-1- propane sulfate or inclusion of the sulfhydryl-reactive agents Hg2+, N-ethylmaleimide, or iodoacetamide did not affect the S-oxidase activities, whereas solubilization of either hepatic or renal microsomes by cholate or heating of renal microsomes in the absence of NADPH significantly reduced the S-oxidase activities. In addition to male rat hepatic and renal microsomes, the S-oxidase activities were detected in lung microsomes of male rats and hepatic and renal microsomes of male mice and female rats and rabbits. The male rat kidney maintained the highest S-oxidase activity of all species and tissues examined. Whereas the aforementioned results provided further evidence for the S-oxidase activities being associated with FMOs, unambiguous evidence for this hypothesis was provided by the purification of the activities from rat liver (580-fold) and kidney (700-fold) microsomes and by the use of the isolated proteins in polyacrylamide gel electrophoresis, flavin content determinations, amino-terminal amino acid sequence analysis, amino acid composition analysis, and substrate kinetic studies. The findings that the S-oxidases were immunoreactive with antibodies raised against the pig liver 1A1 isozyme but not with antibodies raised against the rabbit lung 1B1 isozyme and that the liver S-oxidase amino-terminal amino acid sequence was more comparable to the amino-terminal amino acid sequences of pig and rabbit liver 1A1 isozymes than to those of rabbit lung 1B1 and liver 1D1 isozymes provide evidence that the S-oxidases are related to the known FMO 1A1 isozymes.     

Effects of subsite alterations on substrate-binding mode in the active site of hen egg-white lysozyme

The primary sequence of the low-molecular-mass cadmium-binding protein metalloprotein II of Nereis diversicolor (Hediste diversicolor, recent denomination) has been determined. This protein is composed of 119 amino acids and has 80.8% identity with the N. diversicolor myohemerythrin [Takagi, T. & Cox, J. A. (1991) FEBS Lett. 285, 25-27]. The fact that iron, which normally binds to myohemerythrin, is not found to be associated with the cadmium-binding protein metalloprotein II in cadmium-exposed animals could be the result of the complete abolition of the iron-binding capacity of the protein due to the binding of cadmium.     

Structure and function of the xenobiotic substrate-binding site and location of a potential non-substrate-binding site in a class pi glutathione S-transferase

Complex structures of a naturally occurring variant of human class pi glutathione S-transferase 1-1 (hGSTP1-1) with either S-hexylglutathione or (9R,10R)-9-(S-glutathionyl)-10-hydroxy-9, 10-dihydrophenanthrene [(9R,10R)-GSPhen] have been determined at resolutions of 1.8 and 1.9 A, respectively. The crystal structures reveal that the xenobiotic substrate-binding site (H-site) is located at a position similar to that observed in class mu GST 1-1 from rat liver (rGSTM1-1). In rGSTM1-1, the H-site is a hydrophobic cavity defined by the side chains of Y6, W7, V9, L12, I111, Y115, F208, and S209. In hGSTP1-1, the cavity is approximately half hydrophobic and half hydrophilic and is defined by the side chains of Y7, F8, V10, R13, V104, Y108, N204, and G205 and five water molecules. A hydrogen bond network connects the five water molecules and the side chains of R13 and N204. V104 is positioned such that the introduction of a methyl group (the result of the V104I mutation) disturbs the H-site water structure and alters the substrate-binding properties of the isozyme. The hydroxyl group of Y7 forms a hydrogen bond (3.2 A) with the sulfur atom of the product. There is a short hydrogen bond (2.5 A) between Y108 (OH) and (9R, 10R)-GSPhen (O5), indicating the hydroxyl group of Y108 as an electrophilic participant in the addition of glutathione to epoxides. An N-(2-hydroxethyl)piperazine-N'-2-ethanesulfonic acid (HEPES) molecule is found in the cavity between beta2 and alphaI. The location and properties of this HEPES-binding site fit a possible non-substrate-binding site that is involved in noncompetitive inhibition of the enzyme.     

Orientation domains and texture in hot-dipped galvanized coatings

The crystallographic orientation of galvanized coatings (Zn-0.2Al-0.15Sb in wt pct) has been characterized by Electron-Backscattered Diffraction (EBSD) and optical microscopy. While 80 pct of the nucleation spots in the coating give rise to single-crystal Zn grains, it has been found that about 20 pct of them give rise to two or more orientation domains, each having a specific crystallographic orientation. For such “polycrystalline Zn grains,” the orientations of the domains are crystallographically related: they have a dense crystallographic direction (〈1010〉, 〈1120〉, or 〈0001〉) in common. Moreover, the crystallographic relationships are similar to those observed in snowflakes and can be partially explained by the concept of a coincidence-site lattice (CSL). The EBSD measurements were also used in order to measure quantitatively the crystallographic texture. In particular, it has been evidenced that the (0001) texture of galvanized coatings is the result of two contributions: (1) the nuclei are preferentially oriented with the basal plane parallel to the coating plane (33 pct of the grains have an angle between the basal plane and the coating plane smaller than 22.5 deg), and (2) the grains having the basal plane parallel to the coating plane grow faster (these grains represent 43 pct of the coating surface). This reinforcement of the texture during growth is in agreement with that predicted by growth models, which take into account the effect of the interfaces.     

Structured task analysis in complex domains

This paper describes the application of MUSE, a human factors structured design method, to the design of systems supporting the work of air traffic controllers at European airport control towers. The paper illustrates the derivation of a generalized task model starting from the analyses performed at four airports and the construction of the composite task model of the future system. Other important concerns of the work are the harmonization of the different tools in the airport control tower and the assessment of the impact of these tools on the human operators. The potentialities and limitations of applying the method are discussed in particular with respect to the complexity of the domain and the nonstandard features of the air traffic control as a MUSE application. Indeed, air traffic management is widely acknowledged as a complex domain to design for, and the documented application of human factors structured methods to design are all referred to the modelling of worksystems (as opposed to working positions or workstations) with a single operator. In the present application one has multiple operators and multiple design goals (design, harmonization, assessment). The work presented is a contribution to the extension and better definition of structured methods and their applicability to the analysis of complex domains.     

Constraints on learning in nonprivileged domains

Constraints on learning, rather than being unique to evolutionarily privileged domains, may operate in nonprivileged domains as well. Understanding of the goals that strategies must meet seems to play an especially important role in these domains in constraining the strategies that are generated and in allowing children to evaluate strategies even before they use them. The present experiments showed that children can use their conceptual understanding to accurately evaluate strategies that they not only do not yet use but that are more conceptually advanced than the strategies they do use. In Experiment 1, 5-year-olds who did not yet use the min strategy for adding numbers judged it to be smarter than an equally novel illegitimate strategy, and to be just as smart as their typical strategy of counting from one. In Experiment 2, 9-year-olds who did not yet use the forking strategy to play tic-tac-toe judged it to be even smarter than their own win/block approach. The results demonstrated a large number of commonalities between the functioning of constraints in privileged and nonprivileged domains, as well as suggesting some possible differences.     

Personality and thinking style in different creative domains.

The crucial aspect of creativity in both personality and thinking style may be the ability or tendency to change within personality traits, such as, for example, moving between extraversion and introversion, and within thinking styles, such as moving between heuristic and algorithmic thinking. Such mobility is characteristic of the “complex” personality. On personality and thinking style tests, complexity would be expected to manifest itself in greater variability of responses to items measuring the same overall trait. This issue was investigated with 158 visual art, 136 music, and 309 psychology students. Art students (visual art and music students) showed greater complexity in conscientiousness than psychology and music students, respectively. Visual art students further showed a greater overall complexity (mean complexities across personality and thinking style) than psychology students did. A more traditional analysis revealed that visual art students were more neurotic, more open to experience and more inclined to heuristic thinking than psychology students do, whereas music students were more extraverted and more agreeable than visual art students were, and more inclined to heuristic thinking than psychology students were. Thus, it was possible to distinguish visual art students from music and psychology students by their personality and thinking style. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Identification of the aspartic acid residue located at or near substrate-binding site of rye seed chitinase-c

Two children with acquired immunodeficiency syndrome (AIDS) and progressive encephalopathy underwent MR spectroscopy before and after antiretroviral therapy. Initial MR spectroscopy of the basal ganglia region showed decreased N-acetylaspartate (NAA)/creatine (Cr) and a lactate peak. After therapy, there was improvement in NAA/Cr and an absence of the abnormal lactate peak. We suggest that decreased NAA/Cr in AIDS is reversible, that brain lactate might correlate with inflammation, and that MR spectroscopy can be useful in treatment trials.     

The SWI/SNF complex creates loop domains in DNA and polynucleosome arrays and can disrupt DNA-histone contacts within these domains

To understand the mechanisms by which the chromatin-remodeling SWI/SNF complex interacts with DNA and alters nucleosome organization, we have imaged the SWI/SNF complex with both naked DNA and nucleosomal arrays by using energy-filtered microscopy. By making ATP-independent contacts with DNA at multiple sites on its surface, SWI/SNF creates loops, bringing otherwise-distant sites into close proximity. In the presence of ATP, SWI/SNF action leads to the disruption of nucleosomes within domains that appear to be topologically constrained by the complex. The data indicate that the action of one SWI/SNF complex on an array of nucleosomes can lead to the formation of a region where multiple nucleosomes are disrupted. Importantly, nucleosome disruption by SWI/SNF results in a loss of DNA content from the nucleosomes. This indicates a mechanism by which SWI/SNF unwraps part of the nucleosomal DNA.     

Electrostatic properties deduced from refined structures of NADH-cytochrome b5 reductase and the other flavin-dependent reductases: pyridine nucleotide-binding and interaction with an electron-transfer partner

Electrostatic properties on the protein surface were examined on the basis of the crystal structure of NADH-cytochrome b5 reductase refined to a crystallographic R factor of 0.223 at 2.1 A resolution and of the other three flavin-dependent reductases. A structural comparison of NADH-cytochrome b5 reductase with the other flavin-dependent reductases, ferredoxin-NADP+ reductase, phthalate dioxygenase reductase, and nitrate reductase, showed that the alpha/beta structure is the common motif for binding pyridine nucleotide. Although the amino acid residues associated with pyridine nucleotide-binding are not conserved, the electrostatic properties and the location of the pyridine nucleotide-binding pockets of NADH-requiring reductases were similar to each other. The electrostatic potential of the surface near the flavin-protruding side (dimethylbenzene end of the flavin ring) of NADH-cytochrome b5 reductase was positive over a wide area while that of the surface near the heme-binding site of cytochrome b5 was negative. This implied that the flavin-protruding side of NADH-cytochrome b5 reductase is suitable for interacting with its electron-transfer partner, cytochrome b5. This positive potential area is conserved among four flavin-dependent reductases. A comparison of the electron-transfer partners of four flavin-dependent reductases showed that there are significant differences in the distribution of electrostatic potential between inter-molecular and inter-domain electron-transfer reactions.