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Multiple kinetic isotope effects have been measured for the urease-catalyzed hydrolysis of formamide at pH 6.0 and 25 degrees C. These kinetic isotope effects include the carbonyl-C ((13)k = 1.0241 +/- 0.0009), the carbonyl-O ((18)k = 0.9960 +/- 0.0009), the formyl-H ((D)k = 0.95 +/- 0.01), the leaving-N ((15)k= 1.0327 +/- 0.0006), and the nucleophile-O ((18)k = 0.9778 +/- 0.0005). In addition, the enzyme does not catalyze the exchange of oxygen from the solvent into the carbonyl-O of formamide or the product, formate ion. The isotope effects are consistent with the rate-determining collapse of the tetrahedral intermediate (i.e., C-N bond cleavage). The pH optimum for formamide is at pH 5.3, whereas for urea, it is near 8.0. This is best accommodated by the mechanism proposed by Hausinger and Karplus, in which an active site cysteine binds to the nonleaving nitrogen in urea. For urea, the preference is for the anionic form of the sulfhydryl; for formamide, the neutral form is preferred, leading to the lower pH optimum.
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PMID:Multiple isotope effect study of the hydrolysis of formamide by urease from jack bean (Canavalia ensiformis). 1689 94

The colicin immunity protein Im7 folds from its unfolded state in 6 M urea to its native four-helix structure through an on-pathway intermediate that lacks one of the helices of the native structure (helix III). In order to further characterize the folding mechanism of Im7, we have studied the conformational properties of the protein unfolded in 6 M urea in detail using heteronuclear NMR. Triple-resonance experiments with 13C/15N-labelled Im7 in 6 M urea provided almost complete resonance assignments for the backbone nuclei, and measurement of backbone 15N relaxation parameters allowed dynamic ordering of the unfolded polypeptide chain to be investigated. Reduced spectral density mapping and fitting backbone R2 relaxation rates to a polymer dynamics model identified four clusters of interacting residues, each predicted by the average area buried upon folding for each residue. Chemical shift analyses and measurement of NOEs detected with a long mixing-time 1H-1H-15N NOESY-HSQC spectrum confirmed the formation of four clusters. Each cluster of interacting side-chains in urea-unfolded Im7 occurs in a region of the protein that forms a helix in the protein, with the largest clusters being associated with the three long helices that are formed in the on-pathway folding intermediate, whilst the smallest cluster forms a helix only in the native state. NMR studies of a Phe15Ala Im7 variant and a protein in which residues 51-56 are replaced by three glycine residues (H3G3 Im7*), indicated that the clusters do not interact with each other, possibly because they are solvated by urea, as indicated by analysis of NOEs between the protein and the solvent. Based on these data, we suggest that dilution of the chaotrope to initiate refolding will result in collapse of the clusters, leading to the formation of persistent helical structure and the generation of the three-helix folding intermediate.
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PMID:Characterisation of the conformational properties of urea-unfolded Im7: implications for the early stages of protein folding. 1704 7

As an intermediate filament (IF)-based cytolinker protein, plectin plays a key role in the maintenance of cellular cytoarchitecture and serves at the same time as a scaffolding platform for signaling cascades. Consisting of six structural repeats (R1-6) and harboring binding sites for different IF proteins and proteins involved in signaling, the plectin C-terminal domain is of strategic functional importance. Depending on the species, it contains at least 13 cysteines, 4 of which reside in the R5 domain. To investigate the structural and biological functions of R5 cysteines, we used cysteine-to-serine mutagenesis and spectroscopic, biochemical, and functional analyses. Urea-induced unfolding experiments indicated that wild-type R5 in the oxidized, disulfide bond-mediated conformation was more stable than its cysteine-free mutant derivative. The binding affinity of R5 for vimentin was significantly higher, however, when the protein was in the reduced, more relaxed conformation. Of the four R5 cysteines, one (Cys4) was particularly reactive as reflected by its ability to form disulfide bridges with R5 Cys1 and to serve as a target for nitrosylation in vitro. Using immortalized endothelial cell cultures from mice, we show that endogenous plectin is nitrosylated in vivo, and we found that NO donor-induced IF collapse proceeds dramatically faster in plectin-deficient compared with wild-type cells. Our data suggest an antagonistic role of plectin in nitrosylation (oxidative stress)-mediated alterations of IF cytoarchitecture and a possible role of R5 Cys4 as a regulatory switch.
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PMID:Oxidation and nitrosylation of cysteines proximal to the intermediate filament (IF)-binding site of plectin: effects on structure and vimentin binding and involvement in IF collapse. 1722 53

To obtain quantitative information on the size and dynamics of unfolded proteins we combined single-molecule lifetime and intensity FRET measurements with molecular simulations. We compared the unfolded states of the 64-residue, alpha/beta protein L and the 66-residue, all-beta cold-shock protein CspTm. The average radius of gyration (Rg) calculated from FRET data on freely diffusing molecules was identical for the two unfolded proteins at guanidinium chloride concentrations >3 M, and the FRET-derived Rg of protein L agreed well with the Rg previously measured by equilibrium small-angle x-ray scattering. As the denaturant concentration was lowered, the mean FRET efficiency of the unfolded subpopulation increased, signaling collapse of the polypeptide chain, with protein L being slightly more compact than CspTm. A decrease in Rg with decreasing denaturant was also observed in all-atom molecular dynamics calculations in explicit water/urea solvent, and Langevin simulations of a simplified representation of the polypeptide suggest that collapse can result from either increased interresidue attraction or decreased excluded volume. In contrast to both the FRET and simulation results, previous time-resolved small-angle x-ray scattering experiments showed no collapse for protein L. Analysis of the donor fluorescence decay of the unfolded subpopulation of both proteins gives information about the end-to-end chain distribution and suggests that chain dynamics is slow compared with the donor life-time of approximately 2 ns, whereas the bin-size independence of the small excess width above the shot noise for the FRET efficiency distributions may result from incomplete conformational averaging on even the 1-ms time scale.
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PMID:Characterizing the unfolded states of proteins using single-molecule FRET spectroscopy and molecular simulations. 1725 51

Using small-angle X-ray scattering combined with a continuous-flow mixing device, we monitored the microsecond compaction dynamics in the folding of Escherichia coli dihydrofolate reductase, an alpha/beta-type protein. A significant collapse of the radius of gyration from 30 A to 23.2 A occurs within 300 micros after the initiation of refolding by a urea dilution jump. The subsequent folding after the major chain collapse occurs on a considerably longer time-scale. The protein folding trajectories constructed by comparing the development of the compactness and the secondary structure suggest that the specific hydrophobic collapse model rather than the framework model better explains the experimental observations. The folding trajectory of this alpha/beta-type protein is located between those of alpha-helical and beta-sheet proteins, suggesting that native structure determines the folding landscape.
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PMID:Microsecond hydrophobic collapse in the folding of Escherichia coli dihydrofolate reductase, an alpha/beta-type protein. 1733 39

Despite much study, biomolecule folding cooperativity is not well understood. There are quantitative models for helix-coil transitions and for coil-to-globule transitions, but no accurate models yet treat both chain collapse and secondary structure formation together. We develop here a dynamic programming approach to statistical mechanical partition functions of foldamer chain molecules. We call it the ascending levels model. We apply it to helix-coil and helix-bundle folding and cooperativity. For 14- to 50-mer Baldwin peptides, the model gives good predictions for the heat capacity and helicity versus temperature and urea. The model also gives good fits for the denaturation of Oas's three-helix bundle B domain of protein A (F13W*) and synthetic protein alpha3C by temperature and guanidine. The model predicts the conformational distributions. It shows that these proteins fold with transitions that are two-state, although the transitions in the Baldwin helices are nearly higher order. The model shows that the recently developed three-helix bundle polypeptoids of Lee et al. fold anti-cooperatively, with a predicted value of DeltaHvH/DeltaHcal = 0.72. The model also predicts that two-helix bundles are unstable in proteins but stable in peptoids. Our dynamic programming approach provides a general way to explore cooperativity in complex foldable polymers.
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PMID:Statistical mechanics of helix bundles using a dynamic programming approach. 1736 2

The molecular mechanism of the monooxygenase (phenolase) activity of type 3 copper proteins has been examined in detail both in the model systems and in the enzymatic systems. The reaction of a side-on peroxo dicopper(II) model compound ( A) and neutral phenols proceeds via a proton-coupled electron-transfer (PCET) mechanism to generate phenoxyl radical species, which collapse each other to give the corresponding C-C coupling dimer products. In this reaction, a bis(mu-oxo)dicopper(III) complex ( B) generated by O-O bond homolysis of A is suggested to be a real active species. On the other hand, the reaction of lithium phenolates (deprotonated form of phenols) with the same side-on peroxo dicopper(II) complex proceeds via an electrophilic aromatic substitution mechanism to give the oxygenated products (catechols). The mechanistic difference between these two systems has been discussed on the basis of the Marcus theory of electron transfer and Hammett analysis. Mechanistic details of the monooxygenase activity of tyrosinase have also been examined using a simplified enzymatic reaction system to demonstrate that the enzymatic reaction mechanism is virtually the same as that of the model reaction, that is, an electrophilic aromatic substitution mechanism. In addition, the monooxygenase activity of the oxygen carrier protein hemocyanin has been explored for the first time by employing urea as an additive in the reaction system. In this case as well, the ortho-hydroxylation of phenols to catechols has been demonstrated to involve the same ionic mechanism.
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PMID:Monooxygenase activity of type 3 copper proteins. 1746 41

Copper oxide with various morphologies was synthesized by the hydrolysis of Cu(ac)2 with urea under mild hydrothermal conditions. In the synthesis, a series of organic amines with one or two amine groups (monoamine and diamine), including isobutylamine, octylamine (OLA), dodecylamine, octadecylamine (monoamines), ethylenediamine dihydrochloride, and hexamethylenediamine (diamines), was used as the "structure-directing agent". The monoamines led to the formation of one-dimensional (1D) aggregates of the copper oxide precursor particles (Pre-CuO), while the diamines led to the formation of two-dimensional (2D) aggregates. In both cases, the shorter carbon-chain amine molecules showed a stronger structure-directing function than that of the longer carbon-chain amine molecules. Next, in a series of syntheses, OLA was selected for further study, and the experimental parameters were systematically manipulated. When the hydrolysis was adjusted to a very slow rate by coupling the hydrolysis reaction with an esterification reaction, 1D aggregates of Pre-CuO were formed; when the hydrolysis rate was in the middle range, spherical Pre-CuO architectures composed of smaller linear aggregates were formed. However, under the high hydrolysis rates achieved by increasing the precipitation agent (urea) or by conducting the reaction at high temperatures (>/=120 degrees C), only Pre-CuO nanoparticles with a featureless morphology were formed. The formed spherical Pre-CuO architectures can be converted to a porous structure (CuOx) after removing the OLA molecules via calcination. Compared to the 1D and 2D aggregates, this porous architecture is highly thermally stable and did not collapse even after calcination at 500 degrees C. Preliminary results showed that the porous structure can be used both as a catalyst support and as a catalyst for the oxidation of CO at low temperatures.
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PMID:Manipulating the self-assembling process to obtain control over the morphologies of copper oxide in hydrothermal synthesis and creating pores in the oxide architecture. 1746 56

The rat intestinal fatty acid binding protein (IFABP) primarily comprises two beta-sheet structures surrounding a large internal cavity. The urea denatured WT protein folds within seconds after dilution to nondenaturing conditions. Replacing a glycine with valine in the turn between the last two beta-strands (Gly121Val) slows the folding process by more than three orders of magnitude. After incorporating 4-(19)F-phenylalanine into the mutant protein, we were able to directly monitor the behavior of the eight phenylalanine side chains in real time during folding using (19)F NMR. Specifically, there is a nonnative-like collapse in regions involving three phenylalanine residues (Phe-62, Phe-68, and Phe-93) within milliseconds. At least two distinct NMR peaks were observed, suggesting conformational fluctuations in this region. Formation of this site is followed by formation of native structure of Phe-2 and Phe-17, then by Phe-47, and finally by the cooperative rearrangement of the intermediate structures to the final native structure. It is proposed that the Gly121Val mutation slows the formation of a normal nucleating site, not only slowing overall folding, but also allowing intermediates in regions distant from the mutation to be experimentally observed. Because intermediates involved in protein folding are normally difficult to observe due to their marginal stability, the experimental approach used here may serve as a general method for determining the nature of both early and late steps in protein folding.
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PMID:Observation of sequential steps in the folding of intestinal fatty acid binding protein using a slow folding mutant and 19F NMR. 1761 32

In order to determine the pattern of renal disease and risk factors for renal disease in HIV-infected Nigerians, we studied 400 consecutive HIV/AIDS patients (210 males, 190 females) aged between 18 and 65 years (mean +/- SD; 34.6 +/- 9.4 years), and examined renal disease factors attributable to the infection. Diagnosis of renal disease was based on the consistent presence of at least 1+ albuminuria and/or elevated serum creatinine (>132 micromol/l) as well as the absence of other identifiable causes of chronic kidney disease (CKD). We determined socio-demography and clinical findings, as well as full laboratory work-ups including haemogram, CD4+ cell count, serum electrolytes, urea, creatinine, protein, cholesterol and urine analysis. Renal biopsies were taken in 10 patients who had moderate to massive proteinuria and had consented to the procedure. Finally, we compared HIV/AIDS cases with and without renal disease to determine the risk factors for nephropathy. We observed a high prevalence of renal disease (proteinuria and/or elevated serum creatinine), which was present in 152 (38%) of the patients. This subgroup included 74 males and 78 females with a M:F ratio of 1:1. The mean age (+/-SD) was 35.8 (+/-10.01) years. Systolic and/or diastolic hypertension was seen in 13.2% of these patients while the mean (+/- SD) body mass index (BMI) and packed cell volume (PCV) were 18.5 (+/-3.1) kg/m(2) and 25.26 (+/-6.81)%, respectively. The mean (+/-SD) CD4+ count was 246.49 (+/-192.8) cells/microl, while the mean (+/-SD) serum creatinine and 24-h urine protein excretion rates were 210.11 (+/-337.8) micromol/l and 2.57 (+/- 2.42) g/day, respectively. In subjects with and without nephropathy, there were significant differences in age, BMI, serum cholesterol, serum albumin and CD4+ counts, suggesting that these parameters may be risk factors for nephropathy. Histology revealed mainly focal glomerulosclerosis (FGS) with glomerular collapse. We conclude that the prevalence of proteinuria in HIV-seropositive patients is high in Nigeria. Such subjects show an equal male:female distribution, and glomerular histology revealed that a majority of biopsied patients had the collapsing FSGS variant. The risk factors for renal disease included severity of the HIV infection (inferred from the generally low CD4+ count), anaemia, malnutrition and increasing age.
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PMID:Renal disease in HIV-seropositive patients in Nigeria: an assessment of prevalence, clinical features and risk factors. 1806 7


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