Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.17 (lysozyme)
21,489 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

High-throughput NMR structural biology can play an important role in structural genomics. We report an automated procedure for high-throughput NMR resonance assignment for a protein of known structure, or of a homologous structure. These assignments are a prerequisite for probing protein-protein interactions, protein-ligand binding, and dynamics by NMR. Assignments are also the starting point for structure determination and refinement. A new algorithm, called Nuclear Vector Replacement (NVR) is introduced to compute assignments that optimally correlate experimentally measured NH residual dipolar couplings (RDCs) to a given a priori whole-protein 3D structural model. The algorithm requires only uniform( 15)N-labeling of the protein and processes unassigned H(N)-(15)N HSQC spectra, H(N)-(15)N RDCs, and sparse H(N)-H(N) NOE's (d(NN)s), all of which can be acquired in a fraction of the time needed to record the traditional suite of experiments used to perform resonance assignments. NVR runs in minutes and efficiently assigns the (H(N),(15)N) backbone resonances as well as the d(NN)s of the 3D (15)N-NOESY spectrum, in O(n(3)) time. The algorithm is demonstrated on NMR data from a 76-residue protein, human ubiquitin, matched to four structures, including one mutant (homolog), determined either by x-ray crystallography or by different NMR experiments (without RDCs). NVR achieves an assignment accuracy of 92-100%. We further demonstrate the feasibility of our algorithm for different and larger proteins, using NMR data for hen lysozyme (129 residues, 97-100% accuracy) and streptococcal protein G (56 residues, 100% accuracy), matched to a variety of 3D structural models. Finally, we extend NVR to a second application, 3D structural homology detection, and demonstrate that NVR is able to identify structural homologies between proteins with remote amino acid sequences using a database of structural models.
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PMID:A polynomial-time nuclear vector replacement algorithm for automated NMR resonance assignments. 1528 93

Food allergies represent an important health problem in industrialized countries, such that detection and quantitative analysis of the protein considered to be the main allergen is crucial. A dot-blot fluorescent staining method for the detection and quantitative analysis of protein residues in food grade amino acids and nucleic acids is presented. This method combines fluorescence staining with dot-blotting onto PVDF membrane. Several standard proteins, such as bovine serum albumin (66 kDa), lysozyme (14 kDa), ubiquitin (8.6 kDa), bovine insulin (5.7 kDa), and oxidized insulin B chain (3.5 kDa), were detectable at 0.1 ppm using SYPRO Ruby blot stain. Twenty-five different amino acids and two different nucleic acids of food grade were analyzed using this method combined with an internal standard addition method using BSA as an internal standard. All amino acids and nucleic acids were dissolved in 3.6% aqueous HCl and dot-blotted onto PVDF membrane before a large amount of amino acids and nucleic acid were removed. Protein residues and the internal standard protein immobilized on the membrane were stained using SYPRO ruby blot stain. The internal standard in all samples was detectable at 0.1 ppm. Samples were dissolved at 120 or 70 mg/mL, according to their solubility under acidic conditions. The detection limit of protein residues per weight was 0.8-1.4 ppm in amino acids and nucleic acids; residual protein was not detected in any sample.
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PMID:Detection and quantification of protein residues in food grade amino acids and nucleic acids using a dot-blot fluorescent staining method. 1531 65

Recently, impairment of the ubiquitin-proteasome system is suggested to be responsible for the neuronal death in ageing and Parkinson's disease. The specific degeneration of dopamine neurons containing neuromelanin (NM) suggests that NM itself may be involved in the cellular dysfunction and death, even though the direct link has never been reported. We examined the effects of NM isolated from the human substantia nigra on the proteasome activity in human dopaminergic SH-SY5Y cells. NM reduced the activities of 26S proteasome, as shown in situ using a green fluorescent protein homologue targeted to 26S proteasome and also in vitro using ubiquitinated lysozyme as a substrate. However, NM did not affect 20S proteasome activity in vitro. NM reduced the amount of PA700 regulatory subunit of 26S proteasome, but did not affect that of alpha- and beta-subunits of 20S proteasome. These results suggest that NM may inhibit the ubiquitin-26S proteasome system, and determine the selective vulnerability of dopamine neurons in ageing and related disorders.
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PMID:Neuromelanin inhibits enzymatic activity of 26S proteasome in human dopaminergic SH-SY5Y cells. 1548 Aug 37

A new method to interpret electrospray mass spectral data based on calculating the ratio of mass-to-charge (m/z) values of multiply charged ions is described. The mass-to-charge ratios of any two multiply charged ions corresponding to a single compound are unique numbers that enable the charge states for each ion to be unequivocally identified. The multiply charged ions in electrospray mass spectra originate from the addition or abstraction of protons, cations, or anions to and from a compound under analysis. In contrast to existing deconvolution processes, the charge ratio analysis method (CRAM), identifies the charge states of multiply charged ions without any prior knowledge of the nature of the charge-carrying species. In the case of high-resolution electrospray mass spectral data, in which multiply charged ions are resolved to their isotopic components, the CRAM is capable of correlating the isotope peaks of different multiply charged ions that share the same isotopic composition. This relative ratio method is illustrated here for electrospray mass spectral data of lysozyme and oxidized ubiquitin recorded at low- to high-mass resolution on quadrupole ion trap and Fourier transform ion cyclotron mass spectrometers, and theoretical data for the protein calmodulin based upon a reported spectrum recorded on the latter.
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PMID:Charge ratio analysis method: approach for the deconvolution of electrospray mass spectra. 1562 85

This work was aimed at probing the influence of solvent surface tension on protein ionization by electrospray. In particular, we were interested in testing the previously suggested hypothesis that the charge-state distributions (CSDs) of proteins in electrospray ionization mass spectrometry (ESI-MS) are controlled by the surface tension of the least volatile solvent component. In the attempt to minimize uncontrolled conformational effects, we used acid-sensitive proteins (cytochrome c and myoglobin) at low pH or highly stable proteins (ubiquitin and lysozyme) in the presence of low concentrations of organic solvents. A first set of experiments compared the effect of 1- and 2-propanol. These two alcohols have similar chemico-physical properties but values of vapor pressure below and above that of water, respectively. Both compounds have much lower surface tension than water. The solvents employed allowed testing of the influence of surface tension on protein spectra obtained from similarly denaturing solutions. The compared solvent conditions gave rise to very similar spectra for each tested protein. We then investigated the effect of the addition of dimethyl sulfoxide to acid-unfolded proteins. We observed enhanced ionization in the presence of acetic or formic acid, consistent with the previously described supercharging effect, but almost no shift of the CSD in the presence of HCl. Finally, we analyzed thermally denatured cytochrome c, to obtain reference spectra of the unfolded protein in high-surface-tension solutions. Also in this case, the CSD of the unfolded protein was shifted towards lower m/z values relative to low-surface-tension systems. In contrast to the other results reported here, this effect is consistent with an influence of solvent surface tension on CSD. The magnitude of the effect, however, is much smaller than predicted by the Rayleigh equation. The results presented here are not easy to reconcile with the hypothesis that the maximum charge state exhibited by proteins in ESI-MS reflects the Rayleigh-limit charge of the precursor droplet. The data are discussed with reference to models for the mechanism of electrospray ionization.
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PMID:Testing the role of solvent surface tension in protein ionization by electrospray. 1571 70

The polyglutamine-containing neurodegenerative protein ataxin 3 (AT3) has deubiquitylating activity and binds ubiquitin chains with a preference for chains of four or more ubiquitins. Here we characterize the deubiquitylating activity of AT3 in vitro and show it trims/edits K48-linked ubiquitin chains. AT3 also edits polyubiquitylated (125)I-lysozyme and decreases its degradation by proteasomes. Cellular studies show that endogenous AT3 colocalizes with aggresomes and preaggresome particles of the misfolded cystic fibrosis transmembrane regulator (CFTR) mutant CFTRDeltaF508 and associates with histone deacetylase 6 and dynein, proteins required for aggresome formation and transport of misfolded protein. Small interfering RNA knockdown of AT3 greatly reduces aggresomes formed by CFTRDeltaF508, demonstrating a critical role of AT3 in this process. Wild-type AT3 restores aggresome formation; however, AT3 with mutations in the active site or ubiquitin interacting motifs cannot restore aggresome formation in AT3 knockdown cells. These same mutations decrease the association of AT3 and dynein. These data indicate that the deubiquitylating activity of AT3 and its ubiquitin interacting motifs as well play essential roles in CFTRDeltaF508 aggresome formation.
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PMID:The polyglutamine neurodegenerative protein ataxin 3 regulates aggresome formation. 1576 77

Mass Spectra of charge states of folded proteins were obtained with nanospray and aqueous solution containing 20 microM the protein (ubiquitin, cytochrome c, lysozyme) and one of the NaA salts NaCl, NaI, NaAc (acetate) (1-10 mM). At very low collision activated decomposition (CAD), the mass spectra of a protein with charge z exhibited a replacement of zH+ with zNa+ and also multiple adducts of NaA. Higher CAD converts the NaA adduct peaks to Na minus H peaks. These must be due to loss of HA where the H was provided by the protein. The degree of HA loss with increasing CAD followed the order I < Cl < Ac. Significantly, the intensity of the ions with n (Na minus H) adducts showed a downward break past an n(MAX) which is equal to the number of acidic residues of the protein plus the charge of the protein. All the observations could be rationalized within the framework of the electrospray mechanism and the charge residue model, which predict that due to extensive evaporation of solvent, the solutes will reach very high concentrations in the final charged droplets. At such high concentrations, positive ions such as Na+, NH4+ form ion pairs with ionized acidic residues and the negative A- form ion pairs with ionized basic residues of the protein. Adducts of Na+, and NaA to backbone amide groups occur also. This reaction mechanism fits all the experimental observations and provides predictions that the number of acidic and basic groups at the surface of the gaseous protein that remain ionized can be controlled by the absence or presence of additives to the solution.
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PMID:Ion-ion and ion-molecule reactions at the surface of proteins produced by nanospray. Information on the number of acidic residues and control of the number of ionized acidic and basic residues. 1597 26

The reactivity of arginine residues in model proteins (ubiquitin, cytochrome c, myoglobin, ribonuclease A, lysozyme) was examined using a selective tagging reaction in combination with on-line monitoring of the reaction progress by electrospray ionization mass spectrometry (ESI-MS). The kinetics of this reaction, based on the cyclization of the guanidine group of arginine with 2,3-butanedione and phenylboronic acid at pH 8-10, allow the grouping of arginines in "exposed" or "partially buried" residues, because they differ substantially in their reaction rate constants for the conversion of the guanidine groups. The method allows one to differentiate between different protein conformations as shown for myoglobin and its apo form and native and reduced ribonuclease A: Removal of the heme group in myoglobin resulted in an increased reactivity for the two partially buried arginines. For RNAse A, quantitative reduction of the disulfide bonds lead to the exposure of an additional arginine residue and two different conformations of the reduced protein were observed by ESI-MS that could be distinguished according to their charge-state distribution. Experimentally obtained accessibilities were compared with solvent-accessibility data calculated from 3D structures and substantial agreement between both techniques was observed.
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PMID:Functional probing of arginine residues in proteins using mass spectrometry and an arginine-specific covalent tagging concept. 1601 63

Antimicrobial midgut proteins and peptides that result from blood digestion in feeding American dog ticks Dermacentor variabilis (Say) were identified. Midgut extracts from these ticks showed antimicrobial activity against Micrococcus luteus, regardless of whether they were challenged with peptidoglycan, blood meal components, rabbit blood, Bacillus subtilis, Escherischia coli or Borrelia burgdorferi. However, no peptide band co-migrating with defensin was found in midgut extracts from the challenged ticks. Partial purification of the midgut extracts using C(18) Sep Paks and gel electrophoresis showed the presence of 4 distinct bands with rMW 4.1, 5.3, 5.7 and 8.0 kDa identified by tryptic digestion-mass fingerprinting as digestive fragments of rabbit alpha-, beta-, gamma-chain hemoglobin, and rabbit ubiquitin. No evidence of varisin, a defensin previously identified in the hemolymph of D. variabilis, was found in the tryptic digest, although varisin was found in a hemocyte lysate using the same methods. However, varisin transcript was detected in midgut cell lysates. Also present in all midgut samples was a cluster of 3 overlapping bands with rMW 13.0, 14.1 and 14.7 kDa which were identified by tryptic-digestion LC-MS and MALDI-TOF as rabbit alpha- and beta-chain hemoglobin (undigested) and transtherytin. Lysozyme transcript was detected in midgut cell extracts but the peptide was not. Studies done on other tick species demonstrated that hemoglobin digestion resulted in antimicrobial fragments. Antimicrobial hemoglobin fragments (including fragments larger than any reported previously) also were found in D. variabilis, as well as ubiquitin, a peptide known to occur as part of an antimicrobial complex in vertebrate leukocytes. In addition, we noted that Borrelia burgdorferi spirochetes were not lysed in the midgut lumen, which would be expected if defensin and lysozyme were active in this location. In this respect, the midgut's response to microbial challenge differs from that of the hemolymph. In summary, the midgut's antimicrobial activity appears to be primarily a byproduct of hemoglobin digestion rather than expression of immune peptides and proteins.
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PMID:Host blood proteins and peptides in the midgut of the tick Dermacentor variabilis contribute to bacterial control. 1613 35

Gentle protein electrospray ionization is achieved using the high-velocity gas flow of an air amplifier to improve desolvation in conventional ESI and generate intact folded protein ions in the gas phase. Comparisons are made between the ESI spectra of a number of model proteins, including ubiquitin, cytochrome c, lysozyme, and myoglobin, over a range of pH values under optimized conditions, with and without using an air amplifier to achieve high-velocity gas flow. Previously reported increased ion signals are confirmed. In addition, the peaks recorded using the air amplifier are shown to be narrower, corresponding to more complete desolvation. Significant changes in the charge-state distribution also are observed, with a shift to lower charge state at high-velocity flow. The relationship between the observed charge-state distribution and protein conformation was explored by comparing the charge-state shifts and the distributions of charge states for proteins that are or are not stable in their native conformations in low pH solutions. The data suggest retention of native or nativelike protein conformations using the air amplifier in all cases examined. This is explained by a mechanism in which the air amplifier rapidly creates small droplets from the original large ESI droplets and these microdroplets then desolvate without a significant decrease in pH, resulting in retention of the folded protein conformations. Furthermore, the holoform of ionized myoglobin is visible at pH 3.5, a much lower value than the minimum needed to see this form in conventional ESI. These results provide evidence for the importance of the conditions used in the desolvation process for the preservation of the protein conformation and suggest that the conditions achieved when using high-velocity gas flows to assist droplet evaporation and ion desolvation are much gentler than those in conventional ESI experiments.
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PMID:Gentle protein ionization assisted by high-velocity gas flow. 1619 76


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