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Protein denaturation

Lei Fang, Guoming Li, Ruizeng Gu, Muyi Cai, Jun Lu
BACKGROUND: Shellfish, including oysters, often cause allergic reactions in adults. Thermal treatment is one of the most common technologies to deal with seafood, which may affect biological properties. The aim of this work was to evaluate the impact of heating on conformation and potential allergenicity of oyster-derived tropomyosin (Cra g 1). RESULTS: SDS-PAGE showed that there was an apparent band at 35KD of raw tropomyosin after purification and more significant polymers appeared in heated protein...
April 15, 2018: Journal of the Science of Food and Agriculture
Yoshinori Matsuura, Michiyo Takehira, George I Makhatadze, Yasumasa Joti, Hisashi Naitow, Naoki Kunishima, Katsuhide Yutani
In order to elucidate the contribution of charged residues to protein stabilization at temperatures of over 100 °C, we constructed many mutants of the CutA1 protein ( EcCutA1) from Escherichia coli. The goal was to see if one can achieve the same stability as for a CutA1 from hyperthermophile Pyrococcus horikoshii that has the denaturation temperature near 150 °C. The hydrophobic mutant of EcCutA1 ( Ec0VV) with denaturation temperature (Td) of 113.2 °C was used as a template for mutations. The highest Td of Ec0VV mutants substituted by a single charged residue was 117...
April 12, 2018: Biochemistry
Richard A Campbell, Andrea Tummino, Imre Varga, Olga Yu Milyaeva, Michael M Krycki, Shi-Yow Lin, Valerie Laux, Michael Haertlein, Victor Trevor Forsyth, Boris A Noskov
The application of protein deuteration and high flux neutron reflectometry has allowed a comparison of the adsorption properties of lysozyme at the air-water interface from dilute solutions with and without strong denaturants, urea and guanidine hydrochloride (GuHCl). The surface excess and adsorption layer thickness were resolved and complemented by images of the mesoscopic lateral morphology from Brewster angle microscopy. It was revealed that the thickness of the adsorption layer in the absence of added denaturants is less than the short axial length of the lysozyme molecule, which indicates deformation of the globules at the interface...
April 9, 2018: Langmuir: the ACS Journal of Surfaces and Colloids
Shuichiro Goda, Tomoyuki Koga, Kenichiro Yamashita, Ryo Kuriura, Toshifumi Ueda
In Archaea and Bacteria, surface layer (S-layer) proteins form the cell envelope and are involved in cell protection. In the present study, a putative S-layer protein was purified from the crude extract of Pyrococcus horikoshii using affinity chromatography. The S-layer gene was cloned and expressed in Escherichia coli. Isothermal titration calorimetry analyses showed that the S-layer protein bound N-acetylglucosamine and induced agglutination of the gram-positive bacterium Micrococcus lysodeikticus. The protein comprised a 21-mer structure, with a molecular mass of 1,340 kDa, as determined using small-angle X-ray scattering...
April 8, 2018: Bioscience, Biotechnology, and Biochemistry
Beata Adamczak, Mateusz Kogut, Jacek Czub
Although osmolytes are known to modulate the folding equilibrium, the molecular mechanism of their effect on thermal denaturation of proteins is still poorly understood. Here, we simulated the thermal denaturation of a small model protein (Trp-cage) in the presence of denaturing (urea) and stabilizing (betaine) osmolytes, using the all-atom replica exchange molecular dynamics simulations. We found that urea destabilizes Trp-cage by enthalpically-driven association with the protein, acting synergistically with temperature to induce unfolding...
April 9, 2018: Physical Chemistry Chemical Physics: PCCP
Huanhuan Yang, Haishan Tian, Jiliang Cheng, Jie Zheng, Dezhong Wang, Changye Sun, David Fernig, Taotao Chen, Weiyue Gong, Shen Wang, Xiaokun Li, Chao Jiang
In the 22 member mammalian FGF family, FGF22 belongs to FGF7 subfamily, and its effects are largely confined to the brain and skin. To explore the functions of FGF22 on other tissues and develop a large-scale production of recombinant human FGF22 (rhFGF22) without a fusion tag, a plasmid encoding human FGF22 (pET3a-rhFGF22) was used to express rhFGF22 in E. coli BL21 (DE3) pLysS. A large amount of rhFGF22 inclusion body protein was obtained. A two-step denaturing method successfully solubilized rhFGF22, and it was refolded and then purified in one step via heparin affinity chromatography...
April 5, 2018: Protein Expression and Purification
Prabin Lamichhane, Alan L Kelly, Jeremiah J Sheehan
This study investigated the effect of centrifugation (9,000 × g, 50°C, flow rate = 1,000 L/h), as well as the incorporation of high-heat-treated (HHT) centrifugate into cheese milk on the composition, texture, and ripening characteristics of Maasdam cheese. Neither centrifugation nor incorporation of HHT centrifugate into cheese milk had a pronounced effect on the compositional parameters of any experimental cheeses, except for moisture and moisture in nonfat substance (MNFS) levels. Incorporation of HHT centrifugate at a rate of 6 to 10% of the total milk weight into centrifuged milk increased the level of denatured whey protein in the cheese milk and also increased the level of MNFS in the resultant cheese compared with cheeses made from centrifuged milk and control cheeses; moreover, cheese made from centrifuged milk had ∼3% higher moisture content on average than control cheeses...
April 4, 2018: Journal of Dairy Science
Snehasis Daschakraborty
Glycerol and dimethyl sulphoxide (DMSO) have vital roles in cryoprotection of living cells, tissues, etc. The above action has been directly linked with disruption of hydrogen (H-) bond structure and dynamics of water by these cosolvents at bulk region and around various complex units, such as peptide, amino acid, protein, and lipid membrane. However, the disruption of the local structure of the water solvent around a purely hydrophobic solute is still not studied extensively. The latter is also important in the context of stabilization of protein from cold denaturation...
April 7, 2018: Journal of Chemical Physics
Aditya V Gandhi, Keith J Arlotta, Hsiao-Nung Chen, Shawn C Owen, John F Carpenter
The commercially available antibody-drug conjugate (ADC) product, Kadcyla® is synthesized using a two-step reaction, wherein the linker is conjugated to native lysines on the monoclonal antibody (mAb) in step 1, followed by drug conjugation to the linker-modified antibody in step 2. In our study, we synthesized a lysine conjugated ADC (Syn-ADC) on the same trastuzumab scaffold as Kadcyla® using a one-step reaction. Mass spectrometry of both products revealed a sub-population of Kadcyla® containing free linkers conjugated to the mAb, but not conjugated to the drug, which were absent in the one-step reaction ADC product...
April 4, 2018: Journal of Pharmaceutical Sciences
Emily P Nguyen, Lillian Lee, Amgad R Rezk, Ylias M Sabri, Suresh K Bhargava, Leslie Y Yeo
While many microfluidic devices have been developed for sensing and others for actuation, few devices can perform both tasks effectively and simultaneously on the same platform. In piezoelectric sensors and actuators, this is due to the opposing operating requirements for sensing and actuation. Sensing ideally requires narrow resonant peaks characterized by high quality factors, such as those found in quartz crystals. However, these materials usually have poor electromechanical coupling coefficients that are not ideal for actuation...
April 6, 2018: Analytical Chemistry
Atsushi Shibuya, Hiroaki Kawashima, Masato Tanaka
OBJECTIVES: In order to investigate the pathophysiology of erythrocyte membrane proteins, 10 patients (6 pre- and 4 post-splenectomy) with hereditary spherocytosis (HS) and other patients with haemolytic anaemia were examined. METHODS: The membrane proteins were analysed by biochemical and mass spectrometry. RESULTS: Reductions in the extracellular membrane of band 3 protein by eosin-5'-maleimide (EMA) binding test were greater in patients with pre-splenectomy HS than in patients with post-splenectomy HS, other types of haemolytic anaemia, and controls...
April 6, 2018: Hematology (Amsterdam, Netherlands)
Gang Liu, Dan Yuan, Qi Wang, Wanrong Li, Jie Cai, Shuyi Li, Olusola Lamikanra, Xinguang Qin
Egg white proteins are a potential source of nutrition by their biological and technological values. However, their limited functional properties prevent their widespread industrial applications. In this study, the ovalbumin functionality was improved through purification and its conversion to ovalbumin-lactose conjugate through Maillard reaction. The protein content of the purified ovalbumin was 93%. Denaturing protein sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile and amino acid composition analysis confirmed the glycation...
April 5, 2018: Journal of Agricultural and Food Chemistry
Debabrata Sinha, Rajkrishna Mondal, Avisek Mahapa, Keya Sau, Rajagopal Chattopadhyaya, Subrata Sau
RsbW, an anti-sigma factor possessing kinase activity, is expressed by many Gram-positive bacteria including Staphylococcus aureus. To obtain clues about the domain structure and the folding-unfolding mechanism of RsbW, we have elaborately studied rRsbW, a recombinant S. aureus RsbW. Sequence analysis of the protein fragments, generated by the limited proteolysis of rRsbW, has proposed it to be a single-domain protein. The unfolding of rRsbW in the presence of GdnCl or urea was completely reversible in nature and occurred through the formation of at least two intermediates...
2018: PloS One
Shoeb Ikhlas, Afia Usman, Masood Ahmad
Interaction studies of bisphenol analogues; Biphenol-A (BPA), Bisphenol-B (BPB) and Bisphenol-F (BPF) with bovine serum albumin (BSA) were performed using multi-spectroscopic and molecular docking studies at the protein level. The mechanism of binding of bisphenols with BSA was dynamic in nature. SDS refolding experiments demonstrated no stabilization of BSA structure denatured by BPB, however, BSA denatured by BPA and BPF was found to get stabilized. Also, CD spectra and molecular docking studies revealed that BPB bound more strongly and induced more conformational changes in BSA in comparison to BPA...
April 5, 2018: Journal of Biomolecular Structure & Dynamics
Dayanidhi Mohanta, Madhurima Jana
Monohydric alcohols, such as methanol (MEH), ethanol (ETH) and 2,2,2-trifluoroethanol (TFE), have significant effects on biological processes including the protein folding-unfolding phenomenon. Among the several monohydric alcohols, TFE, a fluorine-substituted alcohol, is known to induce a helical structure in proteins. In this work, we report the heterogeneous unfolding phenomenon of a small protein Chymotrypsin Inhibitor 2 in various concentrations of methanol, ethanol and TFE solutions by performing atomistic molecular dynamics simulation studies...
April 5, 2018: Physical Chemistry Chemical Physics: PCCP
William L Willis, Linan Wang, Takuma Tsuzuki Wada, Mark Gardner, Omar Abdouni, Jeffrey Hampton, Giancarlo Valiente, Nicholas Young, Stacy Ardoin, Sudha Agarwal, Michael A Freitas, Lai-Chu Wu, Wael N Jarjour
High-mobility group box 1 (HMGB1) is a chromatin-associated protein that in response to stress or injury, translocates from the nucleus to the extracellular milieu where it functions as an alarmin. HMGB1's function is in part determined by the complexes (HMGB1c) it forms with other molecules. However, structural modifications in the HMGB1 polypeptide that may regulate HMGB1c formation have not been previously described. In this report, we observed high-molecular weight, denaturing-resistant HMGB1c in the plasma and peripheral blood mononuclear cells of individuals with systemic lupus erythematosus (SLE) and to a much lesser extent in healthy subjects...
April 4, 2018: Journal of Biological Chemistry
Corey E Reed, Jennifer Fournier, Nikolaos Vamvoukas, Stephan M Koza
N-Glycan analysis is routinely performed for biotherapeutic protein characterization. A recently introduced N-glycan analysis kit using RapiFluor-MS (RFMS) labeling provides time savings over reductive amination labeling methods while also providing enhanced fluorescence (FLR) and mass spectrometry (MS) responses. This article demonstrates the semiautomation of this kit using an Andrew Alliance pipetting robot that promises further gains in productivity. This robotic platform uses standard manual pipettors and an optically guided arm to facilitate the automation of manual procedures...
March 1, 2018: SLAS Technology
Betul Uralcan, Sang Beom Kim, Chester E Markwalter, Robert K Prud'homme, Pablo G Debenedetti
Fundamental understanding of protein stability away from physiological conditions is important due to its evolutionary implications and relevance to industrial processing and storage of biological materials. The molecular mechanisms of stabilization/destabilization by environmental perturbations are incompletely understood. We use replica-exchange molecular dynamics simulations and thermodynamic analysis to investigate the effects of ionic liquid-induced perturbations on the folding/unfolding thermodynamics of the Trp-cage miniprotein...
April 4, 2018: Journal of Physical Chemistry. B
Fitsum Getachew, Albert Vandenberg, Judit Smits
BACKGROUND: Faba bean (Vicia faba) vicine/convicine (V-C) aglycones (divicine/isouramil) provoke an acute hemolytic anemia called favism in individuals with a glucose-6-phosphate dehydrogenase (G6PD) enzyme defect in their red blood cells. Geneticists/plant breeders are working with faba bean to decrease V-C levels to improve public acceptance of this high protein pulse crop. Here, we present a fast and simple ex-vivo in vitro bioassay for V-C toxicity testing of faba bean or faba bean food products...
April 2, 2018: Journal of the Science of Food and Agriculture
Giovanni Benelli
The employment of nanoparticles obtained through various synthesis routes as novel pesticides recently attracted high research attention. An impressive number of studies have been conducted to test their toxic potential against a wide number of arthropod pests and vectors, with major emphasis on mosquitoes and ticks. However, precise information on the mechanisms of action of nanoparticles against insects and mites are limited, with the noteworthy exception of silica, alumina, silver, and graphene oxide nanoparticles on insects, while no information is available for mites...
April 3, 2018: Environmental Science and Pollution Research International
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