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Protein denaturation

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https://www.readbyqxmd.com/read/27913224/a-novel-cross-linked-enzyme-aggregates-cleas-of-papain-and-neutrase-production-partial-characterization-and-application
#1
Zhongqin Chen, Yanwei Wang, Wei Liu, Jingya Wang, Haixia Chen
The neutrase (EC 3.4.24.4) and papain (EC 3.4.22.2) were together immobilized ascross-linked enzyme aggregates (N-P-CLEAs) and their properties were characterized. The influence of the precipitant, cross-linking ratio of glutaraldehyde and cross-linking time were investigated. Ethanol was selected as the more efficient precipitant compared with ammonium sulfate. The proper cross-linking ratio of enzyme and glutaraldehyde was 1:5 (v/v) and the optimized cross-linking time was 4h. N-P-CLEAs showed obvious improvement in thermal stability and pH stability than the free enzyme (P<0...
November 29, 2016: International Journal of Biological Macromolecules
https://www.readbyqxmd.com/read/27913143/innate-immune-stimulatory-activity-of-porphyromonas-gingivalis-fimbriae-is-eliminated-by-phase-separation-using-triton-x-114
#2
Kohji Nozoe, Terukazu Sanui, Masaaki Takeshita, Takao Fukuda, Akira Haraguchi, Yoshitomi Aida, Fusanori Nishimura
Fimbriae are virulence factors of Porphyromonas gingivalis (P. gingivalis). In this study, the action of fimbriae on neutrophil respiratory burst and cytokine production by mononuclear cells (MNC) were investigated. Native or denatured form of purified P. gingivalis fimbriae contained endotoxin at an equivalence of 1-3μglipopolysaccharides(LPS)/mg protein. The endotoxin could be reduced to the equivalent of 1ng-LPS/mg protein by phase separation using Triton X-114. Unfractionated fimbriae caused serum-dependent priming of neutrophils for enhanced respiratory burst, but both native and denatured forms of Triton X-114-fractionated fimbriae were not active at 100μg/mL...
November 30, 2016: Journal of Immunological Methods
https://www.readbyqxmd.com/read/27909052/spontaneous-unfolding-refolding-of-fibronectin-type-iii-domains-assayed-by-thiol-exchange-thermodynamic-stability-correlates-with-rates-of-unfolding-rather-than-folding
#3
Riddhi Shah, Tomoo Ohashi, Harold P Erickson, Terrence G Oas
Globular proteins are not permanently folded, but spontaneously unfold and refold on time scales that can span orders of magnitudes for different proteins. A long-standing debate in the protein-folding field is whether unfolding rates or folding rates correlate to the stability of a protein. In the present study we have determined the unfolding and folding kinetics of ten FNIII domains. FNIII domains are one of the most common protein folds, and are present in 2% of animal proteins. FNIII domains are ideal for this study because they have an identical 7-strand beta sandwich structure, but they vary widely in sequence and thermodynamic stability...
November 30, 2016: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/27909048/the-ph-dependence-of-saccharides-influence-on-thermal-denaturation-of-two-model-proteins-supports-an-excluded-volume-model-for-stabilization-generalized-to-allow-for-intramolecular-electrostatic-interactions
#4
Ilyas Beg, Allen P Minton, Asimul Islam, Md Imtaiyaz Hassan, Faizan Ahmad
The reversible thermal denaturation of apo alpha-lactalbumin and lysozyme was measured in the absence and presence of multiple concentrations of each of seven saccharides (glucose, galactose, fructose, sucrose, trehalose, raffinose and stachyose) at multiple pH values. It was observed that with increasing pH, the absolute stability of alpha-lactalbumin decreased while the stabilizing effect per mole of all saccharides increased, and that the absolute stability of lysozyme increased, while the stabilizing effect per mole of all saccharides decreased...
December 1, 2016: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/27908233/procedure-for-purification-of-recombinant-premsk1p-from-e-coli-determines-its-properties-as-a-factor-of-trna-import-into-yeast-mitochondria
#5
E V Smirnova, I V Chicherin, M V Baleva, N S Entelis, I A Tarassov, P A Kamenski
Mitochondrial genomes of many eukaryotic organisms do not code for the full tRNA set necessary for organellar translation. Missing tRNA species are imported from the cytosol. In particular, one out of two cytosolic lysine tRNAs of the yeast Saccharomyces cerevisiae is partially internalized by mitochondria. The key protein factor of this process is the precursor of mitochondrial lysyl-tRNA synthetase, preMsk1p. In this work, we show that recombinant preMsk1p purified from E. coli in native conditions, when used in an in vitro tRNA import system, demonstrates some properties different from those shown by the renatured protein purified from E...
October 2016: Biochemistry. Biokhimii︠a︡
https://www.readbyqxmd.com/read/27907055/effect-of-glu12-his89-interaction-on-dynamic-structures-in-hiv-1-p17-matrix-protein-elucidated-by-nmr
#6
Yuta Konagaya, Rina Miyakawa, Masumi Sato, Akimasa Matsugami, Satoru Watanabe, Fumiaki Hayashi, Takanori Kigawa, Chiaki Nishimura
To test the existence of the salt bridge and stability of the HIV-1 p17 matrix protein, an E12A (mutated at helix 1) was established to abolish possible electrostatic interactions. The chemical shift perturbation from the comparison between wild type and E12A suggested the existence of an electrostatic interaction in wild type between E12 and H89 (located in helix 4). Unexpectedly, the studies using urea denaturation indicated that the E12A substitution slightly stabilized the protein. The dynamic structure of E12A was examined under physiological conditions by both amide proton exchange and relaxation studies...
2016: PloS One
https://www.readbyqxmd.com/read/27906033/the-role-of-the-backbone-torsion-in-protein-folding
#7
EDITORIAL
Irina Sorokina, Arcady Mushegian
BACKGROUND: The set of forces and sequence of events that govern the transition from an unfolded polypeptide chain to a functional protein with correct spatial structure remain incompletely known, despite the importance of the problem and decades of theory development, computer simulations, and laboratory experiments. Information about the correctly folded state of most proteins is likely to be present in their sequences, and yet many proteins fail to attain native structure after overexpression in a non-native environment or upon experimental denaturation and refolding...
December 1, 2016: Biology Direct
https://www.readbyqxmd.com/read/27902336/characterization-of-the-banana-streak-virus-capsid-protein-and-mapping-of-the-immunodominant-continuous-b-cell-epitopes-to-the-surface-exposed-n-terminus
#8
Jenny Vo, Paul Campbell, Nur N Mahfuzc, Ras Ramli, Daniel Pagendam, Ross Barnard, Andrew Geering
This study identified the structural proteins of two badnavirus species, Banana streak MY virus (BSMYV) and Banana streak OL virus (BSOLV), and mapped the distribution of continuous B-cell epitopes. Two different CP isoforms of about 44 and 40 kDa (CP1 and CP2) and the virion-associated protein (VAP) were consistently associated with purified virions. For both viral species, the N-terminus of CP2 was successfully sequenced by Edman degradation but that of CP1 was chemically blocked. De novo peptide sequencing of tryptic digests suggested that CP1 and CP2 derive from the same region of the P3 polyprotein but differ in the length of either the N- or C-terminus...
October 24, 2016: Journal of General Virology
https://www.readbyqxmd.com/read/27899916/estdz3-a-new-esterolytic-enzyme-exhibiting-remarkable-thermostability
#9
Dimitra Zarafeta, Zalan Szabo, Danai Moschidi, Hien Phan, Evangelia D Chrysina, Xu Peng, Colin J Ingham, Fragiskos N Kolisis, Georgios Skretas
Lipolytic enzymes that retain high levels of catalytic activity when exposed to a variety of denaturing conditions are of high importance for a number of biotechnological applications. In this study, we aimed to identify new lipolytic enzymes, which are highly resistant to prolonged exposure to elevated temperatures. To achieve this, we searched for genes encoding for such proteins in the genomes of a microbial consortium residing in a hot spring located in China. After performing functional genomic screening on a bacterium of the genus Dictyoglomus, which was isolated from this hot spring following in situ enrichment, we identified a new esterolytic enzyme, termed EstDZ3...
2016: Frontiers in Microbiology
https://www.readbyqxmd.com/read/27892580/ionic-complex-of-a-rhodamine-dye-with-aggregation-induced-emission-properties
#10
Pei-Yi Huang, Jhen-Yan Gao, Cheng-Yu Song, Jin-Long Hong
An AIE-active rhodamine based luminogen was prepared via a complexation reaction between non-emissive rhodamine hydrazide (RdH) and bulky camphorsulfonic acid (CSA). Besides acting to open the spirolactam ring of RdH, CSA also imposes a rotational restriction on the resultant ionic complex, RdH(CSA)x. Without CSA, the analogous complex RdH(HCl)3 is a luminogen with aggregation-caused quenching (ACQ) properties. The ionic bonds of RdH(CSA)3 are sensitive to several external stimuli and therefore it is a luminescent sensor for metal ions, organic amines and the blood protein bovine serum albumin (BSA)...
November 28, 2016: Faraday Discussions
https://www.readbyqxmd.com/read/27892477/nanomolar-oligomerization-and-selective-co-aggregation-of-%C3%AE-synuclein-pathogenic-mutants-revealed-by-single-molecule-fluorescence
#11
Emma Sierecki, Nichole Giles, Quill Bowden, Mark E Polinkovsky, Janina Steinbeck, Nicholas Arrioti, Diya Rahman, Akshay Bhumkar, Philip R Nicovich, Ian Ross, Robert G Parton, Till Böcking, Yann Gambin
Protein aggregation is a hallmark of many neurodegenerative diseases, notably Alzheimer's and Parkinson's disease. Parkinson's disease is characterized by the presence of Lewy bodies, abnormal aggregates mainly composed of α-synuclein. Moreover, cases of familial Parkinson's disease have been linked to mutations in α-synuclein. In this study, we compared the behavior of wild-type (WT) α-synuclein and five of its pathological mutants (A30P, E46K, H50Q, G51D and A53T). To this end, single-molecule fluorescence detection was coupled to cell-free protein expression to measure precisely the oligomerization of proteins without purification, denaturation or labelling steps...
November 28, 2016: Scientific Reports
https://www.readbyqxmd.com/read/27884606/bag3-is-a-modular-scaffolding-protein-that-physically-links-heat-shock-protein-70-hsp70-to-the-small-heat-shock-proteins
#12
Jennifer N Rauch, Eric Tse, Rebecca Freilich, Sue-Ann Mok, Leah N Makley, Daniel R Southworth, Jason E Gestwicki
Small heat shock proteins (sHsps) are a family of ATP-independent molecular chaperones that are important for binding and stabilizing unfolded proteins. In this task, the sHsps have been proposed to coordinate with ATP-dependent chaperones, including heat shock protein 70 (Hsp70). However, it is not yet clear how these two important components of the chaperone network are linked. We report that the Hsp70 co-chaperone, BAG3, is a modular, scaffolding factor to bring together sHsps and Hsp70s. Using domain deletions and point mutations, we found that BAG3 uses both of its IPV motifs to interact with sHsps, including Hsp27 (HspB1), αB-crystallin (HspB5), Hsp22 (HspB8), and Hsp20 (HspB6)...
November 21, 2016: Journal of Molecular Biology
https://www.readbyqxmd.com/read/27877183/time-resolved-investigation-of-molecular-components-involved-in-the-induction-of-formula-see-text-high-affinity-transport-system-in-maize-roots
#13
Youry Pii, Massimiliano Alessandrini, Luca Dall'Osto, Katia Guardini, Bhakti Prinsi, Luca Espen, Anita Zamboni, Zeno Varanini
The induction, i.e., the rapid increase of nitrate ([Formula: see text]) uptake following the exposure of roots to the anion, was studied integrating physiological and molecular levels in maize roots. Responses to [Formula: see text] treatment were characterized in terms of changes in [Formula: see text] uptake rate and plasma membrane (PM) H(+)-ATPase activity and related to transcriptional and protein profiles of NRT2, NRT3, and PM H(+)-ATPase gene families. The behavior of transcripts and proteins of ZmNRT2s and ZmNRT3s suggested that the regulation of the activity of inducible high-affinity transport system (iHATS) is mainly based on the transcriptional/translational modulation of the accessory protein ZmNRT3...
2016: Frontiers in Plant Science
https://www.readbyqxmd.com/read/27877155/weak-and-saturable-protein-surfactant-interactions-in-the-denaturation-of-apo-%C3%AE-lactalbumin-by-acidic-and-lactonic-sophorolipid
#14
Kell K Andersen, Brian S Vad, Sophie Roelants, Inge N A van Bogaert, Daniel E Otzen
Biosurfactants are of growing interest as sustainable alternatives to fossil-fuel-derived chemical surfactants, particularly for the detergent industry. To realize this potential, it is necessary to understand how they affect proteins which they may encounter in their applications. However, knowledge of such interactions is limited. Here, we present a study of the interactions between the model protein apo-α-lactalbumin (apo-aLA) and the biosurfactant sophorolipid (SL) produced by the yeast Starmerella bombicola...
2016: Frontiers in Microbiology
https://www.readbyqxmd.com/read/27875744/identification-of-protein-secondary-structures-by-laser-induced-autofluorescence-a-study-of-urea-and-gnhcl-induced-protein-denaturation
#15
Manjunath Siddaramaiah, Kapaettu Satyamoorthy, Bola Sadashiva Satish Rao, Suparna Roy, Subhash Chandra, Krishna Kishore Mahato
In the present study an attempt has been made to interrogate the bulk secondary structures of some selected proteins (BSA, HSA, lysozyme, trypsin and ribonuclease A) under urea and GnHCl denaturation using laser induced autofluorescence. The proteins were treated with different concentrations of urea (3M, 6M, 9M) and GnHCl (2M, 4M, 6M) and the corresponding steady state autofluorescence spectra were recorded at 281nm pulsed laser excitations. The recorded fluorescence spectra of proteins were then interpreted based on the existing PDB structures of the proteins and the Trp solvent accessibility (calculated using "Scratch protein predictor" at 30% threshold)...
November 14, 2016: Spectrochimica Acta. Part A, Molecular and Biomolecular Spectroscopy
https://www.readbyqxmd.com/read/27873329/one-step-zymogram-method-for-the-simultaneous-detection-of-cellulase-xylanase-activity-and-molecular-weight-estimation-of-the-enzyme
#16
Claudia Cano-Ramírez, Alejandro Santiago-Hernández, Flor Nohemí Rivera-Orduña, Rosa María Pineda-Mendoza, Gerardo Zúñiga, María Eugenia Hidalgo-Lara
Here, we describe a zymographic method for the simultaneous detection of enzymatic activity and molecular weight (MW) estimation, following a single electrophoresis step. This involved separating cellulase and xylanase activities from bacteria and fungi, obtained from different sources, such as commercial extracts, crude extract and purified proteins, under denaturing conditions, by 10% polyacrylamide gel electrophoresis, using polyacrylamide gels copolymerized with 1% (w/v) carboxymethylcellulose or beechwood xylan as substrates...
November 22, 2016: Electrophoresis
https://www.readbyqxmd.com/read/27872278/reconstitution-of-a-mycobacterium-tuberculosis-proteostasis-network-highlights-essential-cofactor-interactions-with-chaperone-dnak
#17
Tania J Lupoli, Allison Fay, Carolina Adura, Michael S Glickman, Carl F Nathan
During host infection, Mycobacterium tuberculosis (Mtb) encounters several types of stress that impair protein integrity, including reactive oxygen and nitrogen species and chemotherapy. The resulting protein aggregates can be resolved or degraded by molecular machinery conserved from bacteria to eukaryotes. Eukaryotic Hsp104/Hsp70 and their bacterial homologs ClpB/DnaK are ATP-powered chaperones that restore toxic protein aggregates to a native folded state. DnaK is essential in Mycobacterium smegmatis, and ClpB is involved in asymmetrically distributing damaged proteins during cell division as a mechanism of survival in Mtb, commending both proteins as potential drug targets...
November 21, 2016: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/27872216/the-effect-of-temperature-adaptation-on-the-ubiquitin-proteasome-pathway-in-notothenioid-fishes
#18
Anne E Todgham, Timothy A Crombie, Gretchen E Hofmann
There is an accumulating body of evidence suggesting that the sub-zero Antarctic marine environment places physiological constraints on protein homeostasis. Levels of ubiquitin (Ub)-conjugated proteins, 20S proteasome activity and mRNA expression of many proteins involved in both the ubiquitin (Ub) tagging of damaged proteins as well as the different complexes of the 26S proteasome were measured to examine whether there is thermal compensation of the Ub-proteasome pathway in Antarctic fishes to better understand the efficiency of the protein degradation machinery in polar species...
November 21, 2016: Journal of Experimental Biology
https://www.readbyqxmd.com/read/27872164/functional-consequences-of-b-repeat-sequence-variation-in-the-staphylococcal-biofilm-protein-aap-deciphering-the-assembly-code
#19
Catherine L Shelton, Deborah G Conrady, Andrew B Herr
Staphylococcus epidermidis is an opportunistic pathogen that can form robust biofilms that render the bacteria resistant to antibiotic action and immune responses. Intercellular adhesion in S. epidermidis biofilms is mediated by the cell wall-associated Accumulation-Associated Protein (Aap), via zinc-mediated self-assembly of its B-repeat region. This region contains up to 17 nearly-identical sequence repeats, with each repeat assumed to be functionally equivalent. However, Aap B-repeats exist as two subtypes, defined by a cluster of consensus or variant amino acids...
November 21, 2016: Biochemical Journal
https://www.readbyqxmd.com/read/27871884/structural-characterization-of-recombinant-human-fibroblast-growth-factor-receptor-2b-kinase-domain-upon-interaction-with-omega-fatty-acids
#20
Masoumeh Moghadasi, Dariush Ilghari, Majid Sirati-Sabet, Abbas Amini, Hamideh Asghari, Nematollah Gheibi
The mutated recombinant kinase domain of human fibroblast growth factor receptor 2b (hFGFR2b) is overexpressed and purified, and its structural changes upon the interaction with three unsaturated fatty acids (UFAs), oleic, linoleic and α-linolenic are studied. This interaction is investigated to find out about the folding and unfolding effect of unsaturated fatty acids on the kinase domain structure of hFGFR2b. Recombinant pLEICS-01 vectors, containing the mutated coding region of hFGFR2b, are expressed in the standard Escherichia coli BL21 (DE3) host cells and purified by Ni(2+)-NTA affinity chromatography...
November 18, 2016: Chemistry and Physics of Lipids
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