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Protein denaturation

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https://www.readbyqxmd.com/read/28633569/thermal-and-chemical-denaturation-of-colocasia-esculenta-tuber-agglutinin-from-%C3%AE-2%C3%AE-2-to-unfolded-state
#1
Himadri Biswas, Rajagopal Chattopadhyaya
The major tuber storage protein of Colocasia esculenta, is a monocot mannose-binding, widely used dietary lectin, containing two polypeptides of 12.0 and 12.4 kDa. By both gel filtration and dynamic light scattering at pH 7.2, the lectin has a α2β2 form of apparent molecular mass of 48.2 kDa and a hydrodynamic radius of 6.1±0.2 nm; however, at pH 3, it migrates as αβ and has a reduced hydrodynamic radius of 4.6±0.3 nm. Our circular dichroism spectroscopy studies show that the lectin retains approximately 100% of its secondary structure between pH 2-8, going down to ~90% for extreme acidic / alkaline conditions...
June 20, 2017: Journal of Biomolecular Structure & Dynamics
https://www.readbyqxmd.com/read/28629619/slow-interconversion-in-a-heterogeneous-unfolded-state-ensemble-of-outer-membrane-phospholipase-a
#2
Georg Krainer, Pablo Gracia, Erik Frotscher, Andreas Hartmann, Philip Gröger, Sandro Keller, Michael Schlierf
Structural and dynamic investigations of unfolded proteins are important for understanding protein-folding mechanisms as well as the interactions of unfolded polypeptide chains with other cell components. In the case of outer-membrane proteins (OMPs), unfolded-state properties are of particular physiological relevance, because these proteins remain unfolded for extended periods of time during their biogenesis and rely on interactions with binding partners to support proper folding. Using a combination of ensemble and single-molecule spectroscopy, we have scrutinized the unfolded state of outer-membrane phospholipase A (OmpLA) to provide a detailed view of its structural dynamics on timescales from nanoseconds to milliseconds...
June 16, 2017: Biophysical Journal
https://www.readbyqxmd.com/read/28629591/peptide-antibodies-in-clinical-laboratory-diagnostics
#3
Nicole H Trier, Gunnar Houen
Peptide antibodies, with their high specificities and affinities, are invaluable reagents for peptide and protein recognition in biological specimens. Depending on the application and the assay, in which the peptide antibody is to used, several factors influence successful antibody production, including peptide selection and antibody screening. Peptide antibodies have been used in clinical laboratory diagnostics with great success for decades, primarily because they can be produced to multiple targets, recognizing native wildtype proteins, denatured proteins, and newly generated epitopes...
2017: Advances in Clinical Chemistry
https://www.readbyqxmd.com/read/28628309/dissecting-orthosteric-contacts-for-a-reverse-fragment-based-ligand-design
#4
Arun Chandramohan, Nikhil Kumar Tulsian, Ganesh Srinivasan Anand
Orthosteric sites on proteins are formed typically from non-contiguous interacting sites in three dimensional space where the composite binding interaction of a biological ligand is mediated by multiple synergistic interactions of its constituent functional groups. Through these multiple interactions, ligands stabilize both the ligand binding site and the protein fold. However, relative energetic contributions of the individual contacts in these protein-ligand interactions are difficult to resolve. Deconvolution of the contributions of these various functional groups in natural inhibitors/ligand would greatly aid in iterative fragment-based drug discovery (FBDD)...
June 19, 2017: Analytical Chemistry
https://www.readbyqxmd.com/read/28627756/microcontact-peeling-a-cell-micropatterning-technique-for-circumventing-direct-adsorption-of-proteins-to-hydrophobic-pdms
#5
Sho Yokoyama, Tsubasa S Matsui, Shinji Deguchi
Microcontact printing (μCPr) is one of the most popular techniques used for cell micropatterning. In conventional μCPr, a polydimethylsiloxane (PDMS) stamp with microfeatures is used to adsorb extracellular matrix (ECM) proteins onto the featured surface and transfer them onto particular areas of a cell culture substrate. However, some types of functional proteins other than ECM have been reported to denature upon direct adsorption to hydrophobic PDMS. Here we describe a detailed protocol of an alternative technique--microcontact peeling (μCPe)--that allows for cell micropatterning while circumventing the step of adsorbing proteins to bare PDMS...
June 19, 2017: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/28627366/pqbp1-an-intrinsically-disordered-denatured-protein-at-the-crossroad-of-intellectual-disability-and-neurodegenerative-diseases
#6
REVIEW
Hitoshi Okazawa
PQBP1 (polyglutamine binding protein-1) is the earliest identified molecule among the group of disease-related intrinsically disordered/denatured proteins. PQBP1 interacts with splicing-related factors via the disordered/denatured domain and regulates post-transcriptional gene expression. The mutations cause intellectual disability due to decreased dendritic spines and abnormal expression of synapse molecules in neurons, and microcephaly due to elongated cell cycle time and abnormal expression of cell cycle proteins in neural stem progenitor cells...
June 13, 2017: Neurochemistry International
https://www.readbyqxmd.com/read/28627164/thermodynamic-and-structural-adaptation-differences-between-the-mesophilic-and-psychrophilic-lactate-dehydrogenases
#7
Sergei Khrapunov, Eric P Chang, Robert Callender
The thermodynamics of substrate binding and enzymatic activity of a glycolytic enzyme, lactate dehydrogenase (LDH), from both porcine heart, phLDH (Sus scrofa; a mesophile), and from mackerel icefish, cgLDH (Chamapsocephalus gunnari; a psychrophile), were investigated. Using a novel and quite sensitive fluorescence assay which can distinguish protein conformational changes close and distal from the substrate binding pocket, a reversible global protein structural transition preceding the high-temperature transition (denaturation) was surprisingly found to coincide with a marked change in enzymatic activity for both LDHs...
June 19, 2017: Biochemistry
https://www.readbyqxmd.com/read/28624658/optimisation-of-hpmc-ophthalmic-inserts-with-sustained-release-properties-as-a-carrier-for-thermolabile-therapeutics
#8
Arnout Everaert, Yannick Wouters, Eline Melsbach, Nadia Zakaria, Annick Ludwig, Filip Kiekens, Wim Weyenberg
A methodology was developed and optimised for the preparation of a new drug delivery system (DDS) with sustained release properties to allow ocular protein delivery and to limit destructive production steps during manufacturing. Elevated temperatures, shear forces and an oxidative environment should be avoided in order to prevent denaturation or oxidation of proteins. An aqueous HPMC solution was prepared using heat and casted into small semi-rod-shaped PVC blisters. The polymer solution was allowed to cool down and was partially dehydrated at room temperature...
June 15, 2017: International Journal of Pharmaceutics
https://www.readbyqxmd.com/read/28619721/hagfish-slime-exudate-stabilization-and-its-effect-on-slime-formation-and-functionality
#9
L J Böni, R Zurflüh, M Widmer, P Fischer, E J Windhab, P A Rühs, S Kuster
Hagfish produce record breaking amounts of slime when under attack, making it the most dilute hydrogel known to date and a highly interesting material for biomaterial research. The slime forms from a glandular secrete called exudate, which deploys upon contact with seawater. To study the slime formation ex vivo and to characterize its material properties, stabilization of the sensitive slime exudate is crucial. In this study we compared the two main stabilization methods, high osmolarity citrate/PIPES (CP) buffer and immersion in oil and tested the influence of time, temperature, and pH on the stability of the exudate and functionality of the slime...
June 15, 2017: Biology Open
https://www.readbyqxmd.com/read/28616992/coherent-experimental-and-simulation-approach-to-explore-the-underlying-mechanism-of-denaturation-of-stem-bromelain-in-osmolytes
#10
Anjeeta Rani, Mohamed Taha, Pannuru Venkatesu, Ming-Jer Lee
Characterization of a protein in the context of its environment is of crucial importance for a complete understanding of its function. Although biophysical techniques provide powerful tools for studying the stability and activity of the enzyme in the presence of various cosolvents, an approach of combining both experimental techniques and molecular dynamic (MD) simulations may lead to the mechanistic insight into the interactions governing the stability of an enzyme. The knowledge of these interactions can be further utilized for range of modifications in the wild form of an enzyme for various pharmaceutical applications...
June 15, 2017: Journal of Physical Chemistry. B
https://www.readbyqxmd.com/read/28616225/effect-of-three-lactobacilli-with-strain-specific-activities-on-the-growth-performance-faecal-microbiota-and-ileum-mucosa-proteomics-of-piglets
#11
Yating Su, Xingjie Chen, Ming Liu, Xiaohua Guo
BACKGROUND: The beneficial effects of Lactobacillus probiotics in animal production are often strain-related. Different strains from the same species may exert different weight-gain effect on hosts in vivo. Most lactobacilli are selected based on their in vitro activities, and their metabolism and regulation on the intestine based on strain-related characters are largely unexplored. The objective of the present study was to study the in vivo effects of the three lactobacilli on growth performance and to compare the differential effects of the strains on the faecal microbiota and ileum mucosa proteomics of piglets...
2017: Journal of Animal Science and Biotechnology
https://www.readbyqxmd.com/read/28611596/ethidium-bromide-modifies-the-agarose-electrophoretic-mobility-of-cag%C3%A2-ctg-alternative-dna-structures-generated-by-pcr
#12
Mário Gomes-Pereira, Darren G Monckton
The abnormal expansion of unstable simple sequence DNA repeats can cause human disease through a variety of mechanisms, including gene loss-of-function, toxic gain-of-function of the encoded protein and toxicity of the repeat-containing RNA transcript. Disease-associated unstable DNA repeats display unusual biophysical properties, including the ability to adopt non-B-DNA structures. CAG•CTG trinucleotide sequences, in particular, have been most extensively studied and they can fold into slipped-stranded DNA structures, which have been proposed as mutation intermediates in repeat size expansion...
2017: Frontiers in Cellular Neuroscience
https://www.readbyqxmd.com/read/28609478/purification-of-family-b-g-protein-coupled-receptors-using-nanodiscs-application-to-human-glucagon-like-peptide-1-receptor
#13
Yingying Cai, Yuting Liu, Kelly J Culhane, Brian T DeVree, Yang Yang, Roger K Sunahara, Elsa C Y Yan
Family B G protein-coupled receptors (GPCRs) play vital roles in hormone-regulated homeostasis. They are drug targets for metabolic diseases, including type 2 diabetes and osteoporosis. Despite their importance, the signaling mechanisms for family B GPCRs at the molecular level remain largely unexplored due to the challenges in purification of functional receptors in sufficient amount for biophysical characterization. Here, we purified the family B GPCR human glucagon-like peptide-1 (GLP-1) receptor (GLP1R), whose agonists, e...
2017: PloS One
https://www.readbyqxmd.com/read/28608415/crystal-structural-characterization-reveals-novel-oligomeric-interactions-of-human-voltage-dependent-anion-channel-1
#14
Toshiaki Hosaka, Masateru Okazaki, Tomomi Kimura-Someya, Yoshiko Ishizuka-Katsura, Kaori Ito, Shigeyuki Yokoyama, Kosuke Dodo, Mikiko Sodeoka, Mikako Shirouzu
Voltage-dependent anion channel 1 (VDAC1), which is located in the outer mitochondrial membrane, plays important roles in various cellular processes. For example, oligomerization of VDAC1 is involved in the release of cytochrome c to the cytoplasm, leading to apoptosis. However, it is unknown how VDAC1 oligomerization occurs in the membrane. In the present study, we determined high-resolution crystal structures of oligomeric human VDAC1 (hVDAC1) prepared by using an Escherichia coli cell-free protein synthesis system, which avoided the need for denaturation and refolding of the protein...
June 12, 2017: Protein Science: a Publication of the Protein Society
https://www.readbyqxmd.com/read/28608221/ctab-zymography-for-the-analysis-of-aspartic-proteases-from-marine-sponges
#15
Oscar González, Jeff Wilkesman
Electrophoresis under denaturing conditions in the presence of SDS is a standard method for the protein and enzyme scientist. Nevertheless, there are special situations where this method may originate nonoptimal results. SDS may cause protein aggregation or precipitation. Beyond this, depending on the type of protein, some just do not resolve well or migrate abnormally in SDS gels. SDS, an anionic detergent, may be however substituted by a cationic detergent, like CTAB (cetyltrimethylammonium bromide), in order to solubilize and electrophorize proteins...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28608209/simultaneous-detection-of-activity-and-relative-molecular-mass-of-adenylate-kinases-after-sds-page-and-blotting
#16
Silvia Ravera, Isabella Panfoli
Adenylate kinases (AKs) are ubiquitous monomeric phosphotransferases, which play a pivotal role in the energetic metabolism. At the present, nine isoforms are known. AKs catalyze the following reversible reaction: ATP + AMP ↔ 2 ADP, even though isoform 3 uses GTP instead ATP. For many years, the activity of AKs has been detected only after native polyacrylamide gel separations, i.e. in the absence of sodium dodecyl sulfate or methanol. In this work, we report the possibility to detect the activity of the isoforms able to use ATP as substrate, directly onto gel or nitrocellulose sheets, after denaturing SDS-PAGE and electroblotting...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28605192/absolute-protein-quantification-by-mass-spectrometry-not-as-simple-as-advertised
#17
Christopher M Shuford, James J Walters, Patricia M Holland, Uma Sreenivasan, Nadav Askari, Kevin B Ray, Russell P Grant
Stable-isotope labeled (SIL) tryptic peptides, cleavable SIL peptides, and a full-length SIL protein were compared for internal calibration (i.e., as internal calibrators) and external calibration (i.e., as internal standards) when quantifying three forms of unlabeled, human thyroglobulin (Tg) by bottom-up protein analysis. All SIL materials and human proteins were standardized by amino acid analysis to ensure traceability of measurements and allow confident assignment of accuracy. The three forms of human Tg quantified were 1) the primary reference material BCR®457 - a native protein purified from human thyroids - 2) a commercially available form also purified from human thyroids, and 3) a full-length recombinant form expressed and purified from a human embryonic kidney 293 cell-line...
June 12, 2017: Analytical Chemistry
https://www.readbyqxmd.com/read/28602127/sensitive-detection-of-immunoglobulin-g-stability-using-in-real-time-isothermal-differential-scanning-fluorimetry-determinants-of-protein-stability-for-antibody-based-therapeutics
#18
Jason Moggridge, Kyle Biggar, Neal Dawson, Kenneth B Storey
Protein instability is a major obstacle in the production and delivery of monoclonal antibody-based therapies for cancer. This study presents real-time isothermal differential scanning fluorimetry as an emerging method to evaluate the stability of human immunoglobulin G protein with high sensitivity. The stability of polyclonal human immunoglobulin G against urea-induced denaturation was assessed following: (1) oxidation by the free-radical generator 2,2-Azobis[2-amidinopropane]dihydrochloride and (2) in selected storage buffers...
January 1, 2017: Technology in Cancer Research & Treatment
https://www.readbyqxmd.com/read/28600954/high-throughput-thermofluor-based-assays-for-inhibitor-screening-of-stat-sh2-domains
#19
Elvin D de Araujo, Pimyupa Manaswiyoungkul, Johan Israelian, Jisung Park, Karen Yuen, Shiva Farhangi, Angelika Berger-Becvar, Lubna Abu-Jazar, Patrick T Gunning
The development of STAT protein-specific inhibitors has been the focus of a number of drug discovery programs. STAT activation occurs through phosphorylation at the STAT SH2 domain, resulting in dimerization, translocation to the nucleus, and transcription of proliferative genes. Due to the functional significance of the SH2 domain in mediating multiple components of the STAT signalling cascade, many libraries of inhibitors have been designed to target the SH2 domain. This has triggered the requirement for effective high-throughput screening platforms for analyzing binding by larger chemical libraries to STAT proteins...
May 1, 2017: Journal of Pharmaceutical and Biomedical Analysis
https://www.readbyqxmd.com/read/28600316/an-in-vitro-enzyme-system-for-the-production-of-myo-inositol-from-starch
#20
Tomoko Fujisawa, Shohei Fujinaga, Haruyuki Atomi
We developed an in vitro enzyme system to produce myo-inositol from starch. Four enzymes were used, maltodextrin phosphorylase (MalP), phosphoglucomutase (PGM), myo-inositol-3-phosphate synthase (MIPS) and myo-inositol monophosphatase (IMPase). The enzymes were thermostable; MalP and PGM from the hyperthermophilic archaeon Thermococcus kodakarensis, MIPS from the hyperthermophilic archaeon Archaeoglobus fulgidus and IMPase from the hyperthermophilic bacterium Thermotoga maritima Enzymes were individually produced in Escherichia coli, and partially purified by subjecting cell extracts to heat treatment and removing denatured proteins...
June 9, 2017: Applied and Environmental Microbiology
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