Thermal denaturation of egg protein under nanosecond pulsed laser heating of gold nanoparticles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Meshalkin, Yu P; Lapin, I N; Svetlichnyi, Valery A

Thermal denaturation of egg protein in the presence of gold nanoparticles via their heating at the plasmon resonance wavelength by the pulsed radiation of the second harmonic of an Nd:YAG laser (532 nm) is investigated. The experimental dependence of the protein denaturation time on the mean laser power is obtained. The heating temperature of the medium with gold nanoparticles is calculated. The numerical estimates of the temperature of the heated medium containing protein and gold nanoparticles (45.3 deg. C at the moment of protein denaturation) are in good agreement with the literature data on its thermal denaturation and with themore » data of pyrometric measurements (42.0 {+-} 1.5 deg. C). The egg protein may be successfully used to investigate the specific features of laser heating of proteins in the presence of metal nanoparticles under their excitation at the plasmon resonance wavelength. (laser methods in biology)« less

Urea-temperature phase diagrams capture the thermodynamics of denatured state expansion that accompany protein unfolding

PubMed Central

Tischer, Alexander; Auton, Matthew

2013-01-01

We have analyzed the thermodynamic properties of the von Willebrand factor (VWF) A3 domain using urea-induced unfolding at variable temperature and thermal unfolding at variable urea concentrations to generate a phase diagram that quantitatively describes the equilibrium between native and denatured states. From this analysis, we were able to determine consistent thermodynamic parameters with various spectroscopic and calorimetric methods that define the urea–temperature parameter plane from cold denaturation to heat denaturation. Urea and thermal denaturation are experimentally reversible and independent of the thermal scan rate indicating that all transitions are at equilibrium and the van't Hoff and calorimetric enthalpies obtained from analysis of individual thermal transitions are equivalent demonstrating two-state character. Global analysis of the urea–temperature phase diagram results in a significantly higher enthalpy of unfolding than obtained from analysis of individual thermal transitions and significant cross correlations describing the urea dependence of and that define a complex temperature dependence of the m-value. Circular dichroism (CD) spectroscopy illustrates a large increase in secondary structure content of the urea-denatured state as temperature increases and a loss of secondary structure in the thermally denatured state upon addition of urea. These structural changes in the denatured ensemble make up ∼40% of the total ellipticity change indicating a highly compact thermally denatured state. The difference between the thermodynamic parameters obtained from phase diagram analysis and those obtained from analysis of individual thermal transitions illustrates that phase diagrams capture both contributions to unfolding and denatured state expansion and by comparison are able to decipher these contributions. PMID:23813497

Observation of Solvent Penetration during Cold Denaturation of E. coli Phosphofructokinase-2

PubMed Central

Ramírez-Sarmiento, César A.; Baez, Mauricio; Wilson, Christian A.M.; Babul, Jorge; Komives, Elizabeth A.; Guixé, Victoria

2013-01-01

Phosphofructokinase-2 is a dimeric enzyme that undergoes cold denaturation following a highly cooperative N2 2I mechanism with dimer dissociation and formation of an expanded monomeric intermediate. Here, we use intrinsic fluorescence of a tryptophan located at the dimer interface to show that dimer dissociation occurs slowly, over several hours. We then use hydrogen-deuterium exchange mass spectrometry experiments, performed by taking time points over the cold denaturation process, to measure amide exchange throughout the protein during approach to the cold denatured state. As expected, a peptide corresponding to the dimer interface became more solvent exposed over time at 3°C; unexpectedly, amide exchange increased throughout the protein over time at 3°C. The rate of increase in amide exchange over time at 3°C was the same for each region and equaled the rate of dimer dissociation measured by tryptophan fluorescence, suggesting that dimer dissociation and formation of the cold denatured intermediate occur without appreciable buildup of folded monomer. The observation that throughout the protein amide exchange increases as phosphofructokinase-2 cold denatures provides experimental evidence for theoretical predictions that cold denaturation primarily occurs by solvent penetration into the hydrophobic core of proteins in a sequence-independent manner. PMID:23708365

Observation of solvent penetration during cold denaturation of E. coli phosphofructokinase-2.

PubMed

Ramírez-Sarmiento, César A; Baez, Mauricio; Wilson, Christian A M; Babul, Jorge; Komives, Elizabeth A; Guixé, Victoria

2013-05-21

Phosphofructokinase-2 is a dimeric enzyme that undergoes cold denaturation following a highly cooperative N2 2I mechanism with dimer dissociation and formation of an expanded monomeric intermediate. Here, we use intrinsic fluorescence of a tryptophan located at the dimer interface to show that dimer dissociation occurs slowly, over several hours. We then use hydrogen-deuterium exchange mass spectrometry experiments, performed by taking time points over the cold denaturation process, to measure amide exchange throughout the protein during approach to the cold denatured state. As expected, a peptide corresponding to the dimer interface became more solvent exposed over time at 3°C; unexpectedly, amide exchange increased throughout the protein over time at 3°C. The rate of increase in amide exchange over time at 3°C was the same for each region and equaled the rate of dimer dissociation measured by tryptophan fluorescence, suggesting that dimer dissociation and formation of the cold denatured intermediate occur without appreciable buildup of folded monomer. The observation that throughout the protein amide exchange increases as phosphofructokinase-2 cold denatures provides experimental evidence for theoretical predictions that cold denaturation primarily occurs by solvent penetration into the hydrophobic core of proteins in a sequence-independent manner. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Urea-temperature phase diagrams capture the thermodynamics of denatured state expansion that accompany protein unfolding.

PubMed

Tischer, Alexander; Auton, Matthew

2013-09-01

We have analyzed the thermodynamic properties of the von Willebrand factor (VWF) A3 domain using urea-induced unfolding at variable temperature and thermal unfolding at variable urea concentrations to generate a phase diagram that quantitatively describes the equilibrium between native and denatured states. From this analysis, we were able to determine consistent thermodynamic parameters with various spectroscopic and calorimetric methods that define the urea-temperature parameter plane from cold denaturation to heat denaturation. Urea and thermal denaturation are experimentally reversible and independent of the thermal scan rate indicating that all transitions are at equilibrium and the van't Hoff and calorimetric enthalpies obtained from analysis of individual thermal transitions are equivalent demonstrating two-state character. Global analysis of the urea-temperature phase diagram results in a significantly higher enthalpy of unfolding than obtained from analysis of individual thermal transitions and significant cross correlations describing the urea dependence of ΔH0 and ΔCP0 that define a complex temperature dependence of the m-value. Circular dichroism (CD) spectroscopy illustrates a large increase in secondary structure content of the urea-denatured state as temperature increases and a loss of secondary structure in the thermally denatured state upon addition of urea. These structural changes in the denatured ensemble make up ∼40% of the total ellipticity change indicating a highly compact thermally denatured state. The difference between the thermodynamic parameters obtained from phase diagram analysis and those obtained from analysis of individual thermal transitions illustrates that phase diagrams capture both contributions to unfolding and denatured state expansion and by comparison are able to decipher these contributions. © 2013 The Protein Society.

Effects of two solvent conditions on the free energy landscape of the BBL peripheral subunit binding domain.

PubMed

Liu, Hanzhong; Huo, Shuanghong

2012-01-12

BBL is a small independently folding domain with two main parallel helices. The experiment of C(α) secondary shifts has shown that changing the pH from ~7 to ~5 results in the reduced helicity at the C-terminus of helix 2. Combining constant pH molecular dynamics with replica exchange, we sampled the protein conformation space and protonation states extensively under a neutral pH condition and an acidic condition. Our results reveal that the solvent conditions influence the free energy landscape. Under the neutral pH condition, the denatured state and the native state are well separated. The condition of the acidic pH reshapes the free energy surface, leading to a broadly populated denatured-state basin and a low free energy barrier between the denatured state and the native state. The acidic pH shifts the equilibrium between the denatured state and the native state in favor of the denatured state. Caution must be used to interpret experimental data under the acidic condition because the contribution of the denatured state is significant. Our simulation results are supported by the fact that the calculated chemical shifts are in good agreement with the experiment data.

Probing the Action of Chemical Denaturant on an Intrinsically Disordered Protein by Simulation and Experiment.

PubMed

Zheng, Wenwei; Borgia, Alessandro; Buholzer, Karin; Grishaev, Alexander; Schuler, Benjamin; Best, Robert B

2016-09-14

Chemical denaturants are the most commonly used agents for unfolding proteins and are thought to act by better solvating the unfolded state. Improved solvation is expected to lead to an expansion of unfolded chains with increasing denaturant concentration, providing a sensitive probe of the denaturant action. However, experiments have so far yielded qualitatively different results concerning the effects of chemical denaturation. Studies using Förster resonance energy transfer (FRET) and other methods found an increase in radius of gyration with denaturant concentration, but most small-angle X-ray scattering (SAXS) studies found no change. This discrepancy therefore challenges our understanding of denaturation mechanism and more generally the accuracy of these experiments as applied to unfolded or disordered proteins. Here, we use all-atom molecular simulations to investigate the effect of urea and guanidinium chloride on the structure of the intrinsically disordered protein ACTR, which can be studied by experiment over a wide range of denaturant concentration. Using unbiased molecular simulations with a carefully calibrated denaturant model, we find that the protein chain indeed swells with increasing denaturant concentration. This is due to the favorable association of urea or guanidinium chloride with the backbone of all residues and with the side-chains of almost all residues, with denaturant-water transfer free energies inferred from this association in reasonable accord with experimental estimates. Interactions of the denaturants with the backbone are dominated by hydrogen bonding, while interactions with side-chains include other contributions. By computing FRET efficiencies and SAXS intensities at each denaturant concentration, we show that the simulation trajectories are in accord with both experiments on this protein, demonstrating that there is no fundamental inconsistency between the two types of experiment. Agreement with experiment also supports the picture of chemical denaturation described in our simulations, driven by weak association of denaturant with the protein. Our simulations support some assumptions needed for each experiment to accurately reflect changes in protein size, namely, that the commonly used FRET chromophores do not qualitatively alter the results and that possible effects such as preferential solvent partitioning into the interior of the chain do not interfere with the determination of radius of gyration from the SAXS experiments.

Random coil negative control reproduces the discrepancy between scattering and FRET measurements of denatured protein dimensions

PubMed Central

Watkins, Herschel M.; Simon, Anna J.; Sosnick, Tobin R.; Lipman, Everett A.; Hjelm, Rex P.; Plaxco, Kevin W.

2015-01-01

Small-angle scattering studies generally indicate that the dimensions of unfolded single-domain proteins are independent (to within experimental uncertainty of a few percent) of denaturant concentration. In contrast, single-molecule FRET (smFRET) studies invariably suggest that protein unfolded states contract significantly as the denaturant concentration falls from high (∼6 M) to low (∼1 M). Here, we explore this discrepancy by using PEG to perform a hitherto absent negative control. This uncharged, highly hydrophilic polymer has been shown by multiple independent techniques to behave as a random coil in water, suggesting that it is unlikely to expand further on the addition of denaturant. Consistent with this observation, small-angle neutron scattering indicates that the dimensions of PEG are not significantly altered by the presence of either guanidine hydrochloride or urea. smFRET measurements on a PEG construct modified with the most commonly used FRET dye pair, however, produce denaturant-dependent changes in transfer efficiency similar to those seen for a number of unfolded proteins. Given the vastly different chemistries of PEG and unfolded proteins and the significant evidence that dye-free PEG is well-described as a denaturant-independent random coil, this similarity raises questions regarding the interpretation of smFRET data in terms of the hydrogen bond- or hydrophobically driven contraction of the unfolded state at low denaturant. PMID:25964362

Thermodynamics of protein denaturation at temperatures over 100 °C: CutA1 mutant proteins substituted with hydrophobic and charged residues

PubMed Central

Matsuura, Yoshinori; Takehira, Michiyo; Joti, Yasumasa; Ogasahara, Kyoko; Tanaka, Tomoyuki; Ono, Naoko; Kunishima, Naoki; Yutani, Katsuhide

2015-01-01

Although the thermodynamics of protein denaturation at temperatures over 100 °C is essential for the rational design of highly stable proteins, it is not understood well because of the associated technical difficulties. We designed certain hydrophobic mutant proteins of CutA1 from Escherichia coli, which have denaturation temperatures (Td) ranging from 101 to 113 °C and show a reversible heat denaturation. Using a hydrophobic mutant as a template, we successfully designed a hyperthermostable mutant protein (Td = 137 °C) by substituting six residues with charged ones. Thermodynamic analyses of these mutant proteins indicated that the hydrophobic mutants were stabilized by the accumulation of denaturation enthalpy (ΔH) with no entropic gain from hydrophobic solvation around 100 °C, and that the stabilization due to salt bridges resulted from both the increase in ΔH from ion-ion interactions and the entropic effect of the electrostatic solvation over 113 °C. This is the first experimental evidence that has successfully overcome the typical technical difficulties. PMID:26497062

Mesoscopic modeling of DNA denaturation rates: Sequence dependence and experimental comparison

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dahlen, Oda, E-mail: oda.dahlen@ntnu.no; Erp, Titus S. van, E-mail: titus.van.erp@ntnu.no

Using rare event simulation techniques, we calculated DNA denaturation rate constants for a range of sequences and temperatures for the Peyrard-Bishop-Dauxois (PBD) model with two different parameter sets. We studied a larger variety of sequences compared to previous studies that only consider DNA homopolymers and DNA sequences containing an equal amount of weak AT- and strong GC-base pairs. Our results show that, contrary to previous findings, an even distribution of the strong GC-base pairs does not always result in the fastest possible denaturation. In addition, we applied an adaptation of the PBD model to study hairpin denaturation for which experimentalmore » data are available. This is the first quantitative study in which dynamical results from the mesoscopic PBD model have been compared with experiments. Our results show that present parameterized models, although giving good results regarding thermodynamic properties, overestimate denaturation rates by orders of magnitude. We believe that our dynamical approach is, therefore, an important tool for verifying DNA models and for developing next generation models that have higher predictive power than present ones.« less

Correlated parameter fit of arrhenius model for thermal denaturation of proteins and cells.

PubMed

Qin, Zhenpeng; Balasubramanian, Saravana Kumar; Wolkers, Willem F; Pearce, John A; Bischof, John C

2014-12-01

Thermal denaturation of proteins is critical to cell injury, food science and other biomaterial processing. For example protein denaturation correlates strongly with cell death by heating, and is increasingly of interest in focal thermal therapies of cancer and other diseases at temperatures which often exceed 50 °C. The Arrhenius model is a simple yet widely used model for both protein denaturation and cell injury. To establish the utility of the Arrhenius model for protein denaturation at 50 °C and above its sensitivities to the kinetic parameters (activation energy E a and frequency factor A) were carefully examined. We propose a simplified correlated parameter fit to the Arrhenius model by treating E a, as an independent fitting parameter and allowing A to follow dependently. The utility of the correlated parameter fit is demonstrated on thermal denaturation of proteins and cells from the literature as a validation, and new experimental measurements in our lab using FTIR spectroscopy to demonstrate broad applicability of this method. Finally, we demonstrate that the end-temperature within which the denaturation is measured is important and changes the kinetics. Specifically, higher E a and A parameters were found at low end-temperature (50 °C) and reduce as end-temperatures increase to 70 °C. This trend is consistent with Arrhenius parameters for cell injury in the literature that are significantly higher for clonogenics (45-50 °C) vs. membrane dye assays (60-70 °C). Future opportunities to monitor cell injury by spectroscopic measurement of protein denaturation are discussed.

Correlated Parameter Fit of Arrhenius Model for Thermal Denaturation of Proteins and Cells

PubMed Central

Qin, Zhenpeng; Balasubramanian, Saravana Kumar; Wolkers, Willem F.; Pearce, John A.; Bischof, John C.

2014-01-01

Thermal denaturation of proteins is critical to cell injury, food science and other biomaterial processing. For example protein denaturation correlates strongly with cell death by heating, and is increasingly of interest in focal thermal therapies of cancer and other diseases at temperatures which often exceed 50 °C. The Arrhenius model is a simple yet widely used model for both protein denaturation and cell injury. To establish the utility of the Arrhenius model for protein denaturation at 50 °C and above its sensitivities to the kinetic parameters (activation energy Ea and frequency factor A) were carefully examined. We propose a simplified correlated parameter fit to the Arrhenius model by treating Ea, as an independent fitting parameter and allowing A to follow dependently. The utility of the correlated parameter fit is demonstrated on thermal denaturation of proteins and cells from the literature as a validation, and new experimental measurements in our lab using FTIR spectroscopy to demonstrate broad applicability of this method. Finally, we demonstrate that the end-temperature within which the denaturation is measured is important and changes the kinetics. Specifically, higher Ea and A parameters were found at low end-temperature (50°C) and reduce as end-temperatures increase to 70 °C. This trend is consistent with Arrhenius parameters for cell injury in the literature that are significantly higher for clonogenics (45 – 50 °C) vs. membrane dye assays (60 –70 °C). Future opportunities to monitor cell injury by spectroscopic measurement of protein denaturation are discussed. PMID:25205396

Experimental and Modelling Study of the Denaturation of Milk Protein by Heat Treatment

PubMed Central

Qian, Fang; Sun, Jiayue; Cao, Di; Tuo, Yanfeng; Jiang, Shujuan; Mu, Guangqing

2017-01-01

Heat treatment of milk aims to inhibit the growth of microbes, extend the shelf-life of products and improve the quality of the products. Heat treatment also leads to denaturation of whey protein and the formation of whey protein-casein polymer, which has negative effects on milk product. Hence the milk heat treatment conditions should be controlled in milk processing. In this study, the denaturation degree of whey protein and the combination degree of whey protein and casein when undergoing heat treatment were also determined by using the Native-PAGE and SDS-PAGE analysis. The results showed that the denaturation degree of whey protein and the combination degree of whey protein with casein extended with the increase of the heat-treated temperature and time. The effects of the heat-treated temperature and heat-treated time on the denaturation degree of whey protein and on the combination degree of whey protein and casein were well described using the quadratic regression equation. The analysis strategy used in this study reveals an intuitive and effective measure of the denaturation degree of whey protein, and the changes of milk protein under different heat treatment conditions efficiently and accurately in the dairy industry. It can be of great significance for dairy product proteins following processing treatments applied for dairy product manufacturing. PMID:28316470

Urea-induced denaturation of human calcium/calmodulin-dependent protein kinase IV: a combined spectroscopic and MD simulation studies.

PubMed

Naz, Huma; Shahbaaz, Mohd; Haque, Md Anzarul; Bisetty, Krishna; Islam, Asimul; Ahmad, Faizan; Hassan, Md Imtaiyaz

2017-02-01

Calcium/calmodulin-dependent protein kinase IV (CaMKIV) is a multifunctional enzyme which belongs to the Ser/Thr kinase family. CaMKIV plays important role in varieties of biological processes such as gene expression regulation, memory consolidation, bone growth, T-cell maturation, sperm motility, regulation of microtubule dynamics, cell-cycle progression, and apoptosis. To measure stability parameters, urea-induced denaturation of CaMKIV was carried out at pH 7.4 and 25°C, using three different probes, namely far-UV CD, near-UV absorption, and tryptophan fluorescence. A coincidence of normalized denaturation curves of these optical properties suggests that urea-induced denaturation is a two-state process. Analysis of these denaturation curves gave values of 4.20 ± 0.12 kcal mol -1 , 2.95 ± 0.15 M, and 1.42 ± 0.06 kcal mol -1  M -1 for [Formula: see text] (Gibbs free energy change (ΔG D ) in the absence of urea), C m (molar urea concentration ([urea]) at the midpoint of the denaturation curve), and m (=∂ΔG D /∂[urea]), respectively. All these experimental observations have been fully supported by 30 ns molecular dynamics simulation studies.

Sharing Data between Mobile Devices, Connected Vehicles and Infrastructure Task 8 : D2X Hub Proof-of-Concept Test Evaluation Report.

DOT National Transportation Integrated Search

2017-10-25

The Task 8 D2X Hub Proof-of-Concept Test Evaluation Report provides results of the experimental data analysis performed in accordance with the experimental plan for the proof-of-concept version of the prototype system. The data set analyzed includes ...

Probing the free energy landscape of the FBP28WW domain using multiple techniques.

PubMed

Periole, Xavier; Allen, Lucy R; Tamiola, Kamil; Mark, Alan E; Paci, Emanuele

2009-05-01

The free-energy landscape of a small protein, the FBP 28 WW domain, has been explored using molecular dynamics (MD) simulations with alternative descriptions of the molecule. The molecular models used range from coarse-grained to all-atom with either an implicit or explicit treatment of the solvent. Sampling of conformation space was performed using both conventional and temperature-replica exchange MD simulations. Experimental chemical shifts and NOEs were used to validate the simulations, and experimental phi values both for validation and as restraints. This combination of different approaches has provided insight into the free energy landscape and barriers encountered by the protein during folding and enabled the characterization of native, denatured and transition states which are compatible with the available experimental data. All the molecular models used stabilize well defined native and denatured basins; however, the degree of agreement with the available experimental data varies. While the most detailed, explicit solvent model predicts the data reasonably accurately, it does not fold despite a simulation time 10 times that of the experimental folding time. The less detailed models performed poorly relative to the explicit solvent model: an implicit solvent model stabilizes a ground state which differs from the experimental native state, and a structure-based model underestimates the size of the barrier between the two states. The use of experimental phi values both as restraints, and to extract structures from unfolding simulations, result in conformations which, although not necessarily true transition states, appear to share the geometrical characteristics of transition state structures. In addition to characterizing the native, transition and denatured states of this particular system in this work, the advantages and limitations of using varying levels of representation are discussed. 2008 Wiley Periodicals, Inc.

Molecular insight into the counteraction of trehalose on urea-induced protein denaturation using molecular dynamics simulation.

PubMed

Zhang, Na; Liu, Fu-Feng; Dong, Xiao-Yan; Sun, Yan

2012-06-21

Considerable experimental evidence indicates that trehalose can counteract the denaturing effects of urea on proteins. However, its molecular mechanism remains unknown due to the limitations of current experimental techniques. Herein, molecular dynamics simulations were performed to investigate the counteracting effects of trehalose against urea-induced denaturation of chymotrypsin inhibitor 2. The simulations indicate that the protein unfolds in 8 mol/L urea, but at the same condition the protein retains its native structure in the ternary solution of 8 mol/L urea and 1 mol/L trehalose. It is confirmed that the preferential exclusion of trehalose from the protein surface is the origin of its counteracting effects. It is found that trehalose binds urea via hydrogen bonds, so urea molecules are also expelled from the protein surface along with the preferential exclusion of trehalose. The exclusion of urea from the protein surface leads to the alleviation of the Lennard-Jones interactions between urea and the hydrophobic side chains of the protein in the ternary solution. In contrast, the electrostatic interactions between urea and the protein change little in the presence of trehalose because the decrease in the electrostatic interactions between urea and the protein backbone is canceled by the increase in the electrostatic interactions between urea and the charged side chains of the protein. The results have provided molecular explanations for the counteraction of urea-induced protein denaturation by trehalose.

Thermal denaturation of protein studied by terahertz time-domain spectroscopy

NASA Astrophysics Data System (ADS)

Fu, Xiuhua; Li, Xiangjun; Liu, Jianjun; Du, Yong; Hong, Zhi

2012-12-01

In this study, the absorption spectra of native or thermal protein were measured in 0.2-1.4THz using terahertz time-domain spectroscopy (THz-TDS) system at room temperature, their absorption spectra and the refractive spectra were obtained. Experimental results indicate that protein both has strong absorption but their characteristics were not distinct in the THz region, and the absorption decreased during thermal denatured state. In order to prove protein had been denatured, we used Differential scanning calorimeter (DSC) measured their denatured temperature, from their DSC heating traces, collagen Td=101℃, Bovine serum albumin Td=97℃. While we also combined the Fourier transform infrared spectrometer (FTIR) to investigate their secondary and tertiary structure before and after denatuation, but the results did not have the distinct changes. We turned the absorption spectra and the refractive spectra to the dielectric spectra, and used the one-stage Debye model simulated the terahertz dielectric spectra of protein before and after denaturation. This research proved that the terahertz spectrum technology is feasible in testing protein that were affected by temperature or other factors which can provide theoretical foundation in the further study about the THz spectrum of protein and peptide temperature stability.

"Cooperative collapse" of the denatured state revealed through Clausius-Clapeyron analysis of protein denaturation phase diagrams.

PubMed

Tischer, Alexander; Machha, Venkata R; Rösgen, Jörg; Auton, Matthew

2018-02-19

Protein phase diagrams have a unique potential to identify the presence of additional thermodynamic states even when non-2-state character is not readily apparent from the experimental observables used to follow protein unfolding transitions. Two-state analysis of the von Willebrand factor A3 domain has previously revealed a discrepancy in the calorimetric enthalpy obtained from thermal unfolding transitions as compared with Gibbs-Helmholtz analysis of free energies obtained from the Linear Extrapolation Method (Tischer and Auton, Prot Sci 2013; 22(9):1147-60). We resolve this thermodynamic conundrum using a Clausius-Clapeyron analysis of the urea-temperature phase diagram that defines how ΔH and the urea m-value interconvert through the slope of c m versus T, (∂cm/∂T)=ΔH/(mT). This relationship permits the calculation of ΔH at low temperature from m-values obtained through iso-thermal urea denaturation and high temperature m-values from ΔH obtained through iso-urea thermal denaturation. Application of this equation uncovers sigmoid transitions in both cooperativity parameters as temperature is increased. Such residual thermal cooperativity of ΔH and the m-value confirms the presence of an additional state which is verified to result from a cooperative phase transition between urea-expanded and thermally-compact denatured states. Comparison of the equilibria between expanded and compact denatured ensembles of disulfide-intact and carboxyamidated A3 domains reveals that introducing a single disulfide crosslink does not affect the presence of the additional denatured state. It does, however, make a small thermodynamically favorable free energy (∼-13 ± 1 kJ/mol) contribution to the cooperative denatured state collapse transition as temperature is raised and urea concentration is lowered. The thermodynamics of this "cooperative collapse" of the denatured state retain significant compensations between the enthalpy and entropy contributions to the overall free energy. © 2018 Wiley Periodicals, Inc.

Theory of the Protein Equilibrium Population Snapshot by H/D Exchange Electrospray Ionization Mass Spectrometry (PEPS-HDX-ESI-MS) Method used to obtain Protein Folding Energies/Rates and Selected Supporting Experimental Evidence.

PubMed

Liyanage, Rohana; Devarapalli, Nagarjuna; Pyland, Derek B; Puckett, Latisha M; Phan, N H; Starch, Joel A; Okimoto, Mark R; Gidden, Jennifer; Stites, Wesley E; Lay, Jackson O

2012-12-15

Protein equilibrium snapshot by hydrogen/deuterium exchange electrospray ionization mass spectrometry (PEPS-HDX-ESI-MS or PEPS) is a method recently introduced for estimating protein folding energies and rates. Herein we describe the basis for this method using both theory and new experiments. Benchmark experiments were conducted using ubiquitin because of the availability of reference data for folding and unfolding rates from NMR studies. A second set of experiments was also conducted to illustrate the surprising resilience of the PEPS to changes in HDX time, using staphylococcal nuclease and time frames ranging from a few seconds to several minutes. Theory suggests that PEPS experiments should be conducted at relatively high denaturant concentrations, where the protein folding/unfolding rates are slow with respect to HDX and the life times of both the closed and open states are long enough to be sampled experimentally. Upon deliberate denaturation, changes in folding/unfolding are correlated with associated changes in the ESI-MS signal upon fast HDX. When experiments are done quickly, typically within a few seconds, ESI-MS signals, corresponding to the equilibrium population of the native (closed) and denatured (open) states can both be detected. The interior of folded proteins remains largely un-exchanged. Amongst MS methods, the simultaneous detection of both states in the spectrum is unique to PEPS and provides a "snapshot" of these populations. The associated ion intensities are used to estimate the protein folding equilibrium constant (or the free energy change, ΔG). Linear extrapolation method (LEM) plots of derived ΔG values for each denaturant concentration can then be used to calculate ΔG in the absence of denaturant, ΔG(H(2)O). In accordance with the requirement for detection of signals for both the folded and unfolded states, this theoretical framework predicts that PEPS experiments work best at the middle of the denaturation curve where natured and denatured protein molecules are equilibrated at easily detectable ratios, namely 1:1. It also requires that closed and open states have lifetimes measurable in the time frame of the HDX experiment. Because both conditions are met by PEPS, these measurements can provide an accurate assessment of closed/open state populations and thus protein folding energies/rates.

Small-Angle X-ray Scattering and Single-Molecule FRET Spectroscopy Produce Highly Divergent Views of the Low-Denaturant Unfolded State

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yoo, Tae Yeon; Meisburger, Steve P.; Hinshaw, James

2012-10-10

The results of more than a dozen single-molecule Foerster resonance energy transfer (smFRET) experiments suggest that chemically unfolded polypeptides invariably collapse from an expanded random coil to more compact dimensions as the denaturant concentration is reduced. In sharp contrast, small-angle X-ray scattering (SAXS) studies suggest that, at least for single-domain proteins at non-zero denaturant concentrations, such compaction may be rare. Here, we explore this discrepancy by studying protein L, a protein previously studied by SAXS (at 5 C), which suggested fixed unfolded-state dimensions from 1.4 to 5 M guanidine hydrochloride (GuHCl), and by smFRET (at 25 C), which suggested that,more » in contrast, the chain contracts by 15-30% over this same denaturant range. Repeating the earlier SAXS study under the same conditions employed in the smFRET studies, we observe little, if any, evidence that the unfolded state of protein L contracts as the concentration of GuHCl is reduced. For example, scattering profiles (and thus the shape and dimensions) collected within {approx} 4 ms after dilution to as low as 0.67 M GuHCl are effectively indistinguishable from those observed at equilibrium at higher denaturant. Our results thus argue that the disagreement between SAXS and smFRET is statistically significant and that the experimental evidence in favor of obligate polypeptide collapse at low denaturant cannot be considered conclusive yet.« less

Structural stability of Amandin, a major allergen from almond (Prunus dulcis), and its acidic and basic polypeptides.

PubMed

Albillos, Silvia M; Menhart, Nicholas; Fu, Tong-Jen

2009-06-10

Information relating to the resistance of food allergens to thermal and/or chemical denaturation is critical if a reduction in protein allergenicity is to be achieved through food-processing means. This study examined the changes in the secondary structure of an almond allergen, amandin, and its acidic and basic polypeptides as a result of thermal and chemical denaturation. Amandin ( approximately 370 kDa) was purified by cryoprecipitation followed by gel filtration chromatography and subjected to thermal (13-96 degrees C) and chemical (urea and dithiothreitol) treatments. Changes in the secondary structure of the protein were followed using circular dichroism spectroscopy. The secondary structure of the hexameric amandin did not undergo remarkable changes at temperatures up to 90 degrees C, although protein aggregation was observed. In the presence of a reducing agent, irreversible denaturation occurred with the following experimental values: T(m) = 72.53 degrees C (transition temperature), DeltaH = 87.40 kcal/mol (unfolding enthalpy), and C(p) = 2.48 kcal/(mol degrees C) (heat capacity). The concentration of urea needed to achieve 50% denaturation was 2.59 M, and the Gibbs free energy of chemical denaturation was calculated to be DeltaG = 3.82 kcal/mol. The basic and acidic polypeptides of amandin had lower thermal stabilities than the multimeric protein.

Ligand Binding Analysis and Screening by Chemical Denaturation Shift

PubMed Central

Sch n, Arne; Brown, Richard K.; Hutchins, Burleigh M.; Freire, Ernesto

2013-01-01

The identification of small molecule ligands is an important first step in drug development, especially drugs that target proteins with no intrinsic activity. Towards this goal, it is important to have access to technologies that are able to measure binding affinities for a large number of potential ligands in a fast and accurate way. Since ligand binding stabilizes the protein structure in a manner dependent on concentration and binding affinity, the magnitude of the protein stabilization effect elicited by binding can be used to identify and characterize ligands. For example, the shift in protein denaturation temperature (Tm shift) has become a popular approach to identify potential ligands. However, Tm shifts cannot be readily transformed into binding affinities and the ligand rank order obtained at denaturation temperatures (60°C or higher) does not necessarily coincide with the rank order at physiological temperature. An alternative approach is the use of chemical denaturation, which can be implemented at any temperature. Chemical denaturation shifts allow accurate determination of binding affinities with a surprisingly wide dynamic range (high micromolar to sub nanomolar) and in situations in which binding changes the cooperativity of the unfolding transition. In this paper we develop the basic analytical equations and provide several experimental examples. PMID:23994566

Ligand binding analysis and screening by chemical denaturation shift.

PubMed

Schön, Arne; Brown, Richard K; Hutchins, Burleigh M; Freire, Ernesto

2013-12-01

The identification of small molecule ligands is an important first step in drug development, especially drugs that target proteins with no intrinsic activity. Toward this goal, it is important to have access to technologies that are able to measure binding affinities for a large number of potential ligands in a fast and accurate way. Because ligand binding stabilizes the protein structure in a manner dependent on concentration and binding affinity, the magnitude of the protein stabilization effect elicited by binding can be used to identify and characterize ligands. For example, the shift in protein denaturation temperature (Tm shift) has become a popular approach to identify potential ligands. However, Tm shifts cannot be readily transformed into binding affinities, and the ligand rank order obtained at denaturation temperatures (≥60°C) does not necessarily coincide with the rank order at physiological temperature. An alternative approach is the use of chemical denaturation, which can be implemented at any temperature. Chemical denaturation shifts allow accurate determination of binding affinities with a surprisingly wide dynamic range (high micromolar to sub nanomolar) and in situations where binding changes the cooperativity of the unfolding transition. In this article, we develop the basic analytical equations and provide several experimental examples. Copyright © 2013 Elsevier Inc. All rights reserved.

Distribution, transition and thermodynamic stability of protein conformations in the denaturant-induced unfolding of proteins.

PubMed

Bian, Liujiao; Ji, Xu

2014-01-01

Extensive and intensive studies on the unfolding of proteins require appropriate theoretical model and parameter to clearly illustrate the feature and characteristic of the unfolding system. Over the past several decades, four approaches have been proposed to describe the interaction between proteins and denaturants, but some ambiguity and deviations usually occur in the explanation of the experimental data. In this work, a theoretical model was presented to show the dependency of the residual activity ratio of the proteins on the molar denaturant concentration. Through the characteristic unfolding parameters ki and Δmi in this model, the distribution, transition and thermodynamic stability of protein conformations during the unfolding process can be quantitatively described. This model was tested with the two-state unfolding of bovine heart cytochrome c and the three-state unfolding of hen egg white lysozyme induced by both guanidine hydrochloride and urea, the four-state unfolding of bovine carbonic anhydrase b induced by guanidine hydrochloride and the unfolding of some other proteins induced by denaturants. The results illustrated that this model could be used accurately to reveal the distribution and transition of protein conformations in the presence of different concentrations of denaturants and to evaluate the unfolding tendency and thermodynamic stability of different conformations. In most denaturant-induced unfolding of proteins, the unfolding became increasingly hard in next transition step and the proteins became more unstable as they attained next successive stable conformation. This work presents a useful method for people to study the unfolding of proteins and may be used to describe the unfolding and refolding of other biopolymers induced by denaturants, inducers, etc.

The Efficiency of Different Salts to Screen Charge Interactions in Proteins: A Hofmeister Effect?

PubMed Central

Perez-Jimenez, Raul; Godoy-Ruiz, Raquel; Ibarra-Molero, Beatriz; Sanchez-Ruiz, Jose M.

2004-01-01

Understanding the screening by salts of charge-charge interactions in proteins is important for at least two reasons: a), screening by intracellular salt concentration may modulate the stability and interactions of proteins in vivo; and b), the in vitro experimental estimation of the contributions from charge-charge interactions to molecular processes involving proteins is generally carried out on the basis of the salt effect on process energetics, under the assumption that these interactions are screened out by moderate salt concentrations. Here, we explore experimentally the extent to which the screening efficiency depends on the nature of the salt. To this end, we have carried out an energetic characterization of the effect of NaCl (a nondenaturing salt), guanidinium chloride (a denaturing salt), and guanidinium thiocyanate (a stronger denaturant) on the stability of the wild-type form and a T14K variant of Escherichia coli thioredoxin. Our results suggest that the efficiency of different salts to screen charge-charge interactions correlates with their denaturing strength and with the position of the constituent ions in the Hofmeister rankings. This result appears consistent with the plausible relation of the Hofmeister rankings with the extent of solute accumulation/exclusion from protein surfaces. PMID:15041679

Dynamical Scaling and Phase Coexistence in Topologically Constrained DNA Melting.

PubMed

Fosado, Y A G; Michieletto, D; Marenduzzo, D

2017-09-15

There is a long-standing experimental observation that the melting of topologically constrained DNA, such as circular closed plasmids, is less abrupt than that of linear molecules. This finding points to an important role of topology in the physics of DNA denaturation, which is, however, poorly understood. Here, we shed light on this issue by combining large-scale Brownian dynamics simulations with an analytically solvable phenomenological Landau mean field theory. We find that the competition between melting and supercoiling leads to phase coexistence of denatured and intact phases at the single-molecule level. This coexistence occurs in a wide temperature range, thereby accounting for the broadening of the transition. Finally, our simulations show an intriguing topology-dependent scaling law governing the growth of denaturation bubbles in supercoiled plasmids, which can be understood within the proposed mean field theory.

Proof-test-based life prediction of high-toughness pressure vessels

DOE Office of Scientific and Technical Information (OSTI.GOV)

Panontin, T.L.; Hill, M.R.

1996-02-01

The paper examines the problems associated with applying proof-test-based life prediction to vessels made of high-toughness metals. Two A106 Gr B pipe specimens containing long, through-wall circumferential flaws were tested. One failed during hydrostatic testing and the other during tension-tension cycling following a hydrostatic test. Quantitative fractography was used to verify experimentally obtained fatigue crack growth rates and a variety of LEFM and EPFM techniques were used to analyze the experimental results. The results show that: plastic collapse analysis provides accurate predictions of screened (initial) crack size when the flow stress is determined experimentally; LEFM analysis underestimates the crack sizemore » screened by the proof test and overpredicts the subsequent fatigue life of the vessel when retardation effects are small (i.e., low proof levels); and, at a high proof-test level (2.4 {times} operating pressure), the large retardation effect on fatigue crack growth due to the overload overwhelmed the deleterious effect on fatigue life from stable tearing during the proof test and alleviated the problem of screening only long cracks due to the high toughness of the metal.« less

Experimental ladder proof of Hardy's nonlocality for high-dimensional quantum systems

NASA Astrophysics Data System (ADS)

Chen, Lixiang; Zhang, Wuhong; Wu, Ziwen; Wang, Jikang; Fickler, Robert; Karimi, Ebrahim

2017-08-01

Recent years have witnessed a rapidly growing interest in high-dimensional quantum entanglement for fundamental studies as well as towards novel applications. Therefore, the ability to verify entanglement between physical qudits, d -dimensional quantum systems, is of crucial importance. To show nonclassicality, Hardy's paradox represents "the best version of Bell's theorem" without using inequalities. However, so far it has only been tested experimentally for bidimensional vector spaces. Here, we formulate a theoretical framework to demonstrate the ladder proof of Hardy's paradox for arbitrary high-dimensional systems. Furthermore, we experimentally demonstrate the ladder proof by taking advantage of the orbital angular momentum of high-dimensionally entangled photon pairs. We perform the ladder proof of Hardy's paradox for dimensions 3 and 4, both with the ladder up to the third step. Our paper paves the way towards a deeper understanding of the nature of high-dimensionally entangled quantum states and may find applications in quantum information science.

Proof test methodology for composites

NASA Technical Reports Server (NTRS)

Wu, Edward M.; Bell, David K.

1992-01-01

The special requirements for proof test of composites are identified based on the underlying failure process of composites. Two proof test methods are developed to eliminate the inevitable weak fiber sites without also causing flaw clustering which weakens the post-proof-test composite. Significant reliability enhancement by these proof test methods has been experimentally demonstrated for composite strength and composite life in tension. This basic proof test methodology is relevant to the certification and acceptance of critical composite structures. It can also be applied to the manufacturing process development to achieve zero-reject for very large composite structures.

Aqueous ionic liquids and their effects on protein structures: an overview on recent theoretical and experimental results

NASA Astrophysics Data System (ADS)

Smiatek, Jens

2017-06-01

Ionic liquids (ILs) are used in a variety of technological and biological applications. Recent experimental and simulation results reveal the influence of aqueous ionic liquids on the stability of protein and enzyme structures. Depending on different parameters like the concentration and the ion composition, one can observe distinct stabilization or denaturation mechanisms for various ILs. In this review, we summarize the main findings and discuss the implications with regard to molecular theories of solutions and specific ion effects. A preferential binding model is introduced in order to discuss protein-IL effects from a statistical mechanics perspective. The value of the preferential binding coefficient determines the strength of the ion influence and indicates a shift of the chemical equilibrium either to the native or the denatured state of the protein. We highlight the role of water in order to explain the self-association behavior of the IL species and discuss recent experimental and simulation results in the light of the observed binding effects.

Bubbles and denaturation in DNA

NASA Astrophysics Data System (ADS)

van Erp, T. S.; Cuesta-López, S.; Peyrard, M.

2006-08-01

The local opening of DNA is an intriguing phenomenon from a statistical-physics point of view, but is also essential for its biological function. For instance, the transcription and replication of our genetic code cannot take place without the unwinding of the DNA double helix. Although these biological processes are driven by proteins, there might well be a relation between these biological openings and the spontaneous bubble formation due to thermal fluctuations. Mesoscopic models, like the Peyrard-Bishop-Dauxois (PBD) model, have fairly accurately reproduced some experimental denaturation curves and the sharp phase transition in the thermodynamic limit. It is, hence, tempting to see whether these models could be used to predict the biological activity of DNA. In a previous study, we introduced a method that allows to obtain very accurate results on this subject, which showed that some previous claims in this direction, based on molecular-dynamics studies, were premature. This could either imply that the present PBD model should be improved or that biological activity can only be predicted in a more complex framework that involves interactions with proteins and super helical stresses. In this article, we give a detailed description of the statistical method introduced before. Moreover, for several DNA sequences, we give a thorough analysis of the bubble-statistics as a function of position and bubble size and the so-called l-denaturation curves that can be measured experimentally. These show that some important experimental observations are missing in the present model. We discuss how the present model could be improved.

Monte Carlo Simulations of the Kinetics of Protein Adsorption

NASA Astrophysics Data System (ADS)

Zhdanov, V. P.; Kasemo, B.

The past decade has been characterized by rapid progress in Monte Carlo simulations of protein folding in a solution. This review summarizes the main results obtained in the field, as a background to the major topic, namely corresponding advances in simulations of protein adsorption kinetics at solid-liquid interfaces. The latter occur via diffusion in the liquid towards the interface followed by actual adsorption, and subsequent irreversible conformational changes, resulting in more or less pronounced denaturation of the native protein structure. The conventional kinetic models describing these steps are based on the assumption that the denaturation transitions obey the first-order law with a single value of the denaturation rate constant kr. The validity of this assumption has been studied in recent lattice Monte Carlo simulations of denaturation of model protein-like molecules with different types of the monomer-monomer interactions. The results obtained indicate that, due to trapping in metastable states, (i) the transition of a molecule to the denatured state is usually nonexponential in time, i.e. it does not obey the first-order law, and (ii) the denaturation transitions of an ensemble of different molecules are characterized by different time scales, i.e. the denaturation process cannot be described by a single rate constant kr. One should, rather, introduce a distribution of values of this rate constant (physically, different values of kr reflect the fact that the transitions to the altered state occurs via different metastable states). The phenomenological kinetics of irreversible adsorption of proteins with and without a distribution of the denaturation rate constant values have been calculated in the limits where protein diffusion in the solution is, respectively, rapid or slow. In both cases, the adsorption kinetics with a distribution of kr are found to be close to those with a single-valued rate constant kr, provided that the average value of kr in the former case is equal to kr in the latter case. This conclusion holds even for wide distributions of kr. The consequences of this finding for the fitting of global experimental kinetics on the basis of phenomenological equations are briefly discussed.

Using fluorescence correlation spectroscopy to study conformational changes in denatured proteins.

PubMed

Sherman, Eilon; Itkin, Anna; Kuttner, Yosef Yehuda; Rhoades, Elizabeth; Amir, Dan; Haas, Elisha; Haran, Gilad

2008-06-01

Fluorescence correlation spectroscopy (FCS) is a sensitive analytical tool that allows dynamics and hydrodynamics of biomolecules to be studied under a broad range of experimental conditions. One application of FCS of current interest is the determination of the size of protein molecules in the various states they sample along their folding reaction coordinate, which can be accessed through the measurement of diffusion coefficients. It has been pointed out that the analysis of FCS curves is prone to artifacts that may lead to erroneous size determination. To set the stage for FCS studies of unfolded proteins, we first show that the diffusion coefficients of small molecules as well as proteins can be determined accurately even in the presence of high concentrations of co-solutes that change the solution refractive index significantly. Indeed, it is found that the Stokes-Einstein relation between the measured diffusion coefficient and solution viscosity holds even in highly concentrated glycerol or guanidinium hydrochloride (GuHCl) solutions. These measurements form the basis for an investigation of the structure of the denatured state of two proteins, the small protein L and the larger, three-domain protein adenylate kinase (AK). FCS is found useful for probing expansion in the denatured state beyond the unfolding transition. It is shown that the denatured state of protein L expands as the denaturant concentration increases, in a process akin to the transition from a globule to a coil in polymers. This process continues at least up to 5 M GuHCl. On the other hand, the denatured state of AK does not seem to expand much beyond 2 M GuHCl, a result that is in qualitative accord with single-molecule fluorescence histograms. Because both the unfolding transition and the coil-globule transition of AK occur at a much lower denaturant concentration than those of protein L, a possible correlation between the two phenomena is suggested.

Thermal Stabilization of Dihydrofolate Reductase Using Monte Carlo Unfolding Simulations and Its Functional Consequences

PubMed Central

Whitney, Anna; Shakhnovich, Eugene I.

2015-01-01

Design of proteins with desired thermal properties is important for scientific and biotechnological applications. Here we developed a theoretical approach to predict the effect of mutations on protein stability from non-equilibrium unfolding simulations. We establish a relative measure based on apparent simulated melting temperatures that is independent of simulation length and, under certain assumptions, proportional to equilibrium stability, and we justify this theoretical development with extensive simulations and experimental data. Using our new method based on all-atom Monte-Carlo unfolding simulations, we carried out a saturating mutagenesis of Dihydrofolate Reductase (DHFR), a key target of antibiotics and chemotherapeutic drugs. The method predicted more than 500 stabilizing mutations, several of which were selected for detailed computational and experimental analysis. We find a highly significant correlation of r = 0.65–0.68 between predicted and experimentally determined melting temperatures and unfolding denaturant concentrations for WT DHFR and 42 mutants. The correlation between energy of the native state and experimental denaturation temperature was much weaker, indicating the important role of entropy in protein stability. The most stabilizing point mutation was D27F, which is located in the active site of the protein, rendering it inactive. However for the rest of mutations outside of the active site we observed a weak yet statistically significant positive correlation between thermal stability and catalytic activity indicating the lack of a stability-activity tradeoff for DHFR. By combining stabilizing mutations predicted by our method, we created a highly stable catalytically active E. coli DHFR mutant with measured denaturation temperature 7.2°C higher than WT. Prediction results for DHFR and several other proteins indicate that computational approaches based on unfolding simulations are useful as a general technique to discover stabilizing mutations. PMID:25905910

Unravelling the hydrophobicity of urea in water using thermodiffusion: implications for protein denaturation.

PubMed

Niether, Doreen; Di Lecce, Silvia; Bresme, Fernando; Wiegand, Simone

2018-01-03

Urea is widely used as a protein denaturant in aqueous solutions. Experimental and computer simulation studies have shown that it dissolves in water almost ideally at high concentrations, introducing little disruption in the water hydrogen bonded structure. However, at concentrations of the order of 5 M or higher, urea induces denaturation in a wide range of proteins. The origin of this behaviour is not completely understood, but it is believed to stem from a balance between urea-protein and urea-water interactions, with urea becoming possibly hydrophobic at a specific concentration range. The small changes observed in the water structure make it difficult to connect the denaturation effects to the solvation properties. Here we show that the exquisite sensitivity of thermodiffusion to solute-water interactions allows the identification of the onset of hydrophobicity of urea-water mixtures. The hydrophobic behaviour is reflected in a sign reversal of the temperature dependent slope of the Soret coefficient, which is observed, both in experiments and non-equilibrium computer simulations at ∼5 M concentration of urea in water. This concentration regime corresponds to the one where abrupt changes in the denaturation of proteins are commonly observed. We show that the onset of hydrophobicity is intrinsically connected to the urea-water interactions. Our results allow us to identify correlations between the Soret coefficient and the partition coefficient, log P, hence establishing the thermodiffusion technique as a powerful approach to study hydrophobicity.

Structural studies on serum albumins under green light irradiation.

PubMed

Comorosan, Sorin; Polosan, Silviu; Popescu, Irinel; Ionescu, Elena; Mitrica, Radu; Cristache, Ligia; State, Alina Elena

2010-10-01

This paper presents two new experimental results: the protective effect of green light (GL) on ultraviolet (UV) denaturation of proteins, and the effect of GL on protein macromolecular structures. The protective effect of GL was revealed on two serum albumins, bovine (BSA) and human (HSA), and recorded by electrophoresis, absorption, and circular dichroism spectra. The effect of GL irradiation on protein structure was recorded by using fluorescence spectroscopy and electrophoresis. These new effects were modeled by quantum-chemistry computation using Gaussian 03 W, leading to good fit between theoretical and experimental absorption and circular dichroism spectra. A mechanism for these phenomena is suggested, based on a double-photon absorption process. This nonlinear effect may lead to generation of long-lived Rydberg macromolecular systems, capable of long-range interactions. These newly suggested systems, with macroscopic quantum coherence behaviors, may block the UV denaturation processes.

On the ability of trehalose to offset the denaturing activity of urea

NASA Astrophysics Data System (ADS)

Graziano, Giuseppe

2013-01-01

Experimental and computational studies indicate that trehalose offsets the denaturing activity of urea. According to a statistical thermodynamic model, the fundamental stabilizing contribution arises from the difference in the work to create a cavity suitable to host the D-state and the N-state, respectively. The magnitude of this solvent-excluded volume effect increases with aqueous solution density. Trehalose and urea addition to water leads to a density increase, that translates in an increase in the magnitude of the solvent-excluded volume effect which is so large to overwhelm the destabilizing contribution arising from the energetic attractions of urea with protein surface groups.

Effects of reading-oriented tasks on students' reading comprehension of geometry proof

NASA Astrophysics Data System (ADS)

Yang, Kai-Lin; Lin, Fou-Lai

2012-06-01

This study compared the effects of reading-oriented tasks and writing-oriented tasks on students' reading comprehension of geometry proof (RCGP). The reading-oriented tasks were designed with reading strategies and the idea of problem posing. The writing-oriented tasks were consistent with usual proof instruction for writing a proof and applying it. Twenty-two classes of ninth-grade students ( N = 683), aged 14 to 15 years, and 12 mathematics teachers participated in this quasi-experimental classroom study. While the experimental group was instructed to read and discuss the reading tasks in two 45-minute lessons, the control group was instructed to prove and apply the same propositions. Generalised estimating equation (GEE) method was used to compare the scores of the post-test and the delayed post-test with the pre-test scores as covariates. Results showed that the total scores of the delayed post-test of the experimental group were significantly higher than those of the control group. Furthermore, the scores of the experimental group on all facets of reading comprehension except the application facet were significantly higher than those of the control group for both the post-test and delayed post-test.

FACTORS AFFECTING THE UPTAKE OF LISSAMINE GREEN BY SERUM PROTEINS

PubMed Central

Brackenridge, C. J.

1960-01-01

Eight physicochemical factors which affect the uptake of lissamine green on filter paper impregnated with serum proteins have been examined, and their relevance to the staining of electrophoretically separated protein fractions is discussed. It is shown that grade of paper, weight of protein applied, separate and combined denaturation and staining time, temperature and concentration of staining solution, concentration of denaturant, and type of protein all influence the weight of dye absorbed per unit weight of applied protein, and must be rigidly standardized if valid quantitative results are to be obtained. Five sets of conditions are obtained for optimal staining and it is found that separation of denaturant from dye yields the best procedure. It is concluded that lissamine green is an excellent dye for the staining and quantitative estimation of separated protein fractions in paper electrophoresis, and that conditions can usually be arranged to produce a linear relation between dye uptake and protein concentration in an experimentally efficient manner. PMID:13803681

Urea denatured state ensembles contain extensive secondary structure that is increased in hydrophobic proteins.

PubMed

Nick Pace, C; Huyghues-Despointes, Beatrice M P; Fu, Hailong; Takano, Kazufumi; Scholtz, J Martin; Grimsley, Gerald R

2010-05-01

The goal of this article is to gain a better understanding of the denatured state ensemble (DSE) of proteins through an experimental and computational study of their denaturation by urea. Proteins unfold to different extents in urea and the most hydrophobic proteins have the most compact DSE and contain almost as much secondary structure as folded proteins. Proteins that unfold to the greatest extent near pH 7 still contain substantial amounts of secondary structure. At low pH, the DSE expands due to charge-charge interactions and when the net charge per residue is high, most of the secondary structure is disrupted. The proteins in the DSE appear to contain substantial amounts of polyproline II conformation at high urea concentrations. In all cases considered, including staph nuclease, the extent of unfolding by urea can be accounted for using the data and approach developed in the laboratory of Wayne Bolen (Auton et al., Proc Natl Acad Sci 2007; 104:15317-15323).

Protein aggregation induced during glass bead lysis of yeast

PubMed Central

Papanayotou, Irene; Sun, Beimeng; Roth, Amy F.; Davis, Nicholas G.

2013-01-01

Yeast cell lysates produced by mechanical glass bead disruption are widely used in a variety of applications, including for the analysis of native function, e.g. protein–protein interaction, enzyme assays and membrane fractionations. Below, we report a striking case of protein denaturation and aggregation that is induced by this lysis protocol. Most of this analysis focuses on the type 1 casein kinase Yck2, which normally tethers to the plasma membrane through C-terminal palmitoylation. Surprisingly, when cells are subjected to glass bead disruption, non-palmitoylated, cytosolic forms of the kinase denature and aggregate, while membrane-associated forms, whether attached through their native palmitoyl tethers or through a variety of artificial membrane-tethering sequences, are wholly protected from denaturation and aggregation. A wider look at the yeast proteome finds that, while the majority of proteins resist glass bead-induced aggregation, a significant subset does, in fact, succumb to such denaturation. Thus, yeast researchers should be aware of this potential artifact when embarking on biochemical analyses that employ glass bead lysates to look at native protein function. Finally, we demonstrate an experimental utility for glass bead-induced aggregation, using its fine discrimination of membrane-associated from non-associated Yck2 forms to discern fractional palmitoylation states of Yck2 mutants that are partially defective for palmitoylation. PMID:20641011

Glucose oxidase from Penicillium amagasakiense: characterization of the transition state of its denaturation from molecular dynamics simulations.

PubMed

Todde, Guido; Hovmöller, Sven; Laaksonen, Aatto; Mocci, Francesca

2014-10-01

Glucose oxidase (GOx) is a flavoenzyme having applications in food and medical industries. However, GOx, as many other enzymes when extracted from the cells, has relatively short operational lifetimes. Several recent studies (both experimental and theoretical), carried out on small proteins (or small fractions of large proteins), show that a detailed knowledge of how the breakdown process starts and proceeds on molecular level could be of significant help to artificially improve the stability of fragile proteins. We have performed extended molecular dynamics (MD) simulations to study the denaturation of GOx (a protein dimer containing nearly 1200 amino acids) to identify weak points in its structure and in this way gather information to later make it more stable, for example, by mutations. A denaturation of a protein can be simulated by increasing the temperature far above physiological temperature. We have performed a series of MD simulations at different temperatures (300, 400, 500, and 600 K). The exit from the protein's native state has been successfully identified with the clustering method and supported by other methods used to analyze the simulation data. A common set of amino acids is regularly found to initiate the denaturation, suggesting a moiety where the enzyme could be strengthened by a suitable amino acid based modification. © 2014 Wiley Periodicals, Inc.

Thermal denaturation: is solid-state fermentation really a good technology for the production of enzymes?

PubMed

Muller dos Santos, Marcelo; Souza da Rosa, Alexandre; Dal'Boit, Silvia; Mitchell, David A; Krieger, Nadia

2004-07-01

The potential for thermal denaturation to cause enzyme losses during solid-state fermentation processes for the production of enzymes was examined, using the protease of Penicillium fellutanum as a model system. The frequency factor and activation energies for the first-order denaturation of this enzyme were determined as 3.447 x 10(59) h(-1) and 364,070 Jmol(-1), respectively. These values were incorporated into a mathematical model of enzyme deactivation, which was used to investigate the consequences of subjecting this protease to temporal temperature profiles reported in the literature for mid-height in a 34 cm high packed-bed bioreactor of 150 mm diameter. In this literature source, temperature profiles were measured for 5, 15 and 25 liters per minute of air and enzyme activities were measured as a function of time. The enzyme activity profiles predicted by the model were distributed similarly, one relative to the other, as had been found in the experimental study, with substantial amounts of denaturation being predicted when the substrate temperature exceeded 40 degrees C, which occurred at the lower two airflow rates. A mathematical model of a well-mixed bioreactor was used to explore the difficulties that would be faced at large scale. It suggests that even with airflows as high as one volume per volume per minute, up to 85% of the enzyme produced by the microorganism can be denatured by the end of the fermentation. This work highlights the extra care that must be taken in scaling up solid-state fermentation processes for the production of thermolabile products. Copyright 2003 Elsevier Ltd.

27 CFR 19.456 - Adding denaturants.

Code of Federal Regulations, 2010 CFR

2010-04-01

... methods of mixing denaturants and spirits if he deems such denaturation will not hinder effective... Denaturation § 19.456 Adding denaturants. Denaturants and spirits shall be mixed in packages, tanks, or bulk... proprietor shall submit a flow diagram of the intended process or method of adding denaturants. (Sec. 201...

An evaluation of the pressure proof test concept for 2024-T3 aluminium alloy sheet

NASA Technical Reports Server (NTRS)

Dawicke, D. S.; Poe, C. C., Jr.; Newman, J. C.; Harris, C. E.

1991-01-01

The concept of pressure proof testing of fuselage structures with fatigue cracks to insure structural integrity was evaluated from a fracture mechanics viewpoint. A generic analytical and experimental investigation was conducted on uniaxially loaded flat panels with crack configurations and stress levels typical of longitudinal lap splice joints in commercial transport aircraft fuselages. The results revealed that the remaining fatigue life after a proof cycle was longer than that without the proof cycle because of crack growth retardation due to increased crack closure. However, based on a crack length that is slightly less than the critical value at the maximum proof stress, the minimum assured life or proof test interval must be no more than 550 pressure cycles for a 1.33 proof factor and 1530 pressure cycles for a 1.5 proof factor to prevent in-flight failures.

An evaluation of the pressure proof test concept for thin sheet 2024-T3

NASA Technical Reports Server (NTRS)

Dawicke, D. S.; Poe, C. C., Jr.; Newman, J. C., Jr.; Harris, C. E.

1990-01-01

The concept of pressure proof testing of fuselage structures with fatigue cracks to insure structural integrity was evaluated from a fracture mechanics viewpoint. A generic analytical and experimental investigation was conducted on uniaxially loaded flat panels with crack configurations and stress levels typical of longitudinal lap-splice joints in commercial transport aircraft fuselage. The results revealed that the remaining fatigue life after a proof test was longer than that without the proof test because of crack growth retardation due to increased crack closure. However, based on a crack length that is slightly less than the critical value at the maximum proof test stress, the minimum assured life or proof test interval must be no more than 550 pressure cycles for a 1.33 proof factor and 1530 pressure cycles for a 1.5 proof factor to prevent in-flight failures.

An evaluation of the pressure proof test concept for thin sheet 2024-T3

NASA Technical Reports Server (NTRS)

Dawicke, D. S.; Poe, C. C., Jr.; Newman, James C., Jr.; Harris, Charles E.

1990-01-01

The concept of pressure proof testing of fuselage structures with fatigue cracks to insure structural integrity was evaluated from a fracture mechanics viewpoint. A generic analytical and experimental investigation was conducted on uniaxially loaded flat panels with crack configurations and stress levels typical of longitudinal lap splice joints in commercial transport aircraft fuselages. The results revealed that the remaining fatigue life after a proof test was longer than that without the proof test because of crack growth retardation due to increased crack closure. However, based on a crack length that is slightly less than the critical value at the maximum proof test stress, the minimum assured life or proof test interval must be no more than 550 pressure cycles for a 1.33 proof factor and 1530 pressure cycles for a 1.5 proof factor to prevent in-flight failures.

A Novel Multilayered RFID Tagged Cargo Integrity Assurance Scheme

PubMed Central

Yang, Ming Hour; Luo, Jia Ning; Lu, Shao Yong

2015-01-01

To minimize cargo theft during transport, mobile radio frequency identification (RFID) grouping proof methods are generally employed to ensure the integrity of entire cargo loads. However, conventional grouping proofs cannot simultaneously generate grouping proofs for a specific group of RFID tags. The most serious problem of these methods is that nonexistent tags are included in the grouping proofs because of the considerable amount of time it takes to scan a high number of tags. Thus, applying grouping proof methods in the current logistics industry is difficult. To solve this problem, this paper proposes a method for generating multilayered offline grouping proofs. The proposed method provides tag anonymity; moreover, resolving disputes between recipients and transporters over the integrity of cargo deliveries can be expedited by generating grouping proofs and automatically authenticating the consistency between the receipt proof and pick proof. The proposed method can also protect against replay attacks, multi-session attacks, and concurrency attacks. Finally, experimental results verify that, compared with other methods for generating grouping proofs, the proposed method can efficiently generate offline grouping proofs involving several parties in a supply chain using mobile RFID. PMID:26512673

Can violations of Bell's inequalities be considered as a final proof of quantum physics?

NASA Astrophysics Data System (ADS)

Hénault, François

2013-10-01

Nowadays, it is commonly admitted that the experimental violation of Bell's inequalities that was successfully demonstrated in the last decades by many experimenters, are indeed the ultimate proof of quantum physics and of its ability to describe the whole microscopic world and beyond. But the historical and scientific story may not be envisioned so clearly: it starts with the original paper of Einstein, Podolsky and Rosen (EPR) aiming at demonstrating that the formalism of quantum theory is incomplete. It then goes through the works of D. Bohm, to finally proceed to the famous John Bell's relationships providing an experimental setup to solve the EPR paradox. In this communication is proposed an alternative reading of this history, showing that modern experiments based on correlations between light polarizations significantly deviate from the original spirit of the EPR paper. It is concluded that current experimental violations of Bell's inequalities cannot be considered as an ultimate proof of the completeness of quantum physics models.

27 CFR 19.386 - Adjusting pH of denatured spirits.

Code of Federal Regulations, 2014 CFR

2014-04-01

... will counteract or reduce the effect of the denaturants. A proprietor who adjusts the pH of denatured... 27 Alcohol, Tobacco Products and Firearms 1 2014-04-01 2014-04-01 false Adjusting pH of denatured... of Articles Rules for Denaturing Spirits and Testing Denaturants § 19.386 Adjusting pH of denatured...

27 CFR 19.386 - Adjusting pH of denatured spirits.

Code of Federal Regulations, 2011 CFR

2011-04-01

... will counteract or reduce the effect of the denaturants. A proprietor who adjusts the pH of denatured... 27 Alcohol, Tobacco Products and Firearms 1 2011-04-01 2011-04-01 false Adjusting pH of denatured... of Articles Rules for Denaturing Spirits and Testing Denaturants § 19.386 Adjusting pH of denatured...

27 CFR 19.386 - Adjusting pH of denatured spirits.

Code of Federal Regulations, 2012 CFR

2012-04-01

... will counteract or reduce the effect of the denaturants. A proprietor who adjusts the pH of denatured... 27 Alcohol, Tobacco Products and Firearms 1 2012-04-01 2012-04-01 false Adjusting pH of denatured... of Articles Rules for Denaturing Spirits and Testing Denaturants § 19.386 Adjusting pH of denatured...

27 CFR 19.386 - Adjusting pH of denatured spirits.

Code of Federal Regulations, 2013 CFR

2013-04-01

... will counteract or reduce the effect of the denaturants. A proprietor who adjusts the pH of denatured... 27 Alcohol, Tobacco Products and Firearms 1 2013-04-01 2013-04-01 false Adjusting pH of denatured... of Articles Rules for Denaturing Spirits and Testing Denaturants § 19.386 Adjusting pH of denatured...

Thermal denaturing of mutant lysozyme with both the OPLSAA and the CHARMM force fields.

PubMed

Eleftheriou, Maria; Germain, Robert S; Royyuru, Ajay K; Zhou, Ruhong

2006-10-18

Biomolecular simulations enabled by massively parallel supercomputers such as BlueGene/L promise to bridge the gap between the currently accessible simulation time scale and the experimental time scale for many important protein folding processes. In this study, molecular dynamics simulations were carried out for both the wild-type and the mutant hen lysozyme (TRP62GLY) to study the single mutation effect on lysozyme stability and misfolding. Our thermal denaturing simulations at 400-500 K with both the OPLSAA and the CHARMM force fields show that the mutant structure is indeed much less stable than the wild-type, which is consistent with the recent urea denaturing experiment (Dobson et al. Science 2002, 295, 1719-1722; Nature 2003, 424, 783-788). Detailed results also reveal that the single mutation TRP62GLY first induces the loss of native contacts in the beta-domain region of the lysozyme protein at high temperatures, and then the unfolding process spreads into the alpha-domain region through Helix C. Even though the OPLSAA force field in general shows a more stable protein structure than does the CHARMM force field at high temperatures, the two force fields examined here display qualitatively similar results for the misfolding process, indicating that the thermal denaturing of the single mutation is robust and reproducible with various modern force fields.

Radiation-induced polymerization for the immobilization of penicillin acylase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Boccu, E.; Carenza, M.; Lora, S.

The immobilization of Escherichia coli penicillin acylase was investigated by radiation-induced polymerization of 2-hydroxyethyl methacrylate at low temperature. A leak-proof composite that does not swell in water was obtained by adding the cross-linking agent trimethylolpropane trimethacrylate to the monomer-aqueous enzyme mixture. Penicillin acylase, which was immobilized with greater than 70% yield, possessed a higher Km value toward the substrate 6-nitro-3-phenylacetamidobenzoic acid than the free enzyme form (Km = 1.7 X 10(-5) and 1 X 10(-5) M, respectively). The structural stability of immobilized penicillin acylase, as assessed by heat, guanidinium chloride, and pH denaturation profiles, was very similar to that ofmore » the free-enzyme form, thus suggesting that penicillin acylase was entrapped in its native state into aqueous free spaces of the polymer matrix.« less

Revised Robertson's test theory of special relativity

NASA Astrophysics Data System (ADS)

Vargas, José G.

1984-07-01

The only test theory used by workers in the field of testing special relativity to analyze the significance of their experiments is the proof by H. P. Robertson [ Rev. Mod. Phys. 21, 378 (1949)] of the Lorentz transformations from the results of the experimental evidence. Some researchers would argue that the proof contains an unwarranted assumption disguised as a convention about synchronization procedures. Others would say that alternative conventions are possible. In the present paper, no convention is used, but the Lorentz transformations are still obtained using only the results of the experiments in Robertson's proof, namely the Michelson-Morley, Kennedy-Thorndike, and Ives-Stilwell experiments. Thus the revised proof is a valid test theory which is independent of any conventions, since one appeals only to the experimental evidence. The analysis of that evidence shows the directions in which efforts to test special relativity should go. Finally it is shown how the resulting test theory still has to be improved for consistency in the analysis of experiments with complicated experimental setups, how it can be simplified for expediency as to what should be tested, and how it should be completed for a missing step not considered by Robertson.

An integrated experimental and computational approach to material selection for sound proof thermally insulted enclosure of a power generation system

NASA Astrophysics Data System (ADS)

Waheed, R.; Tarar, W.; Saeed, H. A.

2016-08-01

Sound proof canopies for diesel power generators are fabricated with a layer of sound absorbing material applied to all the inner walls. The physical properties of the majority of commercially available sound proofing materials reveal that a material with high sound absorption coefficient has very low thermal conductivity. Consequently a good sound absorbing material is also a good heat insulator. In this research it has been found through various experiments that ordinary sound proofing materials tend to rise the inside temperature of sound proof enclosure in certain turbo engines by capturing the heat produced by engine and not allowing it to be transferred to atmosphere. The same phenomenon is studied by creating a finite element model of the sound proof enclosure and performing a steady state and transient thermal analysis. The prospects of using aluminium foam as sound proofing material has been studied and it is found that inside temperature of sound proof enclosure can be cut down to safe working temperature of power generator engine without compromise on sound proofing.

Chemical Denaturants Smoothen Ruggedness on the Free Energy Landscape of Protein Folding.

PubMed

Malhotra, Pooja; Jethva, Prashant N; Udgaonkar, Jayant B

2017-08-08

To characterize experimentally the ruggedness of the free energy landscape of protein folding is challenging, because the distributed small free energy barriers are usually dominated by one, or a few, large activation free energy barriers. This study delineates changes in the roughness of the free energy landscape by making use of the observation that a decrease in ruggedness is accompanied invariably by an increase in folding cooperativity. Hydrogen exchange (HX) coupled to mass spectrometry was used to detect transient sampling of local energy minima and the global unfolded state on the free energy landscape of the small protein single-chain monellin. Under native conditions, local noncooperative openings result in interconversions between Boltzmann-distributed intermediate states, populated on an extremely rugged "uphill" energy landscape. The cooperativity of these interconversions was increased by selectively destabilizing the native state via mutations, and further by the addition of a chemical denaturant. The perturbation of stability alone resulted in seven backbone amide sites exchanging cooperatively. The size of the cooperatively exchanging and/or unfolding unit did not depend on the extent of protein destabilization. Only upon the addition of a denaturant to a destabilized mutant variant did seven additional backbone amide sites exchange cooperatively. Segmentwise analysis of the HX kinetics of the mutant variants further confirmed that the observed increase in cooperativity was due to the smoothing of the ruggedness of the free energy landscape of folding of the protein by the chemical denaturant.

The molecular basis of the effect of temperature on enzyme activity.

PubMed

Daniel, Roy M; Peterson, Michelle E; Danson, Michael J; Price, Nicholas C; Kelly, Sharon M; Monk, Colin R; Weinberg, Cristina S; Oudshoorn, Matthew L; Lee, Charles K

2009-12-23

Experimental data show that the effect of temperature on enzymes cannot be adequately explained in terms of a two-state model based on increases in activity and denaturation. The Equilibrium Model provides a quantitative explanation of enzyme thermal behaviour under reaction conditions by introducing an inactive (but not denatured) intermediate in rapid equilibrium with the active form. The temperature midpoint (Teq) of the rapid equilibration between the two forms is related to the growth temperature of the organism, and the enthalpy of the equilibrium (DeltaHeq) to its ability to function over various temperature ranges. In the present study, we show that the difference between the active and inactive forms is at the enzyme active site. The results reveal an apparently universal mechanism, independent of enzyme reaction or structure, based at or near the active site, by which enzymes lose activity as temperature rises, as opposed to denaturation which is global. Results show that activity losses below Teq may lead to significant errors in the determination of DeltaG*cat made on the basis of the two-state ('Classical') model, and the measured kcat will then not be a true indication of an enzyme's catalytic power. Overall, the results provide a molecular rationale for observations that the active site tends to be more flexible than the enzyme as a whole, and that activity losses precede denaturation, and provide a general explanation in molecular terms for the effect of temperature on enzyme activity.

Selection for Protein Kinetic Stability Connects Denaturation Temperatures to Organismal Temperatures and Provides Clues to Archaean Life

PubMed Central

Romero-Romero, M. Luisa; Risso, Valeria A.; Martinez-Rodriguez, Sergio; Gaucher, Eric A.; Ibarra-Molero, Beatriz; Sanchez-Ruiz, Jose M.

2016-01-01

The relationship between the denaturation temperatures of proteins (Tm values) and the living temperatures of their host organisms (environmental temperatures: TENV values) is poorly understood. Since different proteins in the same organism may show widely different Tm’s, no simple universal relationship between Tm and TENV should hold, other than Tm≥TENV. Yet, when analyzing a set of homologous proteins from different hosts, Tm’s are oftentimes found to correlate with TENV’s but this correlation is shifted upward on the Tm axis. Supporting this trend, we recently reported Tm’s for resurrected Precambrian thioredoxins that mirror a proposed environmental cooling over long geological time, while remaining a shocking ~50°C above the proposed ancestral ocean temperatures. Here, we show that natural selection for protein kinetic stability (denaturation rate) can produce a Tm↔TENV correlation with a large upward shift in Tm. A model for protein stability evolution suggests a link between the Tm shift and the in vivo lifetime of a protein and, more specifically, allows us to estimate ancestral environmental temperatures from experimental denaturation rates for resurrected Precambrian thioredoxins. The TENV values thus obtained match the proposed ancestral ocean cooling, support comparatively high Archaean temperatures, and are consistent with a recent proposal for the environmental temperature (above 75°C) that hosted the last universal common ancestor. More generally, this work provides a framework for understanding how features of protein stability reflect the environmental temperatures of the host organisms. PMID:27253436

Exact method for numerically analyzing a model of local denaturation in superhelically stressed DNA

NASA Astrophysics Data System (ADS)

Fye, Richard M.; Benham, Craig J.

1999-03-01

Local denaturation, the separation at specific sites of the two strands comprising the DNA double helix, is one of the most fundamental processes in biology, required to allow the base sequence to be read both in DNA transcription and in replication. In living organisms this process can be mediated by enzymes which regulate the amount of superhelical stress imposed on the DNA. We present a numerically exact technique for analyzing a model of denaturation in superhelically stressed DNA. This approach is capable of predicting the locations and extents of transition in circular superhelical DNA molecules of kilobase lengths and specified base pair sequences. It can also be used for closed loops of DNA which are typically found in vivo to be kilobases long. The analytic method consists of an integration over the DNA twist degrees of freedom followed by the introduction of auxiliary variables to decouple the remaining degrees of freedom, which allows the use of the transfer matrix method. The algorithm implementing our technique requires O(N2) operations and O(N) memory to analyze a DNA domain containing N base pairs. However, to analyze kilobase length DNA molecules it must be implemented in high precision floating point arithmetic. An accelerated algorithm is constructed by imposing an upper bound M on the number of base pairs that can simultaneously denature in a state. This accelerated algorithm requires O(MN) operations, and has an analytically bounded error. Sample calculations show that it achieves high accuracy (greater than 15 decimal digits) with relatively small values of M (M<0.05N) for kilobase length molecules under physiologically relevant conditions. Calculations are performed on the superhelical pBR322 DNA sequence to test the accuracy of the method. With no free parameters in the model, the locations and extents of local denaturation predicted by this analysis are in quantitatively precise agreement with in vitro experimental measurements. Calculations performed on the fructose-1,6-bisphosphatase gene sequence from yeast show that this approach can also accurately treat in vivo denaturation.

40 CFR 80.1611 - Standards and requirements for certified ethanol denaturant.

Code of Federal Regulations, 2014 CFR

2014-07-01

... certified ethanol denaturant. 80.1611 Section 80.1611 Protection of Environment ENVIRONMENTAL PROTECTION....1611 Standards and requirements for certified ethanol denaturant. Producers and importers of ethanol denaturant that is suitable for the manufacture of denatured fuel ethanol (DFE) meeting federal quality...

The Maryland Refutation Proof Procedure.

ERIC Educational Resources Information Center

Minker, Jack; And Others

The Maryland Refutation Proof Procedure System (MRPPS) is an interactive experimental system intended for studying deductive search methods. Although the work is oriented towards question-answering, MRPPS provides a general problem solving capability. There are three major components within MRPPS. These are: (1) an inference system, (2) a search…

27 CFR 19.459 - Mixing of denatured spirits.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Mixing of denatured spirits. 19.459 Section 19.459 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE... of Articles Denaturation § 19.459 Mixing of denatured spirits. (a) Denatured spirits produced under...

Techniques to construct New Zealand elk-proof fence.

Treesearch

Larry D. Bryant; Jack W. Thomas; Mary M. Rowland

1993-01-01

An elk-proof fence was built in 1987 at the Starkey Experimental Forest and Range in northeast Oregon. The 25,000-acre research enclosure holds several hundred Rocky Mountain elk (Cervus elaphus nelsoni V. Bailey) and Rocky Mountain mule deer (Odocoileus hemionus hemionus Rafinesque) year round. The fence, constructed with high...

CalFitter: a web server for analysis of protein thermal denaturation data.

PubMed

Mazurenko, Stanislav; Stourac, Jan; Kunka, Antonin; Nedeljkovic, Sava; Bednar, David; Prokop, Zbynek; Damborsky, Jiri

2018-05-14

Despite significant advances in the understanding of protein structure-function relationships, revealing protein folding pathways still poses a challenge due to a limited number of relevant experimental tools. Widely-used experimental techniques, such as calorimetry or spectroscopy, critically depend on a proper data analysis. Currently, there are only separate data analysis tools available for each type of experiment with a limited model selection. To address this problem, we have developed the CalFitter web server to be a unified platform for comprehensive data fitting and analysis of protein thermal denaturation data. The server allows simultaneous global data fitting using any combination of input data types and offers 12 protein unfolding pathway models for selection, including irreversible transitions often missing from other tools. The data fitting produces optimal parameter values, their confidence intervals, and statistical information to define unfolding pathways. The server provides an interactive and easy-to-use interface that allows users to directly analyse input datasets and simulate modelled output based on the model parameters. CalFitter web server is available free at https://loschmidt.chemi.muni.cz/calfitter/.

Improving RNA nearest neighbor parameters for helices by going beyond the two-state model.

PubMed

Spasic, Aleksandar; Berger, Kyle D; Chen, Jonathan L; Seetin, Matthew G; Turner, Douglas H; Mathews, David H

2018-06-01

RNA folding free energy change nearest neighbor parameters are widely used to predict folding stabilities of secondary structures. They were determined by linear regression to datasets of optical melting experiments on small model systems. Traditionally, the optical melting experiments are analyzed assuming a two-state model, i.e. a structure is either complete or denatured. Experimental evidence, however, shows that structures exist in an ensemble of conformations. Partition functions calculated with existing nearest neighbor parameters predict that secondary structures can be partially denatured, which also directly conflicts with the two-state model. Here, a new approach for determining RNA nearest neighbor parameters is presented. Available optical melting data for 34 Watson-Crick helices were fit directly to a partition function model that allows an ensemble of conformations. Fitting parameters were the enthalpy and entropy changes for helix initiation, terminal AU pairs, stacks of Watson-Crick pairs and disordered internal loops. The resulting set of nearest neighbor parameters shows a 38.5% improvement in the sum of residuals in fitting the experimental melting curves compared to the current literature set.

40 CFR 80.1644 - Sampling and testing requirements for producers and importers of certified ethanol denaturant.

Code of Federal Regulations, 2014 CFR

2014-07-01

... producers and importers of certified ethanol denaturant. 80.1644 Section 80.1644 Protection of Environment... ethanol denaturant. (a) Sample and test each batch of certified ethanol denaturant. (1) Producers and importers of certified ethanol denaturant shall collect a representative sample from each batch of certified...

40 CFR 80.1645 - Sample retention requirements for producers and importers of denaturant designated as suitable...

Code of Federal Regulations, 2014 CFR

2014-07-01

... producers and importers of denaturant designated as suitable for the manufacture of denatured fuel ethanol... suitable for the manufacture of denatured fuel ethanol meeting federal quality requirements. Beginning January 1, 2017, or on the first day that any producer or importer of ethanol denaturant designates a...

27 CFR 19.455 - Dissolving of denaturants.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Dissolving of denaturants... Denaturation § 19.455 Dissolving of denaturants. Denaturants which are difficult to dissolve in spirits at... may be liquefied or dissolved in a small quantity of spirits or water in advance of their use in the...

Construction of state-independent proofs for quantum contextuality

NASA Astrophysics Data System (ADS)

Tang, Weidong; Yu, Sixia

2017-12-01

Since the enlightening proofs of quantum contextuality first established by Kochen and Specker, and also by Bell, various simplified proofs have been constructed to exclude the noncontextual hidden variable theory of our nature at the microscopic scale. The conflict between the noncontextual hidden variable theory and quantum mechanics is commonly revealed by Kochen-Specker sets of yes-no tests, represented by projectors (or rays), via either logical contradictions or noncontextuality inequalities in a state-(in)dependent manner. Here we propose a systematic and programmable construction of a state-independent proof from a given set of nonspecific rays in C3 according to their Gram matrix. This approach brings us a greater convenience in the experimental arrangements. Besides, our proofs in C3 can also be generalized to any higher-dimensional systems by a recursive method.

Urea-Induced Unfolding of the Immunity Protein Im9 Monitored by spFRET

PubMed Central

Tezuka-Kawakami, Tomoko; Gell, Chris; Brockwell, David J.; Radford, Sheena E.; Smith, D. Alastair

2006-01-01

We have studied the urea-induced unfolding of the E colicin immunity protein Im9 using diffusion single-pair fluorescence resonance energy transfer. Detailed examination of the proximity ratio of the native and denatured molecules over a wide range of urea concentrations suggests that the conformational properties of both species are denaturant-dependent. Whereas native molecules become gradually more expanded as urea concentration increases, denatured molecules show a dramatic dependence of the relationship between proximity ratio and denaturant concentration, consistent with substantial compaction of the denatured ensemble at low denaturant concentrations. Analysis of the widths of the proximity ratio distributions for each state suggests that whereas the native state ensemble is relatively narrow and homogeneous, the denatured state may possess heterogeneity in mildly denaturing conditions. PMID:16798813

27 CFR 20.216 - Record of shipment.

Code of Federal Regulations, 2010 CFR

2010-04-01

... OF THE TREASURY LIQUORS DISTRIBUTION AND USE OF DENATURED ALCOHOL AND RUM Recovery of Denatured... denatured alcohol, recovered specially denatured rum, or recovered articles to a distilled spirits plant or...

Spatial encoding using the nonlinear field perturbations from magnetic materials.

PubMed

Karimi, Hirad; Dominguez-Viqueira, William; Cunningham, Charles H

2014-08-01

A proof-of-concept study was performed to assess the technical feasibility of using magnetic materials to generate spatial encoding fields. Spatially varying magnetic fields were generated by the placement of markers with different volume susceptibilities within the imaging volume. No linear gradients were used for spatial encoding during the signal acquisition. A signal-encoding model is described for reconstructing the images encoded with these field perturbations. Simulation and proof-of-concept experimental results are presented. Experiments were performed using field perturbations from a cylindrical marker as an example of the new encoding fields. Based on this experimental setup, annular rings were reconstructed from signals encoded with the new fields. Simulation results were presented for different acquisition parameters. Proof-of-concept was supported by the correspondence of regions in an image reconstructed from experimental data compared to those in a conventional gradient-echo image. Experimental results showed that inclusions of dimensions 1.5 mm in size could be resolved with the experimental setup. This study shows the technical feasibility of using magnetic markers to produce encoding fields. Magnetic materials will allow generating spatial encoding fields, which can be tailored to an imaging application with less complexity and at lower cost compared to the use of gradient inserts. Copyright © 2013 Wiley Periodicals, Inc.

27 CFR 19.385 - Making alcohol or water solutions of denaturants.

Code of Federal Regulations, 2014 CFR

2014-04-01

... alcohol or water solutions of denaturants. If a proprietor uses a denaturant that is difficult to dissolve... working temperature, the proprietor may liquefy or dissolve the denaturant in a small amount of spirits or...

27 CFR 19.385 - Making alcohol or water solutions of denaturants.

Code of Federal Regulations, 2012 CFR

2012-04-01

... alcohol or water solutions of denaturants. If a proprietor uses a denaturant that is difficult to dissolve... working temperature, the proprietor may liquefy or dissolve the denaturant in a small amount of spirits or...

Role of Solvation Effects in Protein Denaturation: From Thermodynamics to Single Molecules and Back

PubMed Central

England, Jeremy L.; Haran, Gilad

2011-01-01

Protein stability often is studied in vitro through the use of urea and guanidinium chloride, chemical cosolvents that disrupt protein native structure. Much controversy still surrounds the underlying mechanism by which these molecules denature proteins. Here we review current thinking on various aspects of chemical denaturation. We begin by discussing classic models of protein folding and how the effects of denaturants may fit into this picture through their modulation of the collapse, or coil-globule transition, which typically precedes folding. Subsequently, we examine recent molecular dynamics simulations that have shed new light on the possible microscopic origins of the solvation effects brought on by denaturants. It seems likely that both denaturants operate by facilitating solvation of hydrophobic regions of proteins. Finally, we present recent single-molecule fluorescence studies of denatured proteins, the analysis of which corroborates the role of denaturants in shifting the equilibrium of the coil-globule transition. PMID:21219136

Brownian dynamics simulations of sequence-dependent duplex denaturation in dynamically superhelical DNA

NASA Astrophysics Data System (ADS)

Mielke, Steven P.; Grønbech-Jensen, Niels; Krishnan, V. V.; Fink, William H.; Benham, Craig J.

2005-09-01

The topological state of DNA in vivo is dynamically regulated by a number of processes that involve interactions with bound proteins. In one such process, the tracking of RNA polymerase along the double helix during transcription, restriction of rotational motion of the polymerase and associated structures, generates waves of overtwist downstream and undertwist upstream from the site of transcription. The resulting superhelical stress is often sufficient to drive double-stranded DNA into a denatured state at locations such as promoters and origins of replication, where sequence-specific duplex opening is a prerequisite for biological function. In this way, transcription and other events that actively supercoil the DNA provide a mechanism for dynamically coupling genetic activity with regulatory and other cellular processes. Although computer modeling has provided insight into the equilibrium dynamics of DNA supercoiling, to date no model has appeared for simulating sequence-dependent DNA strand separation under the nonequilibrium conditions imposed by the dynamic introduction of torsional stress. Here, we introduce such a model and present results from an initial set of computer simulations in which the sequences of dynamically superhelical, 147 base pair DNA circles were systematically altered in order to probe the accuracy with which the model can predict location, extent, and time of stress-induced duplex denaturation. The results agree both with well-tested statistical mechanical calculations and with available experimental information. Additionally, we find that sites susceptible to denaturation show a propensity for localizing to supercoil apices, suggesting that base sequence determines locations of strand separation not only through the energetics of interstrand interactions, but also by influencing the geometry of supercoiling.

Denatured states of yeast cytochrome c induced by heat and guanidinium chloride are structurally and thermodynamically different.

PubMed

Zaidi, Sobia; Haque, Md Anzarul; Ubaid-Ullah, Shah; Prakash, Amresh; Hassan, Md Imtaiyaz; Islam, Asimul; Batra, Janendra K; Ahmad, Faizan

2017-05-01

A sequence alignment of mammalian cytochromes c with yeast iso-1-cytochrome c (y-cyt-c) shows that the yeast protein contains five extra N-terminal residues. We have been interested in understanding the question: What is the role of these five extra N-terminal residues in folding and stability of the protein? To answer this question we have prepared five deletants of y-cyt-c by sequentially removing these extra residues. During our studies on the wild type (WT) protein and its deletants, we observed that the amount of secondary structure in the guanidinium chloride (GdmCl)-induced denatured (D) state of each protein is different from that of the heat-induced denatured (H) state. This finding is confirmed by the observation of an additional cooperative transition curve of optical properties between H and D states on the addition of different concentrations of GdmCl to the already heat denatured WT y-cyt-c and its deletants at pH 6.0 and 68°C. For each protein, analysis of transition curves representing processes, native (N) state ↔ D state, N state ↔ H state, and H state ↔ D state, was done to obtain Gibbs free energy changes associated with all the three processes. This analysis showed that, for each protein, thermodynamic cycle accommodates Gibbs free energies associated with transitions between N and D states, N and H states, and H and D states, the characteristics required for a thermodynamic function. All these experimental observations have been supported by our molecular dynamics simulation studies.

Brownian dynamics simulations of sequence-dependent duplex denaturation in dynamically superhelical DNA.

PubMed

Mielke, Steven P; Grønbech-Jensen, Niels; Krishnan, V V; Fink, William H; Benham, Craig J

2005-09-22

The topological state of DNA in vivo is dynamically regulated by a number of processes that involve interactions with bound proteins. In one such process, the tracking of RNA polymerase along the double helix during transcription, restriction of rotational motion of the polymerase and associated structures, generates waves of overtwist downstream and undertwist upstream from the site of transcription. The resulting superhelical stress is often sufficient to drive double-stranded DNA into a denatured state at locations such as promoters and origins of replication, where sequence-specific duplex opening is a prerequisite for biological function. In this way, transcription and other events that actively supercoil the DNA provide a mechanism for dynamically coupling genetic activity with regulatory and other cellular processes. Although computer modeling has provided insight into the equilibrium dynamics of DNA supercoiling, to date no model has appeared for simulating sequence-dependent DNA strand separation under the nonequilibrium conditions imposed by the dynamic introduction of torsional stress. Here, we introduce such a model and present results from an initial set of computer simulations in which the sequences of dynamically superhelical, 147 base pair DNA circles were systematically altered in order to probe the accuracy with which the model can predict location, extent, and time of stress-induced duplex denaturation. The results agree both with well-tested statistical mechanical calculations and with available experimental information. Additionally, we find that sites susceptible to denaturation show a propensity for localizing to supercoil apices, suggesting that base sequence determines locations of strand separation not only through the energetics of interstrand interactions, but also by influencing the geometry of supercoiling.

On the precision of experimentally determined protein folding rates and φ-values

PubMed Central

De Los Rios, Miguel A.; Muralidhara, B.K.; Wildes, David; Sosnick, Tobin R.; Marqusee, Susan; Wittung-Stafshede, Pernilla; Plaxco, Kevin W.; Ruczinski, Ingo

2006-01-01

φ-Values, a relatively direct probe of transition-state structure, are an important benchmark in both experimental and theoretical studies of protein folding. Recently, however, significant controversy has emerged regarding the reliability with which φ-values can be determined experimentally: Because φ is a ratio of differences between experimental observables it is extremely sensitive to errors in those observations when the differences are small. Here we address this issue directly by performing blind, replicate measurements in three laboratories. By monitoring within- and between-laboratory variability, we have determined the precision with which folding rates and φ-values are measured using generally accepted laboratory practices and under conditions typical of our laboratories. We find that, unless the change in free energy associated with the probing mutation is quite large, the precision of φ-values is relatively poor when determined using rates extrapolated to the absence of denaturant. In contrast, when we employ rates estimated at nonzero denaturant concentrations or assume that the slopes of the chevron arms (mf and mu) are invariant upon mutation, the precision of our estimates of φ is significantly improved. Nevertheless, the reproducibility we thus obtain still compares poorly with the confidence intervals typically reported in the literature. This discrepancy appears to arise due to differences in how precision is calculated, the dependence of precision on the number of data points employed in defining a chevron, and interlaboratory sources of variability that may have been largely ignored in the prior literature. PMID:16501226

Determination of Flaw Growth Characteristics of Ti-6Al-4V Sheet in the Solution-Treated and Aged Condition

NASA Technical Reports Server (NTRS)

Hoeppner, David W.; Pettit, Donald E.; Feddersen, Charles E.; Hyler, Walter S.

1968-01-01

The specific experimental investigation undertaken was designed to answer these questions on Ti-6Al-4V in the solution treated and aged condition. The defect growth and fracture characteristics were studied in parent (unwelded) and welded sheet material. The results of the study indicate that cryogenic proof testing will screen smaller size defects than proof testing at ambient conditions. However some unusual crack growth behavior during the proof test simulation suggests that some further study be made of stress and time duration effects.

Predicting the distribution of whey protein fouling in a plate heat exchanger using the kinetic parameters of the thermal denaturation reaction of β-lactoglobulin and the bulk temperature profiles.

PubMed

Blanpain-Avet, P; André, C; Khaldi, M; Bouvier, L; Petit, J; Six, T; Jeantet, R; Croguennec, T; Delaplace, G

2016-12-01

Fouling of plate heat exchangers (PHE) is a severe problem in the dairy industry, notably because the relationship between the build-up of protein fouling deposits and the chemical reactions taking place in the fouling solution has not yet been fully elucidated. Experiments were conducted at pilot scale in a corrugated PHE, and fouling deposits were generated using a model β-lactoglobulin (β-LG) fouling solution for which the β-LG thermal denaturation reaction constants had been previously determined experimentally. Then 18 different bulk temperature profiles within the PHE were imposed. Analysis of the fouling runs shows that the dry deposit mass per channel versus the ratio R=k unf /k agg (with k unf and k agg representing, respectively, the unfolding and aggregation rate constants computed from both the identification of the β-LG thermal denaturation process and knowledge of the imposed bulk temperature profile into the PHE channel) is able to gather reasonably well the experimental fouling mass data into a unique master curve. This type of representation of the results clearly shows that the heat-induced reactions (unfolding and aggregation) of the various β-LG molecular species in the bulk fluid are essential to capture the trend of the fouling mass distribution inside a PHE. This investigation also illustrates unambiguously that the release of the unfolded β-LG (also called β-LG molten globule) within the bulk fluid (and the absence of its consumption in the form of aggregates) is a key phenomenon that controls the extent of protein fouling as well as its location inside the PHE. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Irreversible Denaturation of Maltodextrin Glucosidase Studied by Differential Scanning Calorimetry, Circular Dichroism, and Turbidity Measurements

PubMed Central

Goyal, Megha; Chaudhuri, Tapan K.; Kuwajima, Kunihiro

2014-01-01

Thermal denaturation of Escherichia coli maltodextrin glucosidase was studied by differential scanning calorimetry, circular dichroism (230 nm), and UV-absorption measurements (340 nm), which were respectively used to monitor heat absorption, conformational unfolding, and the production of solution turbidity. The denaturation was irreversible, and the thermal transition recorded at scan rates of 0.5–1.5 K/min was significantly scan-rate dependent, indicating that the thermal denaturation was kinetically controlled. The absence of a protein-concentration effect on the thermal transition indicated that the denaturation was rate-limited by a mono-molecular process. From the analysis of the calorimetric thermograms, a one-step irreversible model well represented the thermal denaturation of the protein. The calorimetrically observed thermal transitions showed excellent coincidence with the turbidity transitions monitored by UV-absorption as well as with the unfolding transitions monitored by circular dichroism. The thermal denaturation of the protein was thus rate-limited by conformational unfolding, which was followed by a rapid irreversible formation of aggregates that produced the solution turbidity. It is thus important to note that the absence of the protein-concentration effect on the irreversible thermal denaturation does not necessarily means the absence of protein aggregation itself. The turbidity measurements together with differential scanning calorimetry in the irreversible thermal denaturation of the protein provided a very effective approach for understanding the mechanisms of the irreversible denaturation. The Arrhenius-equation parameters obtained from analysis of the thermal denaturation were compared with those of other proteins that have been reported to show the one-step irreversible thermal denaturation. Maltodextrin glucosidase had sufficiently high kinetic stability with a half-life of 68 days at a physiological temperature (37°C). PMID:25548918

Irreversible denaturation of maltodextrin glucosidase studied by differential scanning calorimetry, circular dichroism, and turbidity measurements.

PubMed

Goyal, Megha; Chaudhuri, Tapan K; Kuwajima, Kunihiro

2014-01-01

Thermal denaturation of Escherichia coli maltodextrin glucosidase was studied by differential scanning calorimetry, circular dichroism (230 nm), and UV-absorption measurements (340 nm), which were respectively used to monitor heat absorption, conformational unfolding, and the production of solution turbidity. The denaturation was irreversible, and the thermal transition recorded at scan rates of 0.5-1.5 K/min was significantly scan-rate dependent, indicating that the thermal denaturation was kinetically controlled. The absence of a protein-concentration effect on the thermal transition indicated that the denaturation was rate-limited by a mono-molecular process. From the analysis of the calorimetric thermograms, a one-step irreversible model well represented the thermal denaturation of the protein. The calorimetrically observed thermal transitions showed excellent coincidence with the turbidity transitions monitored by UV-absorption as well as with the unfolding transitions monitored by circular dichroism. The thermal denaturation of the protein was thus rate-limited by conformational unfolding, which was followed by a rapid irreversible formation of aggregates that produced the solution turbidity. It is thus important to note that the absence of the protein-concentration effect on the irreversible thermal denaturation does not necessarily means the absence of protein aggregation itself. The turbidity measurements together with differential scanning calorimetry in the irreversible thermal denaturation of the protein provided a very effective approach for understanding the mechanisms of the irreversible denaturation. The Arrhenius-equation parameters obtained from analysis of the thermal denaturation were compared with those of other proteins that have been reported to show the one-step irreversible thermal denaturation. Maltodextrin glucosidase had sufficiently high kinetic stability with a half-life of 68 days at a physiological temperature (37°C).

Fault-tolerant clock synchronization validation methodology. [in computer systems

NASA Technical Reports Server (NTRS)

Butler, Ricky W.; Palumbo, Daniel L.; Johnson, Sally C.

1987-01-01

A validation method for the synchronization subsystem of a fault-tolerant computer system is presented. The high reliability requirement of flight-crucial systems precludes the use of most traditional validation methods. The method presented utilizes formal design proof to uncover design and coding errors and experimentation to validate the assumptions of the design proof. The experimental method is described and illustrated by validating the clock synchronization system of the Software Implemented Fault Tolerance computer. The design proof of the algorithm includes a theorem that defines the maximum skew between any two nonfaulty clocks in the system in terms of specific system parameters. Most of these parameters are deterministic. One crucial parameter is the upper bound on the clock read error, which is stochastic. The probability that this upper bound is exceeded is calculated from data obtained by the measurement of system parameters. This probability is then included in a detailed reliability analysis of the system.

Development of an experimental system for characterization of high-temperature superconductors cooled by liquid hydrogen under the external magnetic field

NASA Astrophysics Data System (ADS)

Tatsumoto, H.; Shirai, Y.; Shiotsu, M.; Naruo, Y.; Kobayashi, H.; Inatani, Y.

2014-05-01

An experimental system has been developed to investigate electro-magnetic properties of high-Tc superconductors cooled by liquid hydrogen under the external magnetic field of up to 7 T. A LH2 cryostat is concentrically mounted on the inside of a LHe cryostat to cool a NbTi superconducting magnet. The experimental system is installed in an explosion-proof room. Explosion proof electrical devices are used and current leads are covered with an enclosure filled with nitrogen gas. A remote control system has been developed. Furthermore, the effects of stray magnetic field on the existing and the new devices are investigated and electro-magnetic shielding panels and enclosure made of iron were designed. It is confirmed through the cryogenic test that the experimental system meets the design requirements.

Proof test criteria for thin-walled 2219 aluminum pressure vessels. Volume 1: Program summary and data analysis

NASA Technical Reports Server (NTRS)

Finger, R. W.

1976-01-01

This experimental program was undertaken to investigate the crack growth behavior of deep surface flaws in 2219 aluminum. The program included tests of uniaxially loaded surface flaw and center crack panels at temperatures ranging from 20K (-423 F) to ambient. The tests were conducted on both the base metal and as-welded weld metal material. The program was designed to provide data on the mechanisms of failure by ligament penetration, and the residual cyclic life, after proof-testing, of a vessel which has been subjected to incipient penetration by the proof test. The results were compared and analyzed with previously developed data to develop guidelines for the proof testing of thin walled 2219 pressure vessels.

40 CFR 80.1600 - Additional definitions for subpart O.

Code of Federal Regulations, 2014 CFR

2014-07-01

... California. Certified ethanol denaturant means ethanol denaturant that meets the requirements of § 80.1611. Certified Sulfur-FRGAS has the meaning given in § 80.1666(a)(5). Denatured fuel ethanol (DFE) means an.... Ethanol denaturant means previously certified gasoline (including previously certified blendstocks for...

Elimination of cannibalistic denaturation by enzyme immobilization or inhibition

PubMed Central

Wu, Hua-Lin; Lace, Daniel A.; Bender, Myron L.

1981-01-01

The cannibalistic denaturation of α-chymotrypsin (EC 3.4.21.1) around neutral pH can be eliminated by immobilization (insolubilization) of the enzyme or by inhibition by specific reversible inhibitors, but the high-pH denaturation cannot be. The denaturation of the immobilized enzyme at high pH follows first-order kinetics, just as the denaturation of the soluble enzyme does. These results lend credence to the description of the denaturation of chymotrypsin as cannibalistic around neutrality and due to a hydroxide ion reaction at high pH; this interpretation followed from kinetic arguments given in the previous article [Wu, H.-L., Wastell, A. & Bender, M. L. (1981) Proc. Natl. Acad. Sci. USA 78, 4116-4117]. Elimination of denaturation around neutrality by immobilization may be the reason why membrane-bound enzymes are so common in vivo. PMID:16593052

Use of anionic denaturing detergents to purify insoluble proteins after overexpression

PubMed Central

2012-01-01

Background Many proteins form insoluble protein aggregates, called “inclusion bodies”, when overexpressed in E. coli. This is the biggest obstacle in biotechnology. Ever since the reversible denaturation of proteins by chaotropic agents such as urea or guanidinium hydrochloride had been shown, these compounds were predominantly used to dissolve inclusion bodies. Other denaturants exist but have received much less attention in protein purification. While the anionic, denaturing detergent sodiumdodecylsulphate (SDS) is used extensively in analytical SDS-PAGE, it has rarely been used in preparative purification. Results Here we present a simple and versatile method to purify insoluble, hexahistidine-tagged proteins under denaturing conditions. It is based on dissolution of overexpressing bacterial cells in a buffer containing sodiumdodecylsulfate (SDS) and whole-lysate denaturation of proteins. The excess of detergent is removed by cooling and centrifugation prior to affinity purification. Host- and overexpressed proteins do not co-precipitate with SDS and the residual concentration of detergent is compatible with affinity purification on Ni/NTA resin. We show that SDS can be replaced with another ionic detergent, Sarkosyl, during purification. Key advantages over denaturing purification in urea or guanidinium are speed, ease of use, low cost of denaturant and the compatibility of buffers with automated FPLC. Conclusion Ionic, denaturing detergents are useful in breaking the solubility barrier, a major obstacle in biotechnology. The method we present yields detergent-denatured protein. Methods to refold proteins from a detergent denatured state are known and therefore we propose that the procedure presented herein will be of general application in biotechnology. PMID:23231964

Applications of Protein Thermodynamic Database for Understanding Protein Mutant Stability and Designing Stable Mutants.

PubMed

Gromiha, M Michael; Anoosha, P; Huang, Liang-Tsung

2016-01-01

Protein stability is the free energy difference between unfolded and folded states of a protein, which lies in the range of 5-25 kcal/mol. Experimentally, protein stability is measured with circular dichroism, differential scanning calorimetry, and fluorescence spectroscopy using thermal and denaturant denaturation methods. These experimental data have been accumulated in the form of a database, ProTherm, thermodynamic database for proteins and mutants. It also contains sequence and structure information of a protein, experimental methods and conditions, and literature information. Different features such as search, display, and sorting options and visualization tools have been incorporated in the database. ProTherm is a valuable resource for understanding/predicting the stability of proteins and it can be accessed at http://www.abren.net/protherm/ . ProTherm has been effectively used to examine the relationship among thermodynamics, structure, and function of proteins. We describe the recent progress on the development of methods for understanding/predicting protein stability, such as (1) general trends on mutational effects on stability, (2) relationship between the stability of protein mutants and amino acid properties, (3) applications of protein three-dimensional structures for predicting their stability upon point mutations, (4) prediction of protein stability upon single mutations from amino acid sequence, and (5) prediction methods for addressing double mutants. A list of online resources for predicting has also been provided.

Sulfur Speciation and Extraction in Jet A (Briefing Charts)

DTIC Science & Technology

2015-08-16

Extraction fluid: denatured ethanol from Fisher Scientific and deionized water – Jet A fuel , approximately 500-800 ppm sulfur by weight – Data...Outline • Background • Experimental Setup – Extraction of sulfur compounds from fuel to alcohol/water extraction fluid – Each rinse is...Hydrophobic / Oleophillic Membrane Oleophobic / Hydrophillic Membrane Emulsion Phase Fuel Phase Water (Extraction Fluid) Phase DISTRIBUTION A

Thermal Unfolding Simulations of Bacterial Flagellin: Insight into its Refolding Before Assembly

PubMed Central

Chng, Choon-Peng; Kitao, Akio

2008-01-01

Flagellin is the subunit of the bacterial filament, the micrometer-long propeller of a bacterial flagellum. The protein is believed to undergo unfolding for transport through the channel of the filament and to refold in a chamber at the end of the channel before being assembled into the growing filament. We report a thermal unfolding simulation study of S. typhimurium flagellin in aqueous solution as an attempt to gain atomic-level insight into the refolding process. Each molecule comprises two filament-core domains {D0, D1} and two hypervariable-region domains {D2, D3}. D2 can be separated into subdomains D2a and D2b. We observed a similar unfolding order of the domains as reported in experimental thermal denaturation. D2a and D3 exhibited high thermal stability and contained persistent three-stranded β-sheets in the denatured state which could serve as folding cores to guide refolding. A recent mutagenesis study on flagellin stability seems to suggest the importance of the folding cores. Using crude size estimates, our data suggests that the chamber might be large enough for either denatured hypervariable-region domains or filament-core domains, but not whole flagellin; this implicates a two-staged refolding process. PMID:18263660

Counteraction of Trehalose on N, N-Dimethylformamide-Induced Candida rugosa Lipase Denaturation: Spectroscopic Insight and Molecular Dynamic Simulation

PubMed Central

Yang, Xin; Jia, Yigang; Hu, Yi; Xu, Qing; Xu, Xian

2016-01-01

Candida rugosa lipase (CRL) has been widely used as a biocatalyst for non-aqueous synthesis in biotechnological applications, which, however, often suffers significant loss of activity in organic solvent. Experimental results show that trehalose could actively counteract the organic-solvent-induced protein denaturation, while the molecular mechanisms still don’t unclear. Herein, CRL was used as a model enzyme to explore the effects of trehalose on the retention of enzymatic activity upon incubation in N,N-dimethylformamide (DMF). Results showed that both catalytic activity and conformation changes of CRL influenced by DMF solvent were inhibited by trehalose in a dose-dependent fashion. The simulations further indicated that the CRL protein unfolded in binary DMF solution, but retained the native state in the ternary DMF/trehalose system. Trehalose as the second osmolyte added into binary DMF solution decreased DMF-CRL hydrogen bonds efficiently, whereas increased the intermolecular hydrogen bondings between DMF and trehalose. Thus, the origin of its denaturing effects of DMF on protein is thought to be due to the preferential exclusion of trehalose as well as the intermolecular hydrogen bondings between trehalose and DMF. These findings suggest that trehalose protect the CRL protein from DMF-induced unfolding via both indirect and direct interactions. PMID:27031946

Deconvolution of complex differential scanning calorimetry profiles for protein transitions under kinetic control.

PubMed

Toledo-Núñez, Citlali; Vera-Robles, L Iraís; Arroyo-Maya, Izlia J; Hernández-Arana, Andrés

2016-09-15

A frequent outcome in differential scanning calorimetry (DSC) experiments carried out with large proteins is the irreversibility of the observed endothermic effects. In these cases, DSC profiles are analyzed according to methods developed for temperature-induced denaturation transitions occurring under kinetic control. In the one-step irreversible model (native → denatured) the characteristics of the observed single-peaked endotherm depend on the denaturation enthalpy and the temperature dependence of the reaction rate constant, k. Several procedures have been devised to obtain the parameters that determine the variation of k with temperature. Here, we have elaborated on one of these procedures in order to analyze more complex DSC profiles. Synthetic data for a heat capacity curve were generated according to a model with two sequential reactions; the temperature dependence of each of the two rate constants involved was determined, according to the Eyring's equation, by two fixed parameters. It was then shown that our deconvolution procedure, by making use of heat capacity data alone, permits to extract the parameter values that were initially used. Finally, experimental DSC traces showing two and three maxima were analyzed and reproduced with relative success according to two- and four-step sequential models. Copyright © 2016 Elsevier Inc. All rights reserved.

Effects of Natural Osmolytes on the Protein Structure in Supercritical CO2: Molecular Level Evidence.

PubMed

Monhemi, Hassan; Housaindokht, Mohammad Reza; Nakhaei Pour, Ali

2015-08-20

Protein instability in supercritical CO2 limits the application of this green solvent in enzyme-catalyzed reactions. CO2 molecules act as a protein denaturant at high pressure under supercritical conditions. Here, for the first time, we show that natural osmolytes could stabilize protein conformation in supercritical CO2. Molecular dynamics simulation is used to monitor the effects of adding different natural osmolytes on the conformation and dynamics of chymotrypsin inhibitor 2 (CI2) in supercritical CO2. Simulations showed that CI2 is denatured at 200 bar in supercritical CO2, which is in agreement with experimental observations. Interestingly, the protein conformation remains native after addition of ∼1 M amino acid- and sugar-based osmolyte models. These molecules stabilize protein through the formation of supramolecular self-assemblies resulting from macromolecule-osmolyte hydrogen bonds. Nevertheless, trimethylamine N-oxide, which is known as a potent osmolyte for protein stabilization in aqueous solutions, amplifies protein denaturation in supercritical CO2. On the basis of our structural analysis, we introduce a new mechanism for the osmolyte effect in supercritical CO2, an "inclusion mechanism". To the best of our knowledge, this is the first study that introduces the application of natural osmolytes in a supercritical fluid and describes mechanistic insights into osmolyte action in nonaqueous media.

Counteraction of Trehalose on N, N-Dimethylformamide-Induced Candida rugosa Lipase Denaturation: Spectroscopic Insight and Molecular Dynamic Simulation.

PubMed

Yang, Xin; Jiang, Ling; Jia, Yigang; Hu, Yi; Xu, Qing; Xu, Xian; Huang, He

2016-01-01

Candida rugosa lipase (CRL) has been widely used as a biocatalyst for non-aqueous synthesis in biotechnological applications, which, however, often suffers significant loss of activity in organic solvent. Experimental results show that trehalose could actively counteract the organic-solvent-induced protein denaturation, while the molecular mechanisms still don't unclear. Herein, CRL was used as a model enzyme to explore the effects of trehalose on the retention of enzymatic activity upon incubation in N,N-dimethylformamide (DMF). Results showed that both catalytic activity and conformation changes of CRL influenced by DMF solvent were inhibited by trehalose in a dose-dependent fashion. The simulations further indicated that the CRL protein unfolded in binary DMF solution, but retained the native state in the ternary DMF/trehalose system. Trehalose as the second osmolyte added into binary DMF solution decreased DMF-CRL hydrogen bonds efficiently, whereas increased the intermolecular hydrogen bondings between DMF and trehalose. Thus, the origin of its denaturing effects of DMF on protein is thought to be due to the preferential exclusion of trehalose as well as the intermolecular hydrogen bondings between trehalose and DMF. These findings suggest that trehalose protect the CRL protein from DMF-induced unfolding via both indirect and direct interactions.

Protein Denaturation on p-T Axes--Thermodynamics and Analysis.

PubMed

Smeller, László

2015-01-01

Proteins are essential players in the vast majority of molecular level life processes. Since their structure is in most cases substantial for their correct function, study of their structural changes attracted great interest in the past decades. The three dimensional structure of proteins is influenced by several factors including temperature, pH, presence of chaotropic and cosmotropic agents, or presence of denaturants. Although pressure is an equally important thermodynamic parameter as temperature, pressure studies are considerably less frequent in the literature, probably due to the technical difficulties associated to the pressure studies. Although the first steps in the high-pressure protein study have been done 100 years ago with Bridgman's ground breaking work, the field was silent until the modern spectroscopic techniques allowed the characterization of the protein structural changes, while the protein was under pressure. Recently a number of proteins were studied under pressure, and complete pressure-temperature phase diagrams were determined for several of them. This review summarizes the thermodynamic background of the typical elliptic p-T phase diagram, its limitations and the possible reasons for deviations of the experimental diagrams from the theoretical one. Finally we show some examples of experimentally determined pressure-temperature phase diagrams.

27 CFR 19.391 - Mixing denatured spirits.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2013-04-01 2013-04-01 false Mixing denatured spirits... Rules for Mixing and Converting Denatured Spirits § 19.391 Mixing denatured spirits. (a) Spirits of the... same formula, the proprietor may mix them on bonded premises. (b) Spirits of different formulas. A...

27 CFR 19.391 - Mixing denatured spirits.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2012-04-01 2012-04-01 false Mixing denatured spirits... Rules for Mixing and Converting Denatured Spirits § 19.391 Mixing denatured spirits. (a) Spirits of the... same formula, the proprietor may mix them on bonded premises. (b) Spirits of different formulas. A...

27 CFR 19.391 - Mixing denatured spirits.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2014-04-01 2014-04-01 false Mixing denatured spirits... Rules for Mixing and Converting Denatured Spirits § 19.391 Mixing denatured spirits. (a) Spirits of the... same formula, the proprietor may mix them on bonded premises. (b) Spirits of different formulas. A...

27 CFR 19.391 - Mixing denatured spirits.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2011-04-01 2011-04-01 false Mixing denatured spirits... Rules for Mixing and Converting Denatured Spirits § 19.391 Mixing denatured spirits. (a) Spirits of the... same formula, the proprietor may mix them on bonded premises. (b) Spirits of different formulas. A...

27 CFR 19.392 - Converting denatured alcohol to a different formula.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2011-04-01 2011-04-01 false Converting denatured alcohol to a different formula. 19.392 Section 19.392 Alcohol, Tobacco Products and Firearms ALCOHOL AND... denatured alcohol to a different formula. (a) General. A proprietor may convert specially denatured alcohol...

27 CFR 21.161 - Weights and specific gravities of specially denatured alcohol.

Code of Federal Regulations, 2010 CFR

2010-04-01

... gravities of specially denatured alcohol. 21.161 Section 21.161 Alcohol, Tobacco Products and Firearms... ALCOHOL AND RUM Weights and Specific Gravities of Specially Denatured Alcohol § 21.161 Weights and specific gravities of specially denatured alcohol. The weight of one gallon of each formula of specially...

27 CFR 21.161 - Weights and specific gravities of specially denatured alcohol.

Code of Federal Regulations, 2011 CFR

2011-04-01

... gravities of specially denatured alcohol. 21.161 Section 21.161 Alcohol, Tobacco Products and Firearms... ALCOHOL AND RUM Weights and Specific Gravities of Specially Denatured Alcohol § 21.161 Weights and specific gravities of specially denatured alcohol. The weight of one gallon of each formula of specially...

27 CFR 21.161 - Weights and specific gravities of specially denatured alcohol.

Code of Federal Regulations, 2013 CFR

2013-04-01

... gravities of specially denatured alcohol. 21.161 Section 21.161 Alcohol, Tobacco Products and Firearms... ALCOHOL AND RUM Weights and Specific Gravities of Specially Denatured Alcohol § 21.161 Weights and specific gravities of specially denatured alcohol. The weight of one gallon of each formula of specially...

27 CFR 21.161 - Weights and specific gravities of specially denatured alcohol.

Code of Federal Regulations, 2012 CFR

2012-04-01

... gravities of specially denatured alcohol. 21.161 Section 21.161 Alcohol, Tobacco Products and Firearms... ALCOHOL AND RUM Weights and Specific Gravities of Specially Denatured Alcohol § 21.161 Weights and specific gravities of specially denatured alcohol. The weight of one gallon of each formula of specially...

27 CFR 21.161 - Weights and specific gravities of specially denatured alcohol.

Code of Federal Regulations, 2014 CFR

2014-04-01

... gravities of specially denatured alcohol. 21.161 Section 21.161 Alcohol, Tobacco Products and Firearms... ALCOHOL AND RUM Weights and Specific Gravities of Specially Denatured Alcohol § 21.161 Weights and specific gravities of specially denatured alcohol. The weight of one gallon of each formula of specially...

27 CFR 19.453 - Testing of denaturants.

Code of Federal Regulations, 2010 CFR

2010-04-01

... Denaturation § 19.453 Testing of denaturants. (a) Testing. Proprietors shall ensure that the materials they... shall be taken in such manner as to represent a true composite of the total lot being sampled. When... part 21, the proprietor shall not use the material unless he treats or manipulates the denaturant to...

Design principles for high–pressure force fields: Aqueous TMAO solutions from ambient to kilobar pressures

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hölzl, Christoph; Horinek, Dominik, E-mail: dominik.horinek@ur.de; Kibies, Patrick

Accurate force fields are one of the major pillars on which successful molecular dynamics simulations of complex biomolecular processes rest. They have been optimized for ambient conditions, whereas high-pressure simulations become increasingly important in pressure perturbation studies, using pressure as an independent thermodynamic variable. Here, we explore the design of non-polarizable force fields tailored to work well in the realm of kilobar pressures – while avoiding complete reparameterization. Our key is to first compute the pressure-induced electronic and structural response of a solute by combining an integral equation approach to include pressure effects on solvent structure with a quantum-chemical treatmentmore » of the solute within the embedded cluster reference interaction site model (EC-RISM) framework. Next, the solute’s response to compression is taken into account by introducing pressure-dependence into selected parameters of a well-established force field. In our proof-of-principle study, the full machinery is applied to N,N,N-trimethylamine-N-oxide (TMAO) in water being a potent osmolyte that counteracts pressure denaturation. EC-RISM theory is shown to describe well the charge redistribution upon compression of TMAO(aq) to 10 kbar, which is then embodied in force field molecular dynamics by pressure-dependent partial charges. The performance of the high pressure force field is assessed by comparing to experimental and ab initio molecular dynamics data. Beyond its broad usefulness for designing non-polarizable force fields for extreme thermodynamic conditions, a good description of the pressure-response of solutions is highly recommended when constructing and validating polarizable force fields.« less

Design principles for high-pressure force fields: Aqueous TMAO solutions from ambient to kilobar pressures.

PubMed

Hölzl, Christoph; Kibies, Patrick; Imoto, Sho; Frach, Roland; Suladze, Saba; Winter, Roland; Marx, Dominik; Horinek, Dominik; Kast, Stefan M

2016-04-14

Accurate force fields are one of the major pillars on which successful molecular dynamics simulations of complex biomolecular processes rest. They have been optimized for ambient conditions, whereas high-pressure simulations become increasingly important in pressure perturbation studies, using pressure as an independent thermodynamic variable. Here, we explore the design of non-polarizable force fields tailored to work well in the realm of kilobar pressures--while avoiding complete reparameterization. Our key is to first compute the pressure-induced electronic and structural response of a solute by combining an integral equation approach to include pressure effects on solvent structure with a quantum-chemical treatment of the solute within the embedded cluster reference interaction site model (EC-RISM) framework. Next, the solute's response to compression is taken into account by introducing pressure-dependence into selected parameters of a well-established force field. In our proof-of-principle study, the full machinery is applied to N,N,N-trimethylamine-N-oxide (TMAO) in water being a potent osmolyte that counteracts pressure denaturation. EC-RISM theory is shown to describe well the charge redistribution upon compression of TMAO(aq) to 10 kbar, which is then embodied in force field molecular dynamics by pressure-dependent partial charges. The performance of the high pressure force field is assessed by comparing to experimental and ab initio molecular dynamics data. Beyond its broad usefulness for designing non-polarizable force fields for extreme thermodynamic conditions, a good description of the pressure-response of solutions is highly recommended when constructing and validating polarizable force fields.

Analysis, design, and testing of a low cost, direct force command linear proof mass actuator for structural control

NASA Technical Reports Server (NTRS)

Slater, G. L.; Shelley, Stuart; Jacobson, Mark

1993-01-01

In this paper, the design, analysis, and test of a low cost, linear proof mass actuator for vibration control is presented. The actuator is based on a linear induction coil from a large computer disk drive. Such disk drives are readily available and provide the linear actuator, current feedback amplifier, and power supply for a highly effective, yet inexpensive, experimental laboratory actuator. The device is implemented as a force command input system, and the performance is virtually the same as other, more sophisticated, linear proof mass systems.

Denaturation of RNA secondary and tertiary structure by urea: simple unfolded state models and free energy parameters account for measured m-values

PubMed Central

Lambert, Dominic; Draper, David E.

2012-01-01

To investigate the mechanism by which urea destabilizes RNA structure, urea-induced unfolding of four different RNA secondary and tertiary structures was quantified in terms of an m-value, the rate at which the free energy of unfolding changes with urea molality. From literature data and our osmometric study of a backbone analog, we derived average interaction potentials (per Å2 of solvent accessible surface) between urea and three kinds of RNA surfaces: phosphate, ribose, and base. Estimates of the increases in solvent accessible surface areas upon RNA denaturation were based on a simple model of unfolded RNA as a combination of helical and single strand segments. These estimates, combined with the three interaction potentials and a term to account for urea interactions with released ions, yield calculated m-values in good agreement with experimental values (200 mm monovalent salt). Agreement was obtained only if single-stranded RNAs were modeled in a highly stacked, A form conformation. The primary driving force for urea induced denaturation is the strong interaction of urea with the large surface areas of bases that become exposed upon denaturation of either RNA secondary or tertiary structure, though urea interactions with backbone and released ions may account for up to a third of the m-value. Urea m-values for all four RNA are salt-dependent, which we attribute to an increased extension (or decreased charge density) of unfolded RNAs with increased urea concentration. The sensitivity of the urea m-value to base surface exposure makes it a potentially useful probe of the conformations of RNA unfolded states. PMID:23088364

27 CFR 19.385 - Making alcohol or water solutions of denaturants.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2011-04-01 2011-04-01 false Making alcohol or water solutions of denaturants. 19.385 Section 19.385 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO... alcohol or water solutions of denaturants. If a proprietor uses a denaturant that is difficult to dissolve...

Toward an Accurate Theoretical Framework for Describing Ensembles for Proteins under Strongly Denaturing Conditions

PubMed Central

Tran, Hoang T.; Pappu, Rohit V.

2006-01-01

Our focus is on an appropriate theoretical framework for describing highly denatured proteins. In high concentrations of denaturants, proteins behave like polymers in a good solvent and ensembles for denatured proteins can be modeled by ignoring all interactions except excluded volume (EV) effects. To assay conformational preferences of highly denatured proteins, we quantify a variety of properties for EV-limit ensembles of 23 two-state proteins. We find that modeled denatured proteins can be best described as follows. Average shapes are consistent with prolate ellipsoids. Ensembles are characterized by large correlated fluctuations. Sequence-specific conformational preferences are restricted to local length scales that span five to nine residues. Beyond local length scales, chain properties follow well-defined power laws that are expected for generic polymers in the EV limit. The average available volume is filled inefficiently, and cavities of all sizes are found within the interiors of denatured proteins. All properties characterized from simulated ensembles match predictions from rigorous field theories. We use our results to resolve between conflicting proposals for structure in ensembles for highly denatured states. PMID:16766618

Salt dependent resistance against chemical denaturation of alkaline protease from a newly isolated haloalkaliphilic Bacillus sp.

PubMed

Dodia, M S; Bhimani, H G; Rawal, C M; Joshi, R H; Singh, S P

2008-09-01

Only few enzymes from haloalkaliphiles are biochemically characterized for their kinetic behaviour and stability. In view of this realization, an alkaline protease from Bacillus sp. AH-6, displaying salt-dependent resistance against chemical denaturation by Urea and Guanidium hydrochloride was investigated for denaturation and in vitro protein folding. The crude enzyme was highly resistant against urea (8 M) denaturation up to 72 h; however, on purification, it turned sensitive and got denatured within 2 h. Interestingly, the purified enzyme regained the resistance in the presence of NaCl. Effective refolding of the purified enzyme was achieved with glycerol; however, other approaches such as lower protein concentrations, rapid dilution and slow removal of the denaturant did not further add to refolding. The results are important from the viewpoint that only few enzymes from haloalkaliphilic bacteria are characterized. Since the resistance against chemical denaturation is a rare phenomenon, the findings would enrich the knowledge on protein stability and denaturation. Besides, such biocatalysts would definitely have novel applications under harsh chemical environments.

On-chip isothermal, chemical cycling polymerase chain reaction (ccPCR)

NASA Astrophysics Data System (ADS)

Persat, Alexandre; Santiago, Juan

2008-11-01

We demonstrate a novel ccPCR technique for microfluidic DNA amplification where temperature is held constant in space and time. The polymerase chain reaction is a platform of choice for biological assays and typically based on a three-step thermal cycling: DNA denaturation, primers annealing and extension by an enzyme. We here demonstrate a novel technique where high concentration chemical denaturants (solvents) denature DNA. We leverage the high electrophoretic mobility of DNA and the electrical neutrality of denaturants to achieve chemical cycling. We focus DNA with isotachophoresis (ITP); a robust electrophoretic preconcentration technique which generates strong electric field gradients and protects the sample from dispersion. We apply a pressure-driven flow to balance electromigration velocity and keep the DNA sample stationary in a microchannel. We drive the DNA through a series of high denaturant concentration zones. DNA denatures at high denaturant concentration. At low denaturant concentration, the enzyme creates complementary strands. DNA reaction kinetics are slower than buffer reactions involved in ITP. We demonstrate successful ccPCR amplification for detection of E. Coli. The ccPCR has the potential for simpler chemistry than traditional PCR.

Effect of heat, pH, ultrasonication and ethanol on the denaturation of whey protein isolate using a newly developed approach in the analysis of difference-UV spectra.

PubMed

Nikolaidis, Athanasios; Andreadis, Marios; Moschakis, Thomas

2017-10-01

A newly developed method of analysis of difference-UV spectra was successfully implemented in the study of the effect of heat, pH, ultrasonication and ethanol on the denaturation of whey protein isolate. It was found that whey proteins exhibit their highest stability against heat denaturation at pH 3.75. At very low pH values, i.e. 2.5, they exhibited considerable cold denaturation, while after heating at this pH value, the supplementary heat denaturation rate was lower compared to that at neutral pH. The highest heat denaturation rates were observed at pH values higher than neutral. High power sonication on whey proteins, previously heated at 90°C for 30min, resulted in a rather small reduction of the fraction of the heat denatured protein aggregates. Finally, when ethanol was used as a cosolvent in the concentration range 20-50%, a sharp increase in the degree of denaturation, compared to the native protein solution, was observed. Copyright © 2017 Elsevier Ltd. All rights reserved.

Structural Characteristic of the Initial Unfolded State on Refolding Determines Catalytic Efficiency of the Folded Protein in Presence of Osmolytes

PubMed Central

Warepam, Marina; Sharma, Gurumayum Suraj; Dar, Tanveer Ali; Khan, Md. Khurshid Alam; Singh, Laishram Rajendrakumar

2014-01-01

Osmolytes are low molecular weight organic molecules accumulated by organisms to assist proper protein folding, and to provide protection to the structural integrity of proteins under denaturing stress conditions. It is known that osmolyte-induced protein folding is brought by unfavorable interaction of osmolytes with the denatured/unfolded states. The interaction of osmolyte with the native state does not significantly contribute to the osmolyte-induced protein folding. We have therefore investigated if different denatured states of a protein (generated by different denaturing agents) interact differently with the osmolytes to induce protein folding. We observed that osmolyte-assisted refolding of protein obtained from heat-induced denatured state produces native molecules with higher enzyme activity than those initiated from GdmCl- or urea-induced denatured state indicating that the structural property of the initial denatured state during refolding by osmolytes determines the catalytic efficiency of the folded protein molecule. These conclusions have been reached from the systematic measurements of enzymatic kinetic parameters (K m and k cat), thermodynamic stability (T m and ΔH m) and secondary and tertiary structures of the folded native proteins obtained from refolding of various denatured states (due to heat-, urea- and GdmCl-induced denaturation) of RNase-A in the presence of various osmolytes. PMID:25313668

Structural stability of DNA origami nanostructures in the presence of chaotropic agents

NASA Astrophysics Data System (ADS)

Ramakrishnan, Saminathan; Krainer, Georg; Grundmeier, Guido; Schlierf, Michael; Keller, Adrian

2016-05-01

DNA origami represent powerful platforms for single-molecule investigations of biomolecular processes. The required structural integrity of the DNA origami may, however, pose significant limitations regarding their applicability, for instance in protein folding studies that require strongly denaturing conditions. Here, we therefore report a detailed study on the stability of 2D DNA origami triangles in the presence of the strong chaotropic denaturing agents urea and guanidinium chloride (GdmCl) and its dependence on concentration and temperature. At room temperature, the DNA origami triangles are stable up to at least 24 h in both denaturants at concentrations as high as 6 M. At elevated temperatures, however, structural stability is governed by variations in the melting temperature of the individual staple strands. Therefore, the global melting temperature of the DNA origami does not represent an accurate measure of their structural stability. Although GdmCl has a stronger effect on the global melting temperature, its attack results in less structural damage than observed for urea under equivalent conditions. This enhanced structural stability most likely originates from the ionic nature of GdmCl. By rational design of the arrangement and lengths of the individual staple strands used for the folding of a particular shape, however, the structural stability of DNA origami may be enhanced even further to meet individual experimental requirements. Overall, their high stability renders DNA origami promising platforms for biomolecular studies in the presence of chaotropic agents, including single-molecule protein folding or structural switching.DNA origami represent powerful platforms for single-molecule investigations of biomolecular processes. The required structural integrity of the DNA origami may, however, pose significant limitations regarding their applicability, for instance in protein folding studies that require strongly denaturing conditions. Here, we therefore report a detailed study on the stability of 2D DNA origami triangles in the presence of the strong chaotropic denaturing agents urea and guanidinium chloride (GdmCl) and its dependence on concentration and temperature. At room temperature, the DNA origami triangles are stable up to at least 24 h in both denaturants at concentrations as high as 6 M. At elevated temperatures, however, structural stability is governed by variations in the melting temperature of the individual staple strands. Therefore, the global melting temperature of the DNA origami does not represent an accurate measure of their structural stability. Although GdmCl has a stronger effect on the global melting temperature, its attack results in less structural damage than observed for urea under equivalent conditions. This enhanced structural stability most likely originates from the ionic nature of GdmCl. By rational design of the arrangement and lengths of the individual staple strands used for the folding of a particular shape, however, the structural stability of DNA origami may be enhanced even further to meet individual experimental requirements. Overall, their high stability renders DNA origami promising platforms for biomolecular studies in the presence of chaotropic agents, including single-molecule protein folding or structural switching. Electronic supplementary information (ESI) available: Melting curves without baseline subtraction, AFM images of DNA origami after 24 h incubation, calculated melting temperatures of all staple strands. See DOI: 10.1039/c6nr00835f

Visualization of early events in acetic acid denaturation of HIV-1 protease: a molecular dynamics study.

PubMed

Borkar, Aditi Narendra; Rout, Manoj Kumar; Hosur, Ramakrishna V

2011-01-01

Protein denaturation plays a crucial role in cellular processes. In this study, denaturation of HIV-1 Protease (PR) was investigated by all-atom MD simulations in explicit solvent. The PR dimer and monomer were simulated separately in 9 M acetic acid (9 M AcOH) solution and water to study the denaturation process of PR in acetic acid environment. Direct visualization of the denaturation dynamics that is readily available from such simulations has been presented. Our simulations in 9 M AcOH reveal that the PR denaturation begins by separation of dimer into intact monomers and it is only after this separation that the monomer units start denaturing. The denaturation of the monomers is flagged off by the loss of crucial interactions between the α-helix at C-terminal and surrounding β-strands. This causes the structure to transit from the equilibrium dynamics to random non-equilibrating dynamics. Residence time calculations indicate that denaturation occurs via direct interaction of the acetic acid molecules with certain regions of the protein in 9 M AcOH. All these observations have helped to decipher a picture of the early events in acetic acid denaturation of PR and have illustrated that the α-helix and the β-sheet at the C-terminus of a native and functional PR dimer should maintain both the stability and the function of the enzyme and thus present newer targets for blocking PR function.

Exploring luminescence-based temperature sensing using protein-passivated gold nanoclusters

NASA Astrophysics Data System (ADS)

Chen, Xi; Essner, Jeremy B.; Baker, Gary A.

2014-07-01

We explore the analytical performance and limitations of optically monitoring aqueous-phase temperature using protein-protected gold nanoclusters (AuNCs). Although not reported elsewhere, we find that these bio-passivated AuNCs show pronounced hysteresis upon thermal cycling. This unwanted behaviour can be eliminated by several strategies, including sol-gel coating and thermal denaturation of the biomolecular template, introducing protein-templated AuNC probes as viable nanothermometers.We explore the analytical performance and limitations of optically monitoring aqueous-phase temperature using protein-protected gold nanoclusters (AuNCs). Although not reported elsewhere, we find that these bio-passivated AuNCs show pronounced hysteresis upon thermal cycling. This unwanted behaviour can be eliminated by several strategies, including sol-gel coating and thermal denaturation of the biomolecular template, introducing protein-templated AuNC probes as viable nanothermometers. Electronic supplementary information (ESI) available: Supplemental figures and experimental details. See DOI: 10.1039/c4nr02069c

Single-Molecule Denaturation Mapping of DNA in Nanofluidic Channels

NASA Astrophysics Data System (ADS)

Reisner, Walter; Larsen, Niels; Silahtaroglu, Asli; Kristensen, Anders; Tommerup, Niels; Tegenfeldt, Jonas O.; Flyvbjerg, Henrik

2010-03-01

Nanochannel based DNA stretching can serve as a platform for a new optical mapping technique based on measuring the pattern of partial melting along the extended molecules. We partially melt DNA extended in nanofluidic channels via a combination of local heating and added chemical denaturants. The melted molecules, imaged via a standard fluorescence videomicroscopy setup, exhibit a nonuniform fluorescence profile corresponding to a series of local dips and peaks in the intensity trace along the stretched molecule. We show that this barcode is consistent with the presence of locally melted regions along the molecule and can be explained by calculations of sequence-dependent melting probability. Specifically, we obtain experimental melting profiles for T4, T7, lambda-phage and bacterial artificial chromosome DNA (from human chromosome 12) and compare these profiles to theory. In addition, we demonstrate that the BAC melting profile can be used to align the BAC to its correct position on chromosome 12.

Energetic rationale for an unexpected and abrupt reversal of guanidinium chloride-induced unfolding of peptide deformylase.

PubMed

Berg, Alexander K; Manokaran, Sumathra; Eiler, Daniel; Kooren, Joel; Mallik, Sanku; Srivastava, D K

2008-01-01

Peptide deformylase (PDF) catalyzes the removal of formyl group from the N-terminal methionine residues of nascent proteins in prokaryotes, and this enzyme is a high priority target for antibiotic design. In pursuit of delineating the structural-functional features of Escherichia coli PDF (EcPDF), we investigated the mechanistic pathway for the guanidinium chloride (GdmCl)-induced unfolding of the enzyme by monitoring the secondary structural changes via CD spectroscopy. The experimental data revealed that EcPDF is a highly stable enzyme, and it undergoes slow denaturation in the presence of varying concentrations of GdmCl. The most interesting aspect of these studies has been the abrupt reversal of the unfolding pathway at low to moderate concentrations of the denaturant, but not at high concentration. An energetic rationale for such an unprecedented feature in protein chemistry is offered.

T1ρ is superior to T2 mapping for the evaluation of articular cartilage denaturalization with osteoarthritis: radiological-pathological correlation after total knee arthroplasty.

PubMed

Takayama, Yukihisa; Hatakenaka, Masamitsu; Tsushima, Hidetoshi; Okazaki, Ken; Yoshiura, Takashi; Yonezawa, Masato; Nishikawa, Kei; Iwamoto, Yukihide; Honda, Hiroshi

2013-04-01

We compared the diagnostic performance of T1ρ and T2 mappings in the evaluation of denatured articular cartilage with osteoarthritis of the knee. 2D-Sagittal T1ρ and T2 mappings of the knee were obtained from 16 patients before total knee arthroplasty. After surgery, specimens of the femur and tibia were regionally segmented according to a 5-point scale of the severity of denaturalization. The T1ρ and T2 values in the full thickness of the articular cartilage in each region were measured by two observers. The two mappings were compared for their ability to differentiate between normal and denatured articular cartilage and also for their usefulness in grading the severity of the denaturalization using the area under receiver operating characteristic curves (Az). A p<0.05 was considered significant for each analysis. The T1ρ mapping showed a significantly higher Az value than the T2 mapping for the differentiation between normal and denatured articular cartilage (p<0.05). Regarding the assessment of the severity of denaturalization, T1ρ mapping could differentiate between normal and mild denaturalization (p<0.05), but T2 mapping could not. However, there were no significant differences between the two mappings in the discrimination of mild versus moderate denaturalization or of moderate versus severe denaturalization. The two observers showed good agreement in the results (intraclass correlation coefficient=0.81 for T1ρ and 0.92 for T2). T1ρ mapping is superior to T2 mapping for the evaluation of denatured articular cartilage with osteoarthritis of the knee. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Quantitative assessments of the distinct contributions of polypeptide backbone amides versus sidechain groups to chain expansion via chemical denaturation

PubMed Central

Holehouse, Alex S.; Garai, Kanchan; Lyle, Nicholas; Vitalis, Andreas; Pappu, Rohit V.

2015-01-01

In aqueous solutions with high concentrations of chemical denaturants such as urea and guanidinium chloride (GdmCl) proteins expand to populate heterogeneous conformational ensembles. These denaturing environments are thought to be good solvents for generic protein sequences because properties of conformational distributions align with those of canonical random coils. Previous studies showed that water is a poor solvent for polypeptide backbones and therefore backbones form collapsed globular structures in aqueous solvents. Here, we ask if polypeptide backbones can intrinsically undergo the requisite chain expansion in aqueous solutions with high concentrations of urea and GdmCl. We answer this question using a combination of molecular dynamics simulations and fluorescence correlation spectroscopy. We find that the degree of backbone expansion is minimal in aqueous solutions with high concentrations denaturants. Instead, polypeptide backbones sample conformations that are denaturant-specific mixtures of coils and globules, with a persistent preference for globules. Therefore, typical denaturing environments cannot be classified as good solvents for polypeptide backbones. How then do generic protein sequences expand in denaturing environments? To answer this question, we investigated the effects of sidechains using simulations of two archetypal sequences with amino acid compositions that are mixtures of charged, hydrophobic, and polar groups. We find that sidechains lower the effective concentration of backbone amides in water leading to an intrinsic expansion of polypeptide backbones in the absence of denaturants. Additional dilution of the effective concentration of backbone amides is achieved through preferential interactions with denaturants. These effects lead to conformational statistics in denaturing environments that are congruent with those of canonical random coils. Our results highlight the role of sidechain-mediated interactions as determinants of the conformational properties of unfolded states in water and in influencing chain expansion upon denaturation. PMID:25664638

Chaperonin-based biolayer interferometry to assess the kinetic stability of metastable, aggregation-prone proteins

PubMed Central

Lea, Wendy A.; Naik, Subhashchandra; Chaudhri, Tapan; Machen, Alexandra J.; O’Neil, Pierce T.; McGinn-Straub, Wesley; Tischer, Alexander; Auton, Matthew T.; Burns, Joshua R.; Baldwin, Michael R.; Khar, Karen R.; Karanicolas, John; Fisher, Mark T.

2017-01-01

Stabilizing the folded state of metastable and/or aggregation-prone proteins through exogenous ligand binding is an appealing strategy to decrease disease pathologies brought on by protein folding defects or deleterious kinetic transitions. Current methods of examining ligand binding to these marginally stable native states are limited, because protein aggregation typically interferes with analysis. Here, we describe a rapid method for assessing the kinetic stability of folded proteins and monitoring the effects of ligand stabilization for both intrinsically stable proteins (monomers, oligomers, multi-domain) and metastable proteins (e.g. low Tm) that uses a new GroEL chaperonin-based biolayer interferometry (BLI) denaturant-pulse platform. A kinetically controlled denaturation isotherm is generated by exposing a target protein immobilized on a BLI biosensor to increasing denaturant concentrations (urea or GnHCl) in a pulsatile manner to induce partial or complete unfolding of the attached protein population. Following the rapid removal of the denaturant, the extent of hydrophobic unfolded/partially folded species that remain is detected by increased GroEL binding. Since this kinetic denaturant pulse is brief, the amplitude of the GroEL binding to the immobilized protein depends on the duration of exposure to denaturant, the concentration of denaturant, wash times, and the underlying protein unfolding/refolding kinetics; fixing all other parameters and plotting GroEL binding amplitude versus denaturant pulse concentration results in a kinetically controlled denaturation isotherm. When folding osmolytes or stabilizing ligands are added to the immobilized target proteins before and during the denaturant pulse, the diminished population of unfolded/partially folded protein is manifested by a decreased GroEL binding and/or a marked shift in these kinetically controlled denaturation profiles to higher denaturant concentrations. This particular platform approach can be used to identify small molecules/solution conditions that can stabilize or destabilize thermally stable proteins, multi-domain proteins, oligomeric proteins, and most importantly, aggregation prone metastable proteins. PMID:27505032

PCR performance of a thermostable heterodimeric archaeal DNA polymerase

PubMed Central

Killelea, Tom; Ralec, Céline; Bossé, Audrey; Henneke, Ghislaine

2014-01-01

DNA polymerases are versatile tools used in numerous important molecular biological core technologies like the ubiquitous polymerase chain reaction (PCR), cDNA cloning, genome sequencing, and nucleic acid based diagnostics. Taking into account the multiple DNA amplification techniques in use, different DNA polymerases must be optimized for each type of application. One of the current tendencies is to reengineer or to discover new DNA polymerases with increased performance and broadened substrate spectra. At present, there is a great demand for such enzymes in applications, e.g., forensics or paleogenomics. Current major limitations hinge on the inability of conventional PCR enzymes, such as Taq, to amplify degraded or low amounts of template DNA. Besides, a wide range of PCR inhibitors can also impede reactions of nucleic acid amplification. Here we looked at the PCR performances of the proof-reading D-type DNA polymerase from P. abyssi, Pab-polD. Fragments, 3 kilobases in length, were specifically PCR-amplified in its optimized reaction buffer. Pab-polD showed not only a greater resistance to high denaturation temperatures than Taq during cycling, but also a superior tolerance to the presence of potential inhibitors. Proficient proof-reading Pab-polD enzyme could also extend a primer containing up to two mismatches at the 3' primer termini. Overall, we found valuable biochemical properties in Pab-polD compared to the conventional Taq, which makes the enzyme ideally suited for cutting-edge PCR-applications. PMID:24847315

40 CFR 80.1642 - Sampling and testing requirements for producers and importers of denatured fuel ethanol and other...

Code of Federal Regulations, 2014 CFR

2014-07-01

... producers and importers of denatured fuel ethanol and other oxygenates for use by oxygenate blenders. 80... requirements for producers and importers of denatured fuel ethanol and other oxygenates for use by oxygenate blenders. Beginning January 1, 2017, producers and importers of denatured fuel ethanol (DFE) and other...

40 CFR 80.1610 - Standards and requirements for producers and importers of denatured fuel ethanol and other...

Code of Federal Regulations, 2014 CFR

2014-07-01

... producers and importers of denatured fuel ethanol and other oxygenates designated for use in transportation... requirements for producers and importers of denatured fuel ethanol and other oxygenates designated for use in transportation fuel. Beginning January 1, 2017, producers and importers of denatured fuel ethanol (DFE) or other...

Guanidinium-Induced Denaturation by Breaking of Salt Bridges.

PubMed

Meuzelaar, Heleen; Panman, Matthijs R; Woutersen, Sander

2015-12-07

Despite its wide use as a denaturant, the mechanism by which guanidinium (Gdm(+) ) induces protein unfolding remains largely unclear. Herein, we show evidence that Gdm(+) can induce denaturation by disrupting salt bridges that stabilize the folded conformation. We study the Gdm(+) -induced denaturation of a series of peptides containing Arg/Glu and Lys/Glu salt bridges that either stabilize or destabilize the folded conformation. The peptides containing stabilizing salt bridges are found to be denatured much more efficiently by Gdm(+) than the peptides containing destabilizing salt bridges. Complementary 2D-infrared measurements suggest a denaturation mechanism in which Gdm(+) binds to side-chain carboxylate groups involved in salt bridges. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Novel microscale approaches for easy, rapid determination of protein stability in academic and commercial settings

PubMed Central

Alexander, Crispin G.; Wanner, Randy; Johnson, Christopher M.; Breitsprecher, Dennis; Winter, Gerhard; Duhr, Stefan; Baaske, Philipp; Ferguson, Neil

2014-01-01

Chemical denaturant titrations can be used to accurately determine protein stability. However, data acquisition is typically labour intensive, has low throughput and is difficult to automate. These factors, combined with high protein consumption, have limited the adoption of chemical denaturant titrations in commercial settings. Thermal denaturation assays can be automated, sometimes with very high throughput. However, thermal denaturation assays are incompatible with proteins that aggregate at high temperatures and large extrapolation of stability parameters to physiological temperatures can introduce significant uncertainties. We used capillary-based instruments to measure chemical denaturant titrations by intrinsic fluorescence and microscale thermophoresis. This allowed higher throughput, consumed several hundred-fold less protein than conventional, cuvette-based methods yet maintained the high quality of the conventional approaches. We also established efficient strategies for automated, direct determination of protein stability at a range of temperatures via chemical denaturation, which has utility for characterising stability for proteins that are difficult to purify in high yield. This approach may also have merit for proteins that irreversibly denature or aggregate in classical thermal denaturation assays. We also developed procedures for affinity ranking of protein–ligand interactions from ligand-induced changes in chemical denaturation data, and proved the principle for this by correctly ranking the affinity of previously unreported peptide–PDZ domain interactions. The increased throughput, automation and low protein consumption of protein stability determinations afforded by using capillary-based methods to measure denaturant titrations, can help to revolutionise protein research. We believe that the strategies reported are likely to find wide applications in academia, biotherapeutic formulation and drug discovery programmes. PMID:25262836

27 CFR 19.41 - Claims on spirits, denatured spirits, articles, or wines lost or destroyed in bond.

Code of Federal Regulations, 2010 CFR

2010-04-01

..., denatured spirits, articles, or wines lost or destroyed in bond. 19.41 Section 19.41 Alcohol, Tobacco... DISTILLED SPIRITS PLANTS Taxes Claims § 19.41 Claims on spirits, denatured spirits, articles, or wines lost..., relating to the destruction or loss of spirits, denatured spirits, articles, or wines in bond, shall be...

Different urea stoichiometries between the dissociation and denaturation of tobacco mosaic virus as probed by hydrostatic pressure.

PubMed

Santos, Jose L R; Aparicio, Ricardo; Joekes, Inés; Silva, Jerson L; Bispo, Jose A C; Bonafe, Carlos F S

2008-05-01

Viruses are very efficient self-assembly structures, but little is understood about the thermodynamics governing their directed assembly. At higher levels of pressure or when pressure is combined with urea, denaturation occurs. For a better understanding of such processes, we investigated the apparent thermodynamic parameters of dissociation and denaturation by assuming a steady-state condition. These processes can be measured considering the decrease of light scattering of a viral solution due to the dissociation process, and the red shift of the fluorescence emission spectra, that occurs with the denaturation process. We determined the apparent urea stoichiometry considering the equilibrium reaction of TMV dissociation and subunit denaturation, which furnished, respectively, 1.53 and 11.1 mol of urea/mol of TMV subunit. The denaturation and dissociation conditions were arrived in a near reversible pathway, allowing the determination of thermodynamic parameters. Gel filtration HPLC, electron microscopy and circular dichroism confirmed the dissociation and denaturation processes. Based on spectroscopic results from earlier papers, the calculation of the apparent urea stoichiometry of dissociation and denaturation of several other viruses resulted in similar values, suggesting a similar virus-urea interaction among these systems.

Pseudomonas aeruginosa cytochrome c551 denaturation by five systematic urea derivatives that differ in the alkyl chain length.

PubMed

Kobayashi, Shinya; Fujii, Sotaro; Koga, Aya; Wakai, Satoshi; Matubayasi, Nobuyuki; Sambongi, Yoshihiro

2017-07-01

Reversible denaturation of Pseudomonas aeruginosa cytochrome c 551 (PAc 551 ) could be followed using five systematic urea derivatives that differ in the alkyl chain length, i.e. urea, N-methylurea (MU), N-ethylurea (EU), N-propylurea (PU), and N-butylurea (BU). The BU concentration was the lowest required for the PAc 551 denaturation, those of PU, EU, MU, and urea being gradually higher. Furthermore, the accessible surface area difference upon PAc 551 denaturation caused by BU was found to be the highest, those by PU, EU, MU, and urea being gradually lower. These findings indicate that urea derivatives with longer alkyl chains are stronger denaturants. In this study, as many as five systematic urea derivatives could be applied for the reversible denaturation of a single protein, PAc 551 , for the first time, and the effects of the alkyl chain length on protein denaturation were systematically verified by means of thermodynamic parameters.

Cold denaturation of α-synuclein amyloid fibrils.

PubMed

Ikenoue, Tatsuya; Lee, Young-Ho; Kardos, József; Saiki, Miyu; Yagi, Hisashi; Kawata, Yasushi; Goto, Yuji

2014-07-21

Although amyloid fibrils are associated with numerous pathologies, their conformational stability remains largely unclear. Herein, we probe the thermal stability of various amyloid fibrils. α-Synuclein fibrils cold-denatured to monomers at 0-20 °C and heat-denatured at 60-110 °C. Meanwhile, the fibrils of β2-microglobulin, Alzheimer's Aβ1-40/Aβ1-42 peptides, and insulin exhibited only heat denaturation, although they showed a decrease in stability at low temperature. A comparison of structural parameters with positive enthalpy and heat capacity changes which showed opposite signs to protein folding suggested that the burial of charged residues in fibril cores contributed to the cold denaturation of α-synuclein fibrils. We propose that although cold-denaturation is common to both native proteins and misfolded fibrillar states, the main-chain dominated amyloid structures may explain amyloid-specific cold denaturation arising from the unfavorable burial of charged side-chains in fibril cores. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Dodine as a Protein Denaturant: The Best of Two Worlds?

PubMed Central

Gelman, Hannah; Perlova, Tatyana; Gruebele, Martin

2013-01-01

Traditional denaturants such as urea and guanidinium ion unfold proteins in a cooperative “all-or-none” fashion. However, their high working concentration in combination with their strong absorption in the far ultraviolet region make it impossible to measure high quality circular dichroism or infrared spectra, which are commonly used to detect changes in protein secondary structure. On the other hand, detergents such as dodecyl sulfate destabilize native protein conformation at low millimolar concentrations and are UV transparent, but they do denature proteins more gradually than guanidinium or urea. In this work we studied the denaturation properties of the fungicide dodecylguanidinium acetate (dodine), which combines both denaturants into one. We show that dodine unfolds some small proteins at millimolar concentrations, facilitates temperature denaturation, and is transparent enough at its working concentration, unlike guanidinium, to measure full range circular dichroism spectra. Our results also suggest that dodine allows fine-tuning of the protein’s unfolded state, unlike traditional “all-or-none” denaturants. PMID:23906507

Amyloid fibrils as a nanoscaffold for enzyme immobilization.

PubMed

Pilkington, Sarah M; Roberts, Sarah J; Meade, Susie J; Gerrard, Juliet A

2010-01-01

Amyloid fibrils are a misfolded state, formed by many proteins when subjected to denaturing conditions. Their constituent amino acids make them ideally suited as a readily functionalized nanoscaffold for enzyme immobilization and their strength, stability, and nanometer size are attractive features for exploitation in the creation of new bionanomaterials. We report successful functionalization of amyloid fibrils by conjugation to glucose oxidase (GOD) using glutaraldehyde. GOD retained activity upon attachment and successful cross-linking was determined using electrophoresis, centrifugation, sucrose gradient centrifugation, and TEM. The resulting functionalized enzyme scaffold was then incorporated into a model poly(vinyl alcohol) (PVOH) film, to create a new bionanomaterial. The antibacterial effect of the functionalized film was then tested on E. coli, the growth of which was inhibited, demonstrating the incorporation of GOD antibacterial activity into the PVOH film. The incorporation of the GOD-functionalized amyloid fibrils into PVOH provides an excellent 'proof of concept' model for the creation of a new bionanomaterial using a functionalized amyloid fibril scaffold.

27 CFR 21.92 - Denaturants listed as U.S.P. or N.F.

Code of Federal Regulations, 2010 CFR

2010-04-01

....P. or N.F. 21.92 Section 21.92 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND... for Denaturants § 21.92 Denaturants listed as U.S.P. or N.F. Denaturing materials and products listed in this part as “U.S.P.” or “N.F.” shall meet the specifications set forth in the current United...

27 CFR 21.92 - Denaturants listed as U.S.P. or N.F.

Code of Federal Regulations, 2014 CFR

2014-04-01

....P. or N.F. 21.92 Section 21.92 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND... for Denaturants § 21.92 Denaturants listed as U.S.P. or N.F. Denaturing materials and products listed in this part as “U.S.P.” or “N.F.” shall meet the specifications set forth in the current United...

27 CFR 21.92 - Denaturants listed as U.S.P. or N.F.

Code of Federal Regulations, 2011 CFR

2011-04-01

....P. or N.F. 21.92 Section 21.92 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND... for Denaturants § 21.92 Denaturants listed as U.S.P. or N.F. Denaturing materials and products listed in this part as “U.S.P.” or “N.F.” shall meet the specifications set forth in the current United...

27 CFR 21.92 - Denaturants listed as U.S.P. or N.F.

Code of Federal Regulations, 2013 CFR

2013-04-01

....P. or N.F. 21.92 Section 21.92 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND... for Denaturants § 21.92 Denaturants listed as U.S.P. or N.F. Denaturing materials and products listed in this part as “U.S.P.” or “N.F.” shall meet the specifications set forth in the current United...

27 CFR 21.92 - Denaturants listed as U.S.P. or N.F.

Code of Federal Regulations, 2012 CFR

2012-04-01

....P. or N.F. 21.92 Section 21.92 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND... for Denaturants § 21.92 Denaturants listed as U.S.P. or N.F. Denaturing materials and products listed in this part as “U.S.P.” or “N.F.” shall meet the specifications set forth in the current United...

Effect of urea and alkylureas on the stability and structural fluctuation of the M80-containing Ω-loop of horse cytochrome c.

PubMed

Kumar, Sandeep; Sharma, Deepak; Kumar, Rajesh

2014-03-01

The effect of denaturants on the structural fluctuation of M80-containing Ω-loop of ferrocytochrome c was determined by measuring the rate coefficient of CO-association with ferrocytochrome c under varying concentrations of urea and alkylureas (methylurea (MU), N,N'-dimethylurea (DMU), ethylurea (EU), tetramethylurea (TMU)) at pH7.0, 25°C. As denaturant concentration is increased within the subdenaturing limit, the CO-association reaction is decelerated indicating that subdenaturing concentrations of denaturant reduce the structural fluctuation of the Ω-loop. Structural fluctuation of the Ω-loop is reduced more for urea and least for TMU. Intermolecular docking between horse cytochrome c and denaturant molecule (urea, MU, DMU, EU and TMU) reveals that polyfunctional interactions between the denaturant and different groups of Ω-loop and other part of protein decrease with an increase of alkyl group on urea molecule, which suggests that the decrease in the extent of restricted dynamics of Ω-loop with a corresponding increase of alkyl groups on urea molecule is due to the decrease of denaturant-mediated cross-linking interactions. These denaturant-mediated interactions are expected to reduce the conformational entropy of protein. Analysis of rate-temperature data shows a progressive decrease in conformational entropy of protein in the native to subdenaturing region. Thermodynamic analysis of denaturant (urea, MU, DMU, EU, TMU) effects on the thermal unfolding of ferrocytochrome c reveals that (i) thermodynamic stability of protein decreases with increasing concentration of denaturant or hydrophobicity of urea derivatives, (ii) water activity plays an important role in stabilization of ferrocytochrome c, and (iii) destabilization of ferrocytochrome c by denaturant occurs through the disturbance of hydrophobic interactions and hydrogen-bonding. Copyright © 2014 Elsevier B.V. All rights reserved.

Electric power generation using geothermal brine resources for a proof of concept facility

NASA Technical Reports Server (NTRS)

Hankin, J. W.

1974-01-01

An exploratory systems study of a geothermal proof-of-concept facility is being conducted. This study is the initial phase (Phase 0) of a project to establish the technical and economic feasibility of using hot brine resources for electric power production and other industrial applications. Phase 0 includes the conceptual design of an experimental test-bed facility and a 10-MWe power generating facility.

40 CFR 80.1662 - Liability for violations.

Code of Federal Regulations, 2014 CFR

2014-07-01

..., retailer, wholesale purchaser-consumer, oxygenate blender, ethanol denaturant producer, or ethanol..., retailer, wholesale purchaser-consumer, oxygenate producer, oxygenate importer, oxygenate blender, ethanol denaturant producer, ethanol denaturant importer, additive manufacturer, or additive blender who owned...

Ovalbumin labeling with p-hydroxymercurybenzoate: The effect of different denaturing agents and the kinetics of reaction.

PubMed

Campanella, Beatrice; Onor, Massimo; Biancalana, Lorenzo; D'Ulivo, Alessandro; Bramanti, Emilia

2015-08-15

The aim of our study was to investigate how denaturing agents commonly used in protein analysis influence the labeling between a reactive molecule and proteins. For this reason, we investigated the labeling of ovalbumin (OVA) as a globular model protein with p-hydroxymercurybenzoate (pHMB) in its native state (phosphate buffer solution) and in different denaturing conditions (8 molL(-1) urea, 3 molL(-1) guanidinium thiocyanate, 6 molL(-1) guanidinium chloride, 0.2% sodium dodecyl sulfate, and 20% methanol). In addition to chemical denaturation, thermal denaturation was also tested. The protein was pre-column simultaneously denatured and derivatized, and the pHMB-labeled denatured OVA complexes were analyzed by size exclusion chromatography (SEC) coupled online with chemical vapor generation-atomic fluorescence spectrometry (CVG-AFS). The number of -SH groups titrated greatly depends on the protein structure in solution. Indeed, we found that, depending on the adopted denaturing conditions, OVA gave different aggregate species that influence the complexation process. The results were compared with those obtained by a common alternative procedure for the titration of -SH groups that employs monobromobimane (mBBr) as tagging molecule and molecular fluorescence spectroscopy as detection technique. We also investigated the labeling kinetics for denatured OVA and pHMB, finding that the 4 thiolic groups of OVA have a very different reactivity toward mercury labeling, in agreement with previous studies. Copyright © 2015 Elsevier Inc. All rights reserved.

Two conformational states in D-shaped DNA: Effects of local denaturation

NASA Astrophysics Data System (ADS)

Lee, O.-Chul; Kim, Cheolhee; Kim, Jae-Yeol; Lee, Nam Ki; Sung, Wokyung

2016-06-01

The bending of double-stranded(ds) DNA on the nano-meter scale plays a key role in many cellular processes such as nucleosome packing, transcription-control, and viral-genome packing. In our recent study, a nanometer-sized dsDNA bent into a D shape was formed by hybridizing a circular single-stranded(ss) DNA and a complementary linear ssDNA. Our fluorescence resonance energy transfer (FRET) measurement of D-DNA revealed two types of conformational states: a less-bent state and a kinked state, which can transform into each other. To understand the origin of the two deformed states of D-DNA, here we study the presence of open base-pairs in the ds portion by using the breathing-DNA model to simulate the system. We provide strong evidence that the two states are due to the emergence of local denaturation, i.e., a bubble in the middle and two forks at ends of the dsDNA portion. We also study the system analytically and find that the free-energy landscape is bistable with two minima representative of the two states. The kink and fork sizes estimated by the analytical calculation are also in excellent agreement with the results of the simulation. Thus, this combined experimental-simulation-analytical study corroborates that highly bent D-DNA reduces bending stress via local denaturation.

The local pathology of interstitial edema: surface tension increases hydration potential in heat-damaged skin.

PubMed

McGee, Maria P; Morykwas, Michael J; Argenta, Louis C

2011-01-01

The local pathogenesis of interstitial edema in burns is incompletely understood. This ex vivo study investigates the forces mediating water-transfer in and out of heat-denatured interstitial matrix. Experimentally, full-thickness dermal samples are heated progressively to disrupt glycosaminoglycans, kill cells, and denature collagen under conditions that prevent water loss/gain; subsequently, a battery of complementary techniques including among others, high-resolution magnetic resonance imaging, equilibrium vapor pressure and osmotic stress are used to compare water-potential parameters of nonheated and heated dermis. The hydration potential (HP) determined by osmotic stress is a measure of the total water-potential defined empirically as the pressure at which no net water influx/efflux into/from the dermis is detected. Results show that after heat denaturation, the HP, the intensity of T2-weighed magnetic resonance images, and the vapor pressure increase indicating higher water activity and necessarily, smaller contributions from colloidosmotic forces to fluid influx in burned relative to healthy dermis. Concomitant increases in HP and in water activity implicate local changes in interfacial and metabolic energy as the source of excess fluid-transfer potential. These ex vivo findings also show that these additional forces contributing to abnormal fluid-transfer in burned skin develop independently of inflammatory and systemic hydrodynamic responses. © 2011 by the Wound Healing Society.

Effects of osmolytes on Pelodiscus sinensis creatine kinase: a study on thermal denaturation and aggregation.

PubMed

Wang, Wei; Lee, Jinhyuk; Jin, Qin-Xin; Fang, Nai-Yun; Si, Yue-Xiu; Yin, Shang-Jun; Qian, Guo-Ying; Park, Yong-Doo

2013-09-01

The protective effect of osmolytes on the thermal denaturation and aggregation of Pelodiscus sinensis muscle creatine kinase (PSCK) was investigated by a combination of spectroscopic methods and thermodynamic analysis. Our results demonstrated that the addition of osmolytes, such as glycine and proline, could prevent thermal denaturation and aggregation of PSCK in a concentration-dependent manner. When the concentration of glycine and proline increased in the denatured system, the relative activation was significantly enhanced; meanwhile, the aggregation of PSCK during thermal denaturation was decreased. Spectrofluorometer results showed that glycine and proline significantly decreased the tertiary structural changes of PSCK and that thermal denaturation directly induced PSCK aggregation. In addition, we also built the 3D structure of PSCK and osmolytes by homology models. The results of computational docking simulations showed that the docking energy was relatively low and that the clustering groups were spread to the surface of PSCK, indicating that osmolytes directly protect the surface of the protein. P. sinensis are poikilothermic and quite sensitive to the change of ambient temperature; however, there were few studies on the thermal denaturation of reptile CK. Our study provides important insight into the protective effects of osmolytes on thermal denaturation and aggregation of PSCK. Copyright © 2013 Elsevier B.V. All rights reserved.

Effects of protein and phosphate buffer concentrations on thermal denaturation of lysozyme analyzed by isoconversional method.

PubMed

Cao, X M; Tian, Y; Wang, Z Y; Liu, Y W; Wang, C X

2016-07-03

Thermal denaturation of lysozymes was studied as a function of protein concentration, phosphate buffer concentration, and scan rate using differential scanning calorimetry (DSC), which was then analyzed by the isoconversional method. The results showed that lysozyme thermal denaturation was only slightly affected by the protein concentration and scan rate. When the protein concentration and scan rate increased, the denaturation temperature (Tm) also increased accordingly. On the contrary, the Tm decreased with the increase of phosphate buffer concentration. The denaturation process of lysozymes was accelatated and the thermal stability was reduced with the increase of phosphate concentration. One part of degeneration process was not reversible where the aggregation occurred. The other part was reversible. The apparent activation energy (Ea) was computed by the isoconversional method. It decreased with the increase of the conversion ratio (α). The observed denaturation process could not be described by a simple reaction mechanism. It was not a process involving 2 standard reversible states, but a multi-step process. The new opportunities for investigating the kinetics process of protein denaturation can be supplied by this novel isoconversional method.

Cation-Induced Stabilization and Denaturation of DNA Origami Nanostructures in Urea and Guanidinium Chloride.

PubMed

Ramakrishnan, Saminathan; Krainer, Georg; Grundmeier, Guido; Schlierf, Michael; Keller, Adrian

2017-11-01

The stability of DNA origami nanostructures under various environmental conditions constitutes an important issue in numerous applications, including drug delivery, molecular sensing, and single-molecule biophysics. Here, the effect of Na + and Mg 2+ concentrations on DNA origami stability is investigated in the presence of urea and guanidinium chloride (GdmCl), two strong denaturants commonly employed in protein folding studies. While increasing concentrations of both cations stabilize the DNA origami nanostructures against urea denaturation, they are found to promote DNA origami denaturation by GdmCl. These inverse behaviors are rationalized by a salting-out of Gdm + to the hydrophobic DNA base stack. The effect of cation-induced DNA origami denaturation by GdmCl deserves consideration in the design of single-molecule studies and may potentially be exploited in future applications such as selective denaturation for purification purposes. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Chemical denaturation as a tool in the formulation optimization of biologics

PubMed Central

Freire, Ernesto; Schön, Arne; Hutchins, Burleigh M.; Brown, Richard K.

2013-01-01

Biologics have become the fastest growing segment in the pharmaceutical industry. As is the case with all proteins, biologics are susceptible to denature or to aggregate; conditions that, if present, preclude their use as pharmaceuticals. Identifying the solvent conditions that maximize their structural stability is crucial during development. Since the structural stability of a protein is susceptible to different chemical and physical conditions, the use of several complementary techniques can be expected to provide the best answers. Stability measurements that rely on temperature or chemical [urea or guanidine hydrochloride (GuHCl)] denaturation have been the preferred ones in research laboratories and together provide a thorough evaluation of protein stability. In this review, we will discuss chemical denaturation as a tool in the optimization of formulation conditions for biologics, and how chemical denaturation complements the role of thermal denaturation for this purpose. PMID:23796912

Estimating conformation content of a protein using citrate-stabilized Au nanoparticles

NASA Astrophysics Data System (ADS)

Deka, Jashmini; Paul, Anumita; Chattopadhyay, Arun

2010-08-01

Herein we report the use of the optical properties of citrate-stabilized gold nanoparticles (Au NPs) for estimation of native or denatured conformation content in a mixture of a protein in solution. The UV-vis extinction spectrum of citrate-stabilized Au NPs is known to broaden differently in the presence of native and denatured states of α-amylase, bovine serum albumin (BSA) or amyloglucosidase (AMG). On the other hand, herein we show that when a mixture of native and denatured protein was present in the medium, the broadening of the spectrum differed for different fractional content of the conformations. Also, the total area under the extinction spectrum varied linearly with the change in the mole fraction content of a state and for a constant total protein concentration. Transmission electron microscopy (TEM) measurements revealed different levels of agglomeration for different fractional contents of the native or denatured state of a protein. In addition, time-dependent denaturation of a protein could be followed using the present method. The rate constants calculated for denaturation indicated a possible fast change in conformation of a protein before complete thermal denaturation. The observations have been explained based on the changes in extinction coefficient (thereby oscillator strength) upon interaction of citrate-stabilized NPs with proteins being in different states and levels of agglomeration.Herein we report the use of the optical properties of citrate-stabilized gold nanoparticles (Au NPs) for estimation of native or denatured conformation content in a mixture of a protein in solution. The UV-vis extinction spectrum of citrate-stabilized Au NPs is known to broaden differently in the presence of native and denatured states of α-amylase, bovine serum albumin (BSA) or amyloglucosidase (AMG). On the other hand, herein we show that when a mixture of native and denatured protein was present in the medium, the broadening of the spectrum differed for different fractional content of the conformations. Also, the total area under the extinction spectrum varied linearly with the change in the mole fraction content of a state and for a constant total protein concentration. Transmission electron microscopy (TEM) measurements revealed different levels of agglomeration for different fractional contents of the native or denatured state of a protein. In addition, time-dependent denaturation of a protein could be followed using the present method. The rate constants calculated for denaturation indicated a possible fast change in conformation of a protein before complete thermal denaturation. The observations have been explained based on the changes in extinction coefficient (thereby oscillator strength) upon interaction of citrate-stabilized NPs with proteins being in different states and levels of agglomeration. Electronic supplementary information (ESI) available: Additional UV-vis and fluorescence spectra and graphs based on UV-vis studies. See DOI: 10.1039/c0nr00154f

High-pressure NMR reveals close similarity between cold and alcohol protein denaturation in ubiquitin.

PubMed

Vajpai, Navratna; Nisius, Lydia; Wiktor, Maciej; Grzesiek, Stephan

2013-01-29

Proteins denature not only at high, but also at low temperature as well as high pressure. These denatured states are not easily accessible for experiment, because usually heat denaturation causes aggregation, whereas cold or pressure denaturation occurs at temperatures well below the freezing point of water or pressures above 5 kbar, respectively. Here we have obtained atomic details of the pressure-assisted, cold-denatured state of ubiquitin at 2,500 bar and 258 K by high-resolution NMR techniques. Under these conditions, a folded, native-like and a disordered state exist in slow exchange. Secondary chemical shifts show that the disordered state has structural propensities for a native-like N-terminal β-hairpin and α-helix and a nonnative C-terminal α-helix. These propensities are very similar to the previously described alcohol-denatured (A-)state. Similar to the A-state, (15)N relaxation data indicate that the secondary structure elements move as independent segments. The close similarity of pressure-assisted, cold-denatured, and alcohol-denatured states with native and nonnative secondary elements supports a hierarchical mechanism of folding and supports the notion that similar to alcohol, pressure and cold reduce the hydrophobic effect. Indeed, at nondenaturing concentrations of methanol, a complete transition from the native to the A-state can be achieved at ambient temperature by varying the pressure from 1 to 2,500 bar. The methanol-assisted pressure transition is completely reversible and can also be induced in protein G. This method should allow highly detailed studies of protein-folding transitions in a continuous and reversible manner.

Metrics that differentiate the origins of osmolyte effects on protein stability: a test of the surface tension proposal.

PubMed

Auton, Matthew; Ferreon, Allan Chris M; Bolen, D Wayne

2006-09-01

Osmolytes that are naturally selected to protect organisms against environmental stresses are known to confer stability to proteins via preferential exclusion from protein surfaces. Solvophobicity, surface tension, excluded volume, water structure changes and electrostatic repulsion are all examples of forces proposed to account for preferential exclusion and the ramifications exclusion has on protein properties. What has been lacking is a systematic way of determining which force(s) is(are) responsible for osmolyte effects. Here, we propose the use of two experimental metrics for assessing the abilities of various proposed forces to account for osmolyte-mediated effects on protein properties. Metric 1 requires prediction of the experimentally determined ability of the osmolyte to bring about folding/unfolding resulting from the application of the force in question (i.e. prediction of the m-value of the protein in osmolyte). Metric 2 requires prediction of the experimentally determined ability of the osmolyte to contract or expand the Stokes radius of the denatured state resulting from the application of the force. These metrics are applied to test separate claims that solvophobicity/solvophilicity and surface tension are driving forces for osmolyte-induced effects on protein stability. The results show clearly that solvophobic/solvophilic forces readily account for protein stability and denatured state dimensional effects, while surface tension alone fails to do so. The agreement between experimental and predicted m-values involves both positive and negative m-values for three different proteins, and as many as six different osmolytes, illustrating that the tests are robust and discriminating. The ability of the two metrics to distinguish which forces account for the effects of osmolytes on protein properties and which do not, provides a powerful means of investigating the origins of osmolyte-protein effects.

Estimation of thermodynamic stability of human carbonic anhydrase IX from urea-induced denaturation and MD simulation studies.

PubMed

Idrees, Danish; Rahman, Safikur; Shahbaaz, Mohd; Haque, Md Anzarul; Islam, Asimul; Ahmad, Faizan; Hassan, Md Imtaiyaz

2017-12-01

Carbonic anhydrase IX (CAIX) is a transmembrane glycoprotein, overexpressed in cancer cells under hypoxia condition. In cancerous cells, CAIX plays an important role to combat the deleterious effects of a high rate of glycolytic metabolism. In order to favor tumor survival, CAIX maintains intracellular pH neutral or slightly alkaline and extracellular acidic pH. The equilibrium unfolding and conformational stability of CAIX were measured in the presence of increasing urea concentrations to understand it's structural features under stressed conditions. Two different spectroscopic techniques were used to follow urea-induced denaturation and observed that urea induces a reversible denaturation of CAIX. Coincidence of the normalized transition curves of both optical properties suggesting that denaturation of CAIX is a two-state process, i.e., native state ↔ denatured state. Each denaturation curve was analyzed to estimate thermodynamic parameters, ΔG D 0 ,value of Gibbs free energy change (ΔG D ) associated with the urea-induced denaturation, C m (midpoint of denaturation) and m (=δΔG D /δ[urea]). We further performed molecular dynamics simulation of CAIX for 50ns to see the dynamics of protein structure in the presence of different urea concentrations. An excellent agreement was observed between in silico and in vitro studies. Copyright © 2017 Elsevier B.V. All rights reserved.

Adsorption induced enzyme denaturation: the role of protein surface in adsorption induced protein denaturation on allyl glycidyl ether (AGE)-ethylene glycol dimethacrylate (EGDM) copolymers.

PubMed

Thudi, Lahari; Jasti, Lakshmi S; Swarnalatha, Y; Fadnavis, Nitin W; Mulani, Khudbudin; Deokar, Sarika; Ponrathnam, Surendra

2012-02-01

The effects of protein size on adsorption and adsorption-induced denaturation of proteins on copolymers of allyl glycidyl ether (AGE)-ethylene glycol dimethacrylate (EGDM) have been studied. Different responses were observed for the amount of protein adsorbed and denatured on the polymer surface for different proteins (trypsin, alchol dehydrogenase from baker's yeast (YADH), glucose dehydrogenase (GDH) from Gluconobacter cerinus, and alkaline phosphates from calf intestinal mucosa (CIAP). Protein adsorption on the copolymer with 25% crosslink density (AGE-25) was dependent not only on the size of the protein but also on the presence of glycoside residues on the protein surface. Adsorption and denaturation of proteins follows the order YADH>trypsin>GDH>CIAP although the molecular weights of the proteins follow the order YADH>CIAP>GDH>trypsin. The lack of correlation between amount of adsorbed protein and its molecular weight was due to the presence of glycoside residues on CIAP and GDH which protect the enzyme surface from denaturation. Enzyme stabilities in aqueous solutions of 1-cyclohexyl-2-pyrrolidinone (CHP) correlate well with the trend in denaturation by the copolymer, strongly suggesting that hydrophobic interactions play a major role in protein binding and the mechanism of protein denaturation is similar to that for water-miscible organic solvents. Copyright © 2011 Elsevier B.V. All rights reserved.

Study on hydraulic characteristics of mine dust-proof water supply network

NASA Astrophysics Data System (ADS)

Deng, Quanlong; Jiang, Zhongan; Han, Shuo; Fu, Enqi

2018-01-01

In order to study the hydraulic characteristics of mine dust-proof water supply network and obtain the change rule of water consumption and water pressure, according to the similarity principle and the fluid continuity equation and energy equation, the similarity criterion of mine dust-proof water supply network is deduced, and a similar model of dust-proof water supply network is established based on the prototype of Kailuan Group, the characteristics of hydraulic parameters in water supply network are studied experimentally. The results show that water pressure at each point is a dynamic process, and there is a negative correlation between water pressure and water consumption. With the increase of water consumption, the pressure of water points show a decreasing trend. According to the structure of the pipe network and the location of the water point, the influence degree on the pressure of each point is different.

Spiral electrode d33 mode piezoelectric diaphragm combined with proof mass as energy harvester

NASA Astrophysics Data System (ADS)

Shen, Zhiyuan; Liu, Shuwei; Miao, Jianmin; Woh, Lye Sun; Wang, Zhihong

2015-03-01

The paper demonstrates an energy harvester using a freestanding piezoelectric diaphragm combined with a proof mass. The diaphragm bearing double-sided spiral electrodes makes use of the d33 piezoelectric effect to realize energy scavenging. The harvester was fabricated by using a MEMS technique. The energy converting performance of the diaphragm was characterized by a shaker system. Proof masses were combined at the center of the diaphragm to tune the resonance of the harvester for the sake of scavenging low frequency vibrational energy. A receptance model was built to explain the vibrational behavior of the combined system. The resonance tuning and energy harvesting performance of the combination system was experimentally verified.

Experimental Demonstration of the Einstein-Podolsky-Rosen Steering Game Based on the All-Versus-Nothing Proof

NASA Astrophysics Data System (ADS)

Sun, Kai; Xu, Jin-Shi; Ye, Xiang-Jun; Wu, Yu-Chun; Chen, Jing-Ling; Li, Chuan-Feng; Guo, Guang-Can

2014-10-01

Einstein-Podolsky-Rosen (EPR) steering, a generalization of the original concept of "steering" proposed by Schrödinger, describes the ability of one system to nonlocally affect another system's states through local measurements. Some experimental efforts to test EPR steering in terms of inequalities have been made, which usually require many measurement settings. Analogy to the "all-versus-nothing" (AVN) proof of Bell's theorem without inequalities, testing steerability without inequalities would be more strong and require less resources. Moreover, the practical meaning of steering implies that it should also be possible to store the state information on the side to be steered, a result that has not yet been experimentally demonstrated. Using a recent AVN criterion for two-qubit entangled states, we experimentally implement a practical steering game using quantum memory. Furthermore, we develop a theoretical method to deal with the noise and finite measurement statistics within the AVN framework and apply it to analyze the experimental data. Our results clearly show the facilitation of the AVN criterion for testing steerability and provide a particularly strong perspective for understanding EPR steering.

Effects of protein and phosphate buffer concentrations on thermal denaturation of lysozyme analyzed by isoconversional method

PubMed Central

Cao, X.M.; Tian, Y.; Wang, Z.Y.; Liu, Y.W.; Wang, C.X.

2016-01-01

ABSTRACT Thermal denaturation of lysozymes was studied as a function of protein concentration, phosphate buffer concentration, and scan rate using differential scanning calorimetry (DSC), which was then analyzed by the isoconversional method. The results showed that lysozyme thermal denaturation was only slightly affected by the protein concentration and scan rate. When the protein concentration and scan rate increased, the denaturation temperature (Tm) also increased accordingly. On the contrary, the Tm decreased with the increase of phosphate buffer concentration. The denaturation process of lysozymes was accelatated and the thermal stability was reduced with the increase of phosphate concentration. One part of degeneration process was not reversible where the aggregation occurred. The other part was reversible. The apparent activation energy (Ea) was computed by the isoconversional method. It decreased with the increase of the conversion ratio (α). The observed denaturation process could not be described by a simple reaction mechanism. It was not a process involving 2 standard reversible states, but a multi-step process. The new opportunities for investigating the kinetics process of protein denaturation can be supplied by this novel isoconversional method. PMID:27459596

Thermodynamic and structural effect of urea and guanidine chloride on the helical and on a hairpin fragment of GB1 from molecular simulations.

PubMed

Meloni, R; Tiana, G

2017-04-01

With the help of molecular-dynamics simulations, we studied the effect of urea and guanidine chloride on the thermodynamic and structural properties of the helical fragment of protein GB1, comparing them with those of its second beta hairpin. We showed that the helical fragment in different solvents populates an ensemble of states that is more complex than that of the hairpin, and thus the associated experimental observables (circular-dichroism spectra, secondary chemical shifts, m values), that we back-calculated from the simulations and compared with the actual data, are more difficult to interpret. We observed that in the case of both peptides, urea binds tightly to their backbone, while guanidine exerts its denaturing effect in a more subtle way, strongly affecting the electrostatic properties of the solution. This difference can have consequences in the way denaturation experiments are interpreted. Proteins 2017; 85:753-763. © 2016 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Synergy in Protein–Osmolyte Mixtures

PubMed Central

2014-01-01

Virtually all taxa use osmolytes to protect cells against biochemical stress. Osmolytes often occur in mixtures, such as the classical combination of urea with TMAO (trimethylamine N-oxide) in cartilaginous fish or the cocktail of at least six different osmolytes in the kidney. The concentration patterns of osmolyte mixtures found in vivo make it likely that synergy between them plays an important role. Using statistical mechanical n-component Kirkwood–Buff theory, we show from first principles that synergy in protein–osmolyte systems can arise from two separable sources: (1) mutual alteration of protein surface solvation and (2) effects mediated through bulk osmolyte chemical activities. We illustrate both effects in a four-component system with the experimental example of the unfolding of a notch ankyrin domain in urea–TMAO mixtures, which make urea a less effective denaturant and TMAO a more effective stabilizer. Protein surface effects are primarily responsible for this synergy. The specific patterns of surface solvation point to denatured state expansion as the main factor, as opposed to direct competition. PMID:25490052

Synthesizing Safety Conditions for Code Certification Using Meta-Level Programming

NASA Technical Reports Server (NTRS)

Eusterbrock, Jutta

2004-01-01

In code certification the code consumer publishes a safety policy and the code producer generates a proof that the produced code is in compliance with the published safety policy. In this paper, a novel viewpoint approach towards an implementational re-use oriented framework for code certification is taken. It adopts ingredients from Necula's approach for proof-carrying code, but in this work safety properties can be analyzed on a higher code level than assembly language instructions. It consists of three parts: (1) The specification language is extended to include generic pre-conditions that shall ensure safety at all states that can be reached during program execution. Actual safety requirements can be expressed by providing domain-specific definitions for the generic predicates which act as interface to the environment. (2) The Floyd-Hoare inductive assertion method is refined to obtain proof rules that allow the derivation of the proof obligations in terms of the generic safety predicates. (3) A meta-interpreter is designed and experimentally implemented that enables automatic synthesis of proof obligations for submitted programs by applying the modified Floyd-Hoare rules. The proof obligations have two separate conjuncts, one for functional correctness and another for the generic safety obligations. Proof of the generic obligations, having provided the actual safety definitions as context, ensures domain-specific safety of program execution in a particular environment and is simpler than full program verification.

The expression and proangiogenic effect of nucleolin during the recovery of heat-denatured HUVECs.

PubMed

Liang, Pengfei; Jiang, Bimei; Lv, Chunliu; Huang, Xu; Sun, Li; Zhang, Pihong; Huang, Xiaoyuan

2013-10-01

The present study aims to examine the expression patterns and roles of nucleolin during the recovery of heat-denatured human umbilical vein endothelial cells (HUVECs). Deep partial thickness burn model in Sprague-Dawley rats and the heat denatured cell model (52°C, 35s) were used. The expression of nucleolin was measured using Western blot analysis and real-time PCR. Angiogenesis was assessed using in vitro parameters including endothelial cell proliferation, transwell migration assay, and scratched wound healing. Gene transfection and RNA interference approaches were employed to investigate the roles of nucleolin. Nucleolin mRNA and protein expression showed a time-dependent increase during the recovery of heat-denatured dermis and HUVECs. Heat-denaturation time-dependently promoted cell growth, adhesion, migration, scratched wound healing and formation of tube-like structures in HUVECs. These effects of heat denaturation on endothelial wound healing and formation of tube-like structures were prevented by knockdown of nucleolin, whereas over-expression of nucleolin increased cell growth, migration, and formation of tube-like structures in cultured HUVEC endothelial cells. In addition, we found that the expression of vascular endothelial growth factor (VEGF) increased during the recovery of heat-denatured dermis and HUVECs, and nucleolin up-regulated VEGF in HUVECs. The present study reveals that the expression of nucleolin is up-regulated, and plays a pro-angiogenic role during the recovery of heat-denatured dermis and its mechanism is probably dependent on production of VEGF. We find a novel and important pro-angiogenic role of nucleolin during the recovery of heat-denatured dermis. Copyright © 2013 Elsevier B.V. All rights reserved.

40 CFR 80.1660 - Prohibited acts.

Code of Federal Regulations, 2014 CFR

2014-07-01

..., supply, offer for supply, store or transport gasoline, oxygenate, or ethanol denaturant that does not...) Causing violating gasoline, oxygenate, or ethanol denaturant to be in the distribution system. Cause gasoline, oxygenate, or ethanol denaturant to be in the distribution system which does not comply with an...

Stability of HAMLET--a kinetically trapped alpha-lactalbumin oleic acid complex.

PubMed

Fast, Jonas; Mossberg, Ann-Kristin; Svanborg, Catharina; Linse, Sara

2005-02-01

The stability toward thermal and urea denaturation was measured for HAMLET (human alpha-lactalbumin made lethal to tumor cells) and alpha-lactalbumin, using circular dichroism and fluorescence spectroscopy as well as differential scanning calorimetry. Under all conditions examined, HAMLET appears to have the same or lower stability than alpha-lactalbumin. The largest difference is seen for thermal denaturation of the calcium free (apo) forms, where the temperature at the transition midpoint is 15 degrees C lower for apo HAMLET than for apo alpha-lactalbumin. The difference becomes progressively smaller as the calcium concentration increases. Denaturation of HAMLET was found to be irreversible. Samples of HAMLET that have been renatured after denaturation have lost the specific biological activity toward tumor cells. Three lines of evidence indicate that HAMLET is a kinetic trap: (1) It has lower stability than alpha-lactalbumin, although it is a complex of alpha-lactalbumin and oleic acid; (2) its denaturation is irreversible and HAMLET is lost after denaturation; (3) formation of HAMLET requires a specific conversion protocol.

How a protein can remain stable in a solvent with high content of urea: insights from molecular dynamics simulation of Candida antarctica lipase B in urea : choline chloride deep eutectic solvent.

PubMed

Monhemi, Hassan; Housaindokht, Mohammad Reza; Moosavi-Movahedi, Ali Akbar; Bozorgmehr, Mohammad Reza

2014-07-28

Deep eutectic solvents (DESs) are utilized as green and inexpensive alternatives to classical ionic liquids. It has been known that some of DESs can be used as solvent in the enzymatic reactions to obtain very green chemical processes. DESs are quite poorly understood at the molecular level. Moreover, we do not know much about the enzyme microstructure in such systems. For example, how some hydrolase can remain active and stable in a deep eutectic solvent including 9 M of urea? In this study, the molecular dynamics of DESs as a liquid was simulated at the molecular level. Urea : choline chloride as a well-known eutectic mixture was chosen as a model DES. The behavior of the lipase as a biocatalyst was studied in this system. For comparison, the enzyme structure was also simulated in 8M urea. The thermal stability of the enzyme was also evaluated in DESs, water, and 8M urea. The enzyme showed very good conformational stability in the urea : choline chloride mixture with about 66% urea (9 M) even at high temperatures. The results are in good agreement with recent experimental observations. In contrast, complete enzyme denaturation occurred in 8M urea with only 12% urea in water. It was found that urea molecules denature the enzyme by interrupting the intra-chain hydrogen bonds in a "direct denaturation mechanism". However, in a urea : choline chloride deep eutectic solvent, as a result of hydrogen bonding with choline and chloride ions, urea molecules have a low diffusion coefficient and cannot reach the protein domains. Interestingly, urea, choline, and chloride ions form hydrogen bonds with the surface residues of the enzyme which, instead of lipase denaturation, leads to greater enzyme stability. To the best of our knowledge, this is the first study in which the microstructural properties of a macromolecule are examined in a deep eutectic solvent.

The expression of miR-125b regulates angiogenesis during the recovery of heat-denatured HUVECs.

PubMed

Zhou, Situo; Zhang, Pihong; Liang, Pengfei; Huang, Xiaoyuan

2015-06-01

In previous studies we found that miR-125b was down-regulated in denatured dermis of deep partial thickness burn patients. Moreover, miR-125b inhibited tumor-angiogenesis associated with the decrease of ERBB2 and VEGF expression in ovarian cancer cells and breast cancer cells, etc. In this study, we investigated the expression patterns and roles of miR-125b during the recovery of denatured dermis and heat-denatured human umbilical vein endothelial cells (HUVECs). Deep partial thickness burns in Sprague-Dawley rats and the heat-denatured cells (52°C, 35 s) were used for analysis. Western blot analysis and real-time PCR were applied to evaluate the expression of miR-125b and ERBB2 and VEGF. The ability of angiogenesis in heat-denatured HUVECs was analyzed by scratch wound healing and tube formation assay after pri-miR-125b or anti-miR-125b transfection. miR-125b expression was time-dependent during the recovery of heat-denatured dermis and HUVECs. Moreover, miR-125b regulated ERBB2 mRNA and Protein Expression and regulated angiogenesis association with regulating the expression of VEGF in heat-denatured HUVECs. Taken together our results show that the expression of miR-125b is time-dependent and miR-125b plays a regulatory role of angiogenesis during wound healing after burns. Copyright © 2014 Elsevier Ltd and ISBI. All rights reserved.

27 CFR 19.457 - Neutralizing denatured spirits.

Code of Federal Regulations, 2010 CFR

2010-04-01

... BUREAU, DEPARTMENT OF THE TREASURY LIQUORS DISTILLED SPIRITS PLANTS Denaturing Operations and Manufacture... quantities of compounds such as caustics or acids to certain formulas of denatured spirits to neutralize such... spirits must record, for each formula the kinds and quantities of compounds used, and the formula number...

Attendance Policies, Student Attendance, and Instructor Verbal Aggressiveness

ERIC Educational Resources Information Center

Snyder, Jason; Forbus, Robert; Cistulli, Mark

2012-01-01

The authors utilized an experimental design across six sections of a managerial communications course (N = 173) to test the impact of instructor verbal aggressiveness and class attendance policies on student class attendance. The experimental group received a policy based on the principle of social proof (R. B. Cialdini, 2001), which indicated…

Thermal and chemical denaturation of Bacillus circulans xylanase: A biophysical chemistry laboratory module.

PubMed

Raabe, Richard; Gentile, Lisa

2008-11-01

A number of institutions have been, or are in the process of, modifying their biochemistry major to include some emphasis on the quantitative physical chemistry of biomolecules. Sometimes this is done as a replacement for part for the entire physical chemistry requirement, while at other institutions this is incorporated as a component into the traditional two-semester biochemistry series. The latter is the model used for biochemistry and molecular biology majors at the University of Richmond, whose second semester of biochemistry is a course entitled Proteins: Structure, Function, and Biophysics. What is described herein is a protein thermodynamics laboratory module, using the protein Bacillus circulans xylanase, which reinforces many lecture concepts, including: (i) the denatured (D) state ensemble of a protein can be different, depending on how it was populated; (ii) intermediate states may be detected by some spectroscopic techniques but not by others; (iii) the use and assumptions of the van't Hoff approach to calculate ΔH(o) , ΔS(o) , and ΔG(o) (T) for thermal protein unfolding transitions; and (iv) the use and assumptions of an approach that allows determination of the Gibb's free energy of a protein unfolding transition based on the linear dependence of ΔG(o) on the concentration of denaturant used. This module also requires students to design their own experimental protocols and spend time in the primary literature, both important parts of an upper division lab. Copyright © 2008 International Union of Biochemistry and Molecular Biology, Inc.

Structural stability of DNA origami nanostructures in the presence of chaotropic agents.

PubMed

Ramakrishnan, Saminathan; Krainer, Georg; Grundmeier, Guido; Schlierf, Michael; Keller, Adrian

2016-05-21

DNA origami represent powerful platforms for single-molecule investigations of biomolecular processes. The required structural integrity of the DNA origami may, however, pose significant limitations regarding their applicability, for instance in protein folding studies that require strongly denaturing conditions. Here, we therefore report a detailed study on the stability of 2D DNA origami triangles in the presence of the strong chaotropic denaturing agents urea and guanidinium chloride (GdmCl) and its dependence on concentration and temperature. At room temperature, the DNA origami triangles are stable up to at least 24 h in both denaturants at concentrations as high as 6 M. At elevated temperatures, however, structural stability is governed by variations in the melting temperature of the individual staple strands. Therefore, the global melting temperature of the DNA origami does not represent an accurate measure of their structural stability. Although GdmCl has a stronger effect on the global melting temperature, its attack results in less structural damage than observed for urea under equivalent conditions. This enhanced structural stability most likely originates from the ionic nature of GdmCl. By rational design of the arrangement and lengths of the individual staple strands used for the folding of a particular shape, however, the structural stability of DNA origami may be enhanced even further to meet individual experimental requirements. Overall, their high stability renders DNA origami promising platforms for biomolecular studies in the presence of chaotropic agents, including single-molecule protein folding or structural switching.

Theoretical analysis of Lumry-Eyring models in differential scanning calorimetry

PubMed Central

Sanchez-Ruiz, Jose M.

1992-01-01

A theoretical analysis of several protein denaturation models (Lumry-Eyring models) that include a rate-limited step leading to an irreversibly denatured state of the protein (the final state) has been carried out. The differential scanning calorimetry transitions predicted for these models can be broadly classified into four groups: situations A, B, C, and C′. (A) The transition is calorimetrically irreversible but the rate-limited, irreversible step takes place with significant rate only at temperatures slightly above those corresponding to the transition. Equilibrium thermodynamics analysis is permissible. (B) The transition is distorted by the occurrence of the rate-limited step; nevertheless, it contains thermodynamic information about the reversible unfolding of the protein, which could be obtained upon the appropriate data treatment. (C) The heat absorption is entirely determined by the kinetics of formation of the final state and no thermodynamic information can be extracted from the calorimetric transition; the rate-determining step is the irreversible process itself. (C′) same as C, but, in this case, the rate-determining step is a previous step in the unfolding pathway. It is shown that ligand and protein concentration effects on transitions corresponding to situation C (strongly rate-limited transitions) are similar to those predicted by equilibrium thermodynamics for simple reversible unfolding models. It has been widely held in recent literature that experimentally observed ligand and protein concentration effects support the applicability of equilibrium thermodynamics to irreversible protein denaturation. The theoretical analysis reported here disfavors this claim. PMID:19431826

27 CFR 19.464 - Denatured spirits inventories.

Code of Federal Regulations, 2010 CFR

2010-04-01

... of Articles Inventories § 19.464 Denatured spirits inventories. Each proprietor shall take a physical inventory of all denatured spirits in the processing account at the close of each calendar quarter and at... inventories. 19.464 Section 19.464 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE...

27 CFR 20.144 - Packages of completely denatured alcohol.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Packages of completely denatured alcohol. 20.144 Section 20.144 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY LIQUORS DISTRIBUTION AND USE OF DENATURED ALCOHOL AND RUM Sale...

27 CFR 20.144 - Packages of completely denatured alcohol.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2011-04-01 2011-04-01 false Packages of completely denatured alcohol. 20.144 Section 20.144 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY LIQUORS DISTRIBUTION AND USE OF DENATURED ALCOHOL AND RUM Sale...

27 CFR 19.493 - Caution label for completely denatured alcohol.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2011-04-01 2011-04-01 false Caution label for completely denatured alcohol. 19.493 Section 19.493 Alcohol, Tobacco Products and Firearms ALCOHOL AND... Marks Marking Requirements for Spirits § 19.493 Caution label for completely denatured alcohol. A...

27 CFR 20.261 - Records of completely denatured alcohol.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Records of completely denatured alcohol. 20.261 Section 20.261 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY LIQUORS DISTRIBUTION AND USE OF DENATURED ALCOHOL AND RUM...

27 CFR 19.492 - Marks on containers of completely denatured alcohol.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2011-04-01 2011-04-01 false Marks on containers of completely denatured alcohol. 19.492 Section 19.492 Alcohol, Tobacco Products and Firearms ALCOHOL AND... Marks Marking Requirements for Spirits § 19.492 Marks on containers of completely denatured alcohol...

27 CFR 20.261 - Records of completely denatured alcohol.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2011-04-01 2011-04-01 false Records of completely denatured alcohol. 20.261 Section 20.261 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY LIQUORS DISTRIBUTION AND USE OF DENATURED ALCOHOL AND RUM...

Calcium Binding and Disulfide Bonds Regulate the Stability of Secretagogin towards Thermal and Urea Denaturation

PubMed Central

Weiffert, Tanja; Ní Mhurchú, Niamh; O’Connell, David; Linse, Sara

2016-01-01

Secretagogin is a calcium-sensor protein with six EF-hands. It is widely expressed in neurons and neuro-endocrine cells of a broad range of vertebrates including mammals, fishes and amphibia. The protein plays a role in secretion and interacts with several vesicle-associated proteins. In this work, we have studied the contribution of calcium binding and disulfide-bond formation to the stability of the secretagogin structure towards thermal and urea denaturation. SDS-PAGE analysis of secretagogin in reducing and non-reducing conditions identified a tendency of the protein to form dimers in a redox-dependent manner. The denaturation of apo and Calcium-loaded secretagogin was studied by circular dichroism and fluorescence spectroscopy under conditions favoring monomer or dimer or a 1:1 monomer: dimer ratio. This analysis reveals significantly higher stability towards urea denaturation of Calcium-loaded secretagogin compared to the apo protein. The secondary and tertiary structure of the Calcium-loaded form is not completely denatured in the presence of 10 M urea. Reduced and Calcium-loaded secretagogin is found to refold reversibly after heating to 95°C, while both oxidized and reduced apo secretagogin is irreversibly denatured at this temperature. Thus, calcium binding greatly stabilizes the structure of secretagogin towards chemical and heat denaturation. PMID:27812162

Mutation of charged residues to neutral ones accelerates urea denaturation of HP-35.

PubMed

Wei, Haiyan; Yang, Lijiang; Gao, Yi Qin

2010-09-16

Following the studies of urea denaturation of β-hairpins using molecular dynamics, in this paper, molecular dynamics simulations of two peptides, a 35 residue three helix bundle villin headpiece protein HP-35 and its doubly norleucine-substituent mutant (Lys24Nle/Lys29Nle) HP-35 NleNle, were undertaken in urea solutions to understand the molecular mechanism of urea denaturation of α-helices. The mutant HP-35 NleNle was found to denature more easily than the wild type. During the expansion of the small hydrophobic core, water penetration occurs first, followed by that of urea molecules. It was also found that the initial hydration of the peptide backbone is achieved through water hydrogen bonding with the backbone CO groups during the denaturation of both polypeptides. The mutation of the two charged lysine residues to apolar norleucine enhances the accumulation of urea near the hydrophobic core and facilitates the denaturation process. Urea also interacts directly with the peptide backbone as well as side chains, thereby stabilizing nonnative conformations. The mechanism revealed here is consistent with the previous study on secondary structure of β-hairpin polypeptide, GB1, PEPTIDE 1, and TRPZIP4, suggesting that there is a general mechanism in the denaturation of protein backbone hydrogen bonds by urea.

Deriving robust noncontextuality inequalities from algebraic proofs of the Kochen-Specker theorem: the Peres-Mermin square

NASA Astrophysics Data System (ADS)

Krishna, Anirudh; Spekkens, Robert W.; Wolfe, Elie

2017-12-01

When a measurement is compatible with each of two other measurements that are incompatible with one another, these define distinct contexts for the given measurement. The Kochen-Specker theorem rules out models of quantum theory that satisfy a particular assumption of context-independence: that sharp measurements are assigned outcomes both deterministically and independently of their context. This notion of noncontextuality is not suited to a direct experimental test because realistic measurements always have some degree of unsharpness due to noise. However, a generalized notion of noncontextuality has been proposed that is applicable to any experimental procedure, including unsharp measurements, but also preparations as well, and for which a quantum no-go result still holds. According to this notion, the model need only specify a probability distribution over the outcomes of a measurement in a context-independent way, rather than specifying a particular outcome. It also implies novel constraints of context-independence for the representation of preparations. In this article, we describe a general technique for translating proofs of the Kochen-Specker theorem into inequality constraints on realistic experimental statistics, the violation of which witnesses the impossibility of a noncontextual model. We focus on algebraic state-independent proofs, using the Peres-Mermin square as our illustrative example. Our technique yields the necessary and sufficient conditions for a particular set of correlations (between the preparations and the measurements) to admit a noncontextual model. The inequalities thus derived are demonstrably robust to noise. We specify how experimental data must be processed in order to achieve a test of these inequalities. We also provide a criticism of prior proposals for experimental tests of noncontextuality based on the Peres-Mermin square.

Tight finite-key analysis for quantum cryptography

PubMed Central

Tomamichel, Marco; Lim, Charles Ci Wen; Gisin, Nicolas; Renner, Renato

2012-01-01

Despite enormous theoretical and experimental progress in quantum cryptography, the security of most current implementations of quantum key distribution is still not rigorously established. One significant problem is that the security of the final key strongly depends on the number, M, of signals exchanged between the legitimate parties. Yet, existing security proofs are often only valid asymptotically, for unrealistically large values of M. Another challenge is that most security proofs are very sensitive to small differences between the physical devices used by the protocol and the theoretical model used to describe them. Here we show that these gaps between theory and experiment can be simultaneously overcome by using a recently developed proof technique based on the uncertainty relation for smooth entropies. PMID:22252558

Tight finite-key analysis for quantum cryptography.

PubMed

Tomamichel, Marco; Lim, Charles Ci Wen; Gisin, Nicolas; Renner, Renato

2012-01-17

Despite enormous theoretical and experimental progress in quantum cryptography, the security of most current implementations of quantum key distribution is still not rigorously established. One significant problem is that the security of the final key strongly depends on the number, M, of signals exchanged between the legitimate parties. Yet, existing security proofs are often only valid asymptotically, for unrealistically large values of M. Another challenge is that most security proofs are very sensitive to small differences between the physical devices used by the protocol and the theoretical model used to describe them. Here we show that these gaps between theory and experiment can be simultaneously overcome by using a recently developed proof technique based on the uncertainty relation for smooth entropies.

Simple proof of the quantum benchmark fidelity for continuous-variable quantum devices

DOE Office of Scientific and Technical Information (OSTI.GOV)

Namiki, Ryo

2011-04-15

An experimental success criterion for continuous-variable quantum teleportation and memory is to surpass the limit of the average fidelity achieved by classical measure-and-prepare schemes with respect to a Gaussian-distributed set of coherent states. We present an alternative proof of the classical limit based on the familiar notions of state-channel duality and partial transposition. The present method enables us to produce a quantum-domain criterion associated with a given set of measured fidelities.

Proof of Concept for Efficient Application of Quantum Chemical Techniques to Model Enviromental Mercury Depletion Reactions Through Transition State Theory

DTIC Science & Technology

2018-01-02

Army Research Office P.O. Box 12211 Research Triangle Park, NC 27709-2211 REPORT DOCUMENTATION PAGE 11. SPONSOR/MONITOR’S REPORT NUMBER(S) 10...Collaborators: Award Information Award Number ARO W911NF-16-1-0486 Title of Research Proof of Concept for Efficient Application of Quantum...figure out the feasibility of the interconversion between the two forms of mercury. For this purpose, we mainly deal with the experimental studies of

Differential Denaturation of Serum Proteome Reveals a Significant Amount of Hidden Information in Complex Mixtures of Proteins

PubMed Central

Polci, Maria Letizia; Rossi, Stefania; Cordella, Martina; Carlucci, Giuseppe; Marchetti, Paolo; Antonini-Cappellini, Giancarlo; Facchiano, Antonio; D'Arcangelo, Daniela; Facchiano, Francesco

2013-01-01

Recently developed proteomic technologies allow to profile thousands of proteins within a high-throughput approach towards biomarker discovery, although results are not as satisfactory as expected. In the present study we demonstrate that serum proteome denaturation is a key underestimated feature; in fact, a new differential denaturation protocol better discriminates serum proteins according to their electrophoretic mobility as compared to single-denaturation protocols. Sixty nine different denaturation treatments were tested and the 3 most discriminating ones were selected (TRIDENT analysis) and applied to human sera, showing a significant improvement of serum protein discrimination as confirmed by MALDI-TOF/MS and LC-MS/MS identification, depending on the type of denaturation applied. Thereafter sera from mice and patients carrying cutaneous melanoma were analyzed through TRIDENT. Nine and 8 protein bands were found differentially expressed in mice and human melanoma sera, compared to healthy controls (p<0.05); three of them were found, for the first time, significantly modulated: α2macroglobulin (down-regulated in melanoma, p<0.001), Apolipoprotein-E and Apolipoprotein-A1 (both up-regulated in melanoma, p<0.04), both in mice and humans. The modulation was confirmed by immunological methods. Other less abundant proteins (e.g. gelsolin) were found significantly modulated (p<0.05). Conclusions: i) serum proteome contains a large amount of information, still neglected, related to proteins folding; ii) a careful serum denaturation may significantly improve analytical procedures involving complex protein mixtures; iii) serum differential denaturation protocol highlights interesting proteomic differences between cancer and healthy sera. PMID:23533572

Pressure-assisted cold denaturation of hen egg white lysozyme: the influence of co-solvents probed by hydrogen exchange nuclear magnetic resonance.

PubMed

Vogtt, K; Winter, R

2005-08-01

COSY proton nuclear magnetic resonance was used to measure the exchange rates of amide protons of hen egg white lysozyme (HEWL) in the pressure-assisted cold-denatured state and in the heat-denatured state. After dissolving lysozyme in deuterium oxide buffer, labile protons exchange for deuterons in such a way that exposed protons are substituted rapidly, whereas "protected" protons within structured parts of the protein are substituted slowly. The exchange rates k obs were determined for HEWL under heat treatment (80 degrees C) and under high pressure conditions at low temperature (3.75 kbar, -13 degrees C). Moreover, the influence of co-solvents (sorbitol, urea) on the exchange rate was examined under pressure-assisted cold denaturation conditions, and the corresponding protection factors, P, were determined. The exchange kinetics upon heat treatment was found to be a two-step process with initial slow exchange followed by a fast one, showing residual protection in the slow-exchange state and P-factors in the random-coil-like range for the final temperature-denatured state. Addition of sorbitol (500 mM) led to an increase of P-factors for the pressure-assisted cold denatured state, but not for the heat-denatured state. The presence of 2 M urea resulted in a drastic decrease of the P-factors of the pressure-assisted cold denatured state. For both types of co-solvents, the effect they exert appears to be cooperative, i.e., no particular regions within the protein can be identified with significantly diverse changes of P-factors.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sobol', E N; Vorobieva, N N; Omel'chenko, A I

A new approach is proposed for correcting the eye refraction by controlled variation of the mechanical properties of the sclera and cornea upon nondestructive laser heating. Experimental ex vivo studies of rabbit and pig eyes show that laser-induced local denaturation of the sclera changes the refraction of the cornea by 3 diopters on the average, and the subsequent nondestructive irradiation of the cornea increases its plasticity, which leads to a further increase in its radius of curvature and a decrease in refraction down to 7 diopters.

27 CFR 20.148 - Manufacture of articles with completely denatured alcohol.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Manufacture of articles with completely denatured alcohol. 20.148 Section 20.148 Alcohol, Tobacco Products and Firearms ALCOHOL... ALCOHOL AND RUM Sale and Use of Completely Denatured Alcohol § 20.148 Manufacture of articles with...

19 CFR 10.56 - Vegetable oils, denaturing; release.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 19 Customs Duties 1 2014-04-01 2014-04-01 false Vegetable oils, denaturing; release. 10.56 Section... Vegetable Oils § 10.56 Vegetable oils, denaturing; release. (a) Olive, palm-kernel, rapeseed, sunflower, and sesame oil shall be classifiable under subheadings 1509.10.20, 1509.10.40, 1509.90.20, 1509.90.40, 1510...

19 CFR 10.56 - Vegetable oils, denaturing; release.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 19 Customs Duties 1 2012-04-01 2012-04-01 false Vegetable oils, denaturing; release. 10.56 Section... Vegetable Oils § 10.56 Vegetable oils, denaturing; release. (a) Olive, palm-kernel, rapeseed, sunflower, and sesame oil shall be classifiable under subheadings 1509.10.20, 1509.10.40, 1509.90.20, 1509.90.40, 1510...

19 CFR 10.56 - Vegetable oils, denaturing; release.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 19 Customs Duties 1 2013-04-01 2013-04-01 false Vegetable oils, denaturing; release. 10.56 Section... Vegetable Oils § 10.56 Vegetable oils, denaturing; release. (a) Olive, palm-kernel, rapeseed, sunflower, and sesame oil shall be classifiable under subheadings 1509.10.20, 1509.10.40, 1509.90.20, 1509.90.40, 1510...

19 CFR 10.56 - Vegetable oils, denaturing; release.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 19 Customs Duties 1 2010-04-01 2010-04-01 false Vegetable oils, denaturing; release. 10.56 Section... Vegetable Oils § 10.56 Vegetable oils, denaturing; release. (a) Olive, palm-kernel, rapeseed, sunflower, and sesame oil shall be classifiable under subheadings 1509.10.20, 1509.10.40, 1509.90.20, 1509.90.40, 1510...

19 CFR 10.56 - Vegetable oils, denaturing; release.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 19 Customs Duties 1 2011-04-01 2011-04-01 false Vegetable oils, denaturing; release. 10.56 Section... Vegetable Oils § 10.56 Vegetable oils, denaturing; release. (a) Olive, palm-kernel, rapeseed, sunflower, and sesame oil shall be classifiable under subheadings 1509.10.20, 1509.10.40, 1509.90.20, 1509.90.40, 1510...

In vitro anti-denaturation and anti-hyaluronidase activities of extracts and galactolipids from leaves of Impatiens parviflora DC.

PubMed

Grabowska, Karolina; Podolak, Irma; Galanty, Agnieszka; Załuski, Daniel; Makowska-Wąs, Justyna; Sobolewska, Danuta; Janeczko, Zbigniew; Żmudzki, Paweł

2016-01-01

The in vitro anti-denaturation and anti-hyaluronidase activities of Impatiens parviflora extracts and isolated galactolipids (MGDG-1, DGDG-1) were investigated. This is the first report on these compounds in I. parviflora. All extracts showed anti-hyaluronidase activity, but only methanolic extract from fresh leaves exhibited significant activity against heat-induced denaturation of BSA in a dose-dependent manner. At 500 μg/mL, the extract and the reference drug showed 79.05% and 99.81% inhibition of protein denaturation, respectively. These results indicate that fresh leaves of I. parviflora may be beneficial in inflammatory conditions, especially those associated with protein denaturation, such as rheumatoid arthritis. The study revealed that only MGDG-1 showed weak activity in anti-denaturation assay but both galactolipids were potent inhibitors of hyaluronidase. MGDG-1 completely inhibited the enzyme activity at the concentration of 127.9 μg/mL. These results indicate the potential of galactolipids in the treatment of diseases associated with the loss of hyaluronic acid.

The effects of disulfide bonds on the denatured state of barnase.

PubMed Central

Clarke, J.; Hounslow, A. M.; Bond, C. J.; Fersht, A. R.; Daggett, V.

2000-01-01

The effects of engineered disulfide bonds on protein stability are poorly understood because they can influence the structure, dynamics, and energetics of both the native and denatured states. To explore the effects of two engineered disulfide bonds on the stability of barnase, we have conducted a combined molecular dynamics and NMR study of the denatured state of the two mutants. As expected, the disulfide bonds constrain the denatured state. However, specific extended beta-sheet structure can also be detected in one of the mutant proteins. This mutant is also more stable than would be predicted. Our study suggests a possible cause of the very high stability conferred by this disulfide bond: the wild-type denatured ensemble is stabilized by a nonnative hydrophobic cluster, which is constrained from occurring in the mutant due to the formation of secondary structure. PMID:11206061

Radioprotective Thiolamines WR-1065 and WR-33278 Selectively Denature Nonhistone Nuclear Proteins

NASA Technical Reports Server (NTRS)

Booth, Valerie K.; Roberts, Jeanette C.; Warters, Raymond L.; Wilmore, Britta H.; Lepock, James R.

2000-01-01

Differential scanning calorimetry was used to study the interactions of nuclei isolated from Chinese hamster V79 cells with the radioprotector WR-1065, other thiol compounds, and polyamines. Differential scanning calorimetry monitors denaturation of macromolecules and resolves the major nuclear components (e.g. constrained and relaxed DNA, nucleosome core, and nuclear matrix) of intact nuclei on the basis of thermal stability. WR-1065 treatment (0.5-10 mM) of isolated nuclei led to the irreversible denaturation of nuclear proteins, a fraction of which are nuclear matrix proteins. Denaturation of 50% of the total nonhistone nuclear protein content of isolated nuclei occurred after exposure to 4.7 mM WR-1065 for 20 min at 23 C. In addition, a 22% increase in the insoluble protein content of nuclei isolated from V79 cells that had been treated with 4 mM WR-1065 for 30 min at 37 C was observed, indicating that WR-1065-induced protein denaturation occurs not only in isolated nuclei but also in the nuclei of intact cells. From the extent of the increase in insoluble protein in the nucleus, protein denaturation by WR-1065 is expected to contribute to drug toxicity at concentrations greater than approximately 4 mM. WR-33278, the disulfide form of WR1065, was approximately twice as effective as the free thiol at denaturing nuclear proteins. The proposed mechanism for nucleoprotein denaturation is through direct interactions with protein cysteine groups with the formation of destabilizing protein-WR-1065 disulfides. In comparison to its effect on nuclear proteins in isolated nuclei, WR-1065 had only a very small effect on non-nuclear proteins of whole cells, isolated nuclear matrix, or the thiol-rich Ca (2+) ATPase of sarcoplasmic reticulum, indicating that WR-1065 can effectively denature protein only inside an intact nucleus, probably due to the increased concentration of the positively charged drug in the vicinity of DNA.

The Influence of Fluorocarbon and Hydrocarbon Acyl Groups at the Surface of Bovine Carbonic Anhydrase II on the Kinetics of Denaturation by Sodium Dodecyl Sulfate

PubMed Central

Lee, Andrew; Mirica, Katherine A.; Whitesides, George M.

2011-01-01

This paper examines the influence of acylation of the Lys-ε-NH3+ groups of bovine carbonic anhydrase (BCA, E.C. 4.2.1.1) to Lys-ε-NHCOR (R = -CH3, -CH2CH3, and -CH(CH3)2, -CF3) on the rate of denaturation of this protein in buffer containing sodium dodecyl sulfate (SDS). Analysis of the rates suggested separate effects due to electrostatic charge and hydrophobic interactions. Rates of denaturation (kAc,n) of each series of acylated derivatives depended on the number of acylations (n). Plots of log kAc,n vs. n followed U-shaped curves. Within each series of derivatives, rates of denaturation decreased as n increased to ~7; this decrease was compatible with increasingly unfavorable electrostatic interactions between SDS and protein. In this range of n, rates of denaturation also depended on the choice of the acyl group as n increased to ~7, in a manner compatible with favorable hydrophobic interactions between SDS and the -NHCOR groups. As n increased in the range 7 < n < 14 however, rates of denaturation stayed approximately constant; analysis suggested these rates were compatible with an increasingly important contribution to denaturation that depended both on the net negative charge of the protein and on the hydrophobicity of the R group. The mechanism of denaturation thus seems to change with the extent of acylation of the protein. For derivatives with the same net electrostatic charge, rates of denaturation increased with the acyl group (by a factor of ~3 for n ~ 14) in the order CH3CONH- < CH3CH2CONH- < (CH3)2CHCONH- < CF3CONH-. These results suggested that the hydrophobicity of CF3CONH- is slightly greater (by a factor of < 2) than that of RHCONH- similar in surface area. PMID:21182314

Hyperthermophile Protein Behavior: Partially-Structured Conformations of Pyrococcus furiosus Rubredoxin Monomers Generated through Forced Cold-Denaturation and Refolding

PubMed Central

Ahmed, Shubbir; Guptasarma, Purnananda

2014-01-01

Some years ago, we showed that thermo-chemically denatured, partially-unfolded forms of Pyrococcus furiosus triosephosphateisomerase (PfuTIM) display cold-denaturation upon cooling, and heat-renaturation upon reheating, in proportion with the extent of initial partial unfolding achieved. This was the first time that cold-denaturation was demonstrated for a hyperthermophile protein, following unlocking of surface salt bridges. Here, we describe the behavior of another hyperthermophile protein, the small, monomeric, 53 residues-long rubredoxin from Pyrococcus furiosus (PfRd), which is one of the most thermostable proteins known to man. Like PfuTIM, PfRd too displays cold-denaturation after initial thermo-chemical perturbation, however, with two differences: (i) PfRd requires considerably higher temperatures as well as higher concentrations of guanidium hydrochloride (Gdm.HCl) than PfuTIM; (ii) PfRd's cold-denaturation behavior during cooling after thermo-chemical perturbation is incompletely reversible, unlike PfuTIM's, which was clearly reversible (from each different conformation generated). Differential cold-denaturation treatments allow PfRd to access multiple partially-unfolded states, each of which is clearly highly kinetically-stable. We refer to these as ‘Trishanku’ unfolding intermediates (or TUIs). Fascinatingly, refolding of TUIs through removal of Gdm.HCl generates multiple partially-refolded, monomeric, kinetically-trapped, non-native ‘Trishanku’ refolding intermediates (or TRIs), which differ from each other and from native PfRd and TUIs, in structural content and susceptibility to proteolysis. We find that the occurrence of cold denaturation and observations of TUI and TRI states is contingent on the oxidation status of iron, with redox agents managing to modulate the molecule's behavior upon gaining access to PfRd's iron atom. Mass spectrometric examination provides no evidence of the formation of disulfide bonds, but other experiments suggest that the oxidation status of iron (and its extent of burial) together determine whether or not PfRd shows cold denaturation, and also whether redox agents are able to modulate its behavior. PMID:24603413

Influence of fluorocarbon and hydrocarbon acyl groups at the surface of bovine carbonic anhydrase II on the kinetics of denaturation by sodium dodecyl sulfate.

PubMed

Lee, Andrew; Mirica, Katherine A; Whitesides, George M

2011-02-10

This paper examines the influence of acylation of the Lys-ε-NH(3)(+) groups of bovine carbonic anhydrase (BCA, EC 4.2.1.1) to Lys-ε-NHCOR (R = -CH(3), -CH(2)CH(3), and -CH(CH(3))(2), -CF(3)) on the rate of denaturation of this protein in buffer containing sodium dodecyl sulfate (SDS). Analysis of the rates suggested separate effects due to electrostatic charge and hydrophobic interactions. Rates of denaturation (k(Ac,n)) of each series of acylated derivatives depended on the number of acylations (n). Plots of log k(Ac,n) vs n followed U-shaped curves. Within each series of derivatives, rates of denaturation decreased as n increased to ∼7; this decrease was compatible with increasingly unfavorable electrostatic interactions between SDS and protein. In this range of n, rates of denaturation also depended on the choice of the acyl group as n increased to ∼7, in a manner compatible with favorable hydrophobic interactions between SDS and the -NHCOR groups. As n increased in the range 7 < n < 14, however, rates of denaturation stayed approximately constant; analysis suggested that these rates were compatible with an increasingly important contribution to denaturation that depended both on the net negative charge of the protein and on the hydrophobicity of the R group. The mechanism of denaturation thus seems to change with the extent of acylation of the protein. For derivatives with the same net electrostatic charge, rates of denaturation increased with the acyl group (by a factor of ∼3 for n ∼ 14) in the order CH(3)CONH- < CH(3)CH(2)CONH- < (CH(3))(2)CHCONH- < CF(3)CONH-. These results suggested that the hydrophobicity of CF(3)CONH- is slightly greater (by a factor of <2) than that of RHCONH- with similar surface area.

Complement-fixing antibodies against denatured HLA and MICA antigens are associated with antibody mediated rejection.

PubMed

Cai, Junchao; Terasaki, Paul I; Zhu, Dong; Lachmann, Nils; Schönemann, Constanze; Everly, Matthew J; Qing, Xin

2016-02-01

We have found antibodies against denatured HLA class I antigens in the serum of allograft recipients which were not significantly associated with graft failure. It is unknown whether transplant recipients also have denatured HLA class II and MICA antibodies. The effects of denatured HLA class I, class II, and MICA antibodies on long-term graft outcome were further investigated based on their ability to fix complement c1q. In this 4-year retrospective cohort study, post-transplant sera from 975 kidney transplant recipients were tested for antibodies against denatured HLA/MICA antigens and these antibodies were further classified based on their ability to fix c1q. Thirty percent of patients had antibodies against denatured HLA class I, II, or MICA antigens. Among them, 8.5% and 21.5% of all patients had c1q-fixing and non c1q-fixing antibodies respectively. There was no significant difference on graft survival between patients with or without antibodies against denatured HLA/MICA. However, when these antibodies were further classified according to their ability to fix c1q, patients with c1q-fixing antibodies had a significantly lower graft survival rate than patients without antibodies or patients with non c1q-fixing antibodies (p=0.008). In 169 patients who lost renal grafts, 44% of them had c1q-fixing antibodies against denatured HLA/MICA antigens, which was significantly higher than that in patients with functioning renal transplants (25%, p<0.0001). C1q-fixing antibodies were more significantly associated with graft failure caused by AMR (72.73%) or mixed AMR/CMR (61.9%) as compared to failure due to CMR (35.3%) or other causes (39.2%) (p=0.026). Transplant recipients had antibodies against denatured HLA class I, II, and MICA antigens. However, only c1q-fixing antibodies were associated with graft failure which was related to antibody mediated rejection. Copyright © 2015 Elsevier Inc. All rights reserved.

All-Versus-Nothing Proof of Einstein-Podolsky-Rosen Steering

PubMed Central

Chen, Jing-Ling; Ye, Xiang-Jun; Wu, Chunfeng; Su, Hong-Yi; Cabello, Adán; Kwek, L. C.; Oh, C. H.

2013-01-01

Einstein-Podolsky-Rosen steering is a form of quantum nonlocality intermediate between entanglement and Bell nonlocality. Although Schrödinger already mooted the idea in 1935, steering still defies a complete understanding. In analogy to “all-versus-nothing” proofs of Bell nonlocality, here we present a proof of steering without inequalities rendering the detection of correlations leading to a violation of steering inequalities unnecessary. We show that, given any two-qubit entangled state, the existence of certain projective measurement by Alice so that Bob's normalized conditional states can be regarded as two different pure states provides a criterion for Alice-to-Bob steerability. A steering inequality equivalent to the all-versus-nothing proof is also obtained. Our result clearly demonstrates that there exist many quantum states which do not violate any previously known steering inequality but are indeed steerable. Our method offers advantages over the existing methods for experimentally testing steerability, and sheds new light on the asymmetric steering problem. PMID:23828242

Protein nanocrystallography: growth mechanism and atomic structure of crystals induced by nanotemplates.

PubMed

Pechkova, E; Vasile, F; Spera, R; Fiordoro, S; Nicolini, C

2005-11-01

Protein nanocrystallography, a new technology for crystal growth based on protein nanotemplates, has recently been shown to produce diffracting, stable and radiation-resistant lysozyme crystals. This article, by computing these lysozyme crystals' atomic structures, obtained by the diffraction patterns of microfocused synchrotron radiation, provides a possible mechanism for this increased stability, namely a significant decrease in water content accompanied by a minor but significant alpha-helix increase. These data are shown to be compatible with the circular dichroism and two-dimensional Fourier transform spectra of high-resolution H NMR of proteins dissolved from the same nanotemplate-based crystal versus those from a classical crystal. Finally, evidence for protein direct transfer from the nanotemplate to the drop and the participation of the template proteins in crystal nucleation and growth is provided by high-resolution NMR spectrometry and mass spectrometry. Furthermore, the lysozyme nanotemplate appears stable up to 523 K, as confirmed by a thermal denaturation study using spectropolarimetry. The overall data suggest that heat-proof lysozyme presence in the crystal provides a possible explanation of the crystal's resistance to synchrotron radiation.

Fast and easy enzyme immobilization by photoinitiated polymerization for efficient bioelectrochemical devices.

PubMed

Suraniti, Emmanuel; Studer, Vincent; Sojic, Neso; Mano, Nicolas

2011-04-01

Immobilization and electrical wiring of enzymes is of particular importance for the elaboration of efficient biosensors and can be cumbersome. Here, we report a fast and easy protocol for enzyme immobilization, and as a proof of concept, we applied it to the immobilization of bilirubin oxidase, a labile enzyme. In the first step, bilirubin oxidase is mixed with a redox hydrogel "wiring" the enzyme reaction centers to electrodes. Then, this adduct is covered by an outer layer of PEGDA made by photoinitiated polymerization of poly(ethylene-glycol) diacrylate (PEGDA) and a photoclivable precursor, DAROCUR. This two-step protocol is 18 times faster than the current state-of-the-art protocol and leads to currents 25% higher. In addition, the outer layer of PEGDA acts as a protective layer increasing the lifetime of the electrode by 100% when operating continuously for 2000 s and by 60% when kept in dry state for 24 h. This new protocol is particularly appropriate for labile enzymes that quickly denaturate. In addition, by tuning the ratio PEGDA/DAROCUR, it is possible to make the enzyme electrodes even more active or more stable.

Probing the Intermediacy of Covalent RNA Enzyme Complexes in RNA Modification Enzymes

PubMed Central

Chervin, Stephanie M.; Kittendorf, Jeffrey D.; Garcia, George A.

2009-01-01

Within the large and diverse group of RNA-modifying enzymes, a number of enzymes seem to form stable covalent linkages to their respective RNA substrates. A complete understanding of the chemical and kinetic mechanisms of these enzymes, some of which have identified pathological roles, is lacking. As part of our ongoing work studying the posttranscriptional modification of tRNA with queuine, we wish to understand fully the chemical and kinetic mechanisms involved in this key transglycosylation reaction. In our previous investigations, we have used a gel mobility-shift assay to characterize an apparent covalent enzyme-RNA intermediate believed to be operative in the catalytic pathway. However, the simple observation of a covalent complex is not sufficient to prove intermediacy. To be a true intermediate, the complex must be both chemically and kinetically competent. As a case study for the proof of intermediacy, we report the use of this gel-shift assay under mildly denaturing conditions to probe the kinetic competency of the covalent association between RNA and the tRNA modifying enzyme tRNA-guanine transglycosylase (TGT). PMID:17673081

l-Proline and RNA Duplex m-Value Temperature Dependence.

PubMed

Schwinefus, Jeffrey J; Baka, Nadia L; Modi, Kalpit; Billmeyer, Kaylyn N; Lu, Shutian; Haase, Lucas R; Menssen, Ryan J

2017-08-03

The temperature dependence of l-proline interactions with the RNA dodecamer duplex surface exposed after unfolding was quantified using thermal and isothermal titration denaturation monitored by uv-absorbance. The m-value quantifying proline interactions with the RNA duplex surface area exposed after unfolding was measured using RNA duplexes with GC content ranging between 17 and 83%. The m-values from thermal denaturation decreased with increasing GC content signifying increasingly favorable proline interactions with the exposed RNA surface area. However, m-values from isothermal titration denaturation at 25.0 °C were independent of GC content and less negative than those from thermal denaturation. The m-value from isothermal titration denaturation for a 50% GC RNA duplex decreased (became more negative) as the temperature increased and was in nearly exact agreement with the m-value from thermal denaturation. Since RNA duplex transition temperatures increased with GC content, the more favorable proline interactions with the high GC content duplex surface area observed from thermal denaturation resulted from the temperature dependence of proline interactions rather than the RNA surface chemical composition. The enthalpy contribution to the m-value was positive and small (indicating a slight increase in duplex unfolding enthalpy with proline) while the entropic contribution to the m-value was positive and increased with temperature. Our results will facilitate proline's use as a probe of solvent accessible surface area changes during biochemical reactions at different reaction temperatures.

Heat-denatured lysozyme could be a novel disinfectant for reducing hepatitis A virus and murine norovirus on berry fruit.

PubMed

Takahashi, Michiko; Okakura, Yumiko; Takahashi, Hajime; Imamura, Minami; Takeuchi, Akira; Shidara, Hiroyuki; Kuda, Takashi; Kimura, Bon

2018-02-02

Hepatitis A virus (HAV) is well known worldwide as a causative virus of acute hepatitis. In recent years, numerous cases of HAV infection caused by HAV-contaminated berries have occurred around the world. Because berries are often consumed without prior heating, reliable disinfection of the raw fruit is important in order to prevent HAV outbreaks. Previous studies have found that murine norovirus strain 1 (MNV-1) and human norovirus GII.4 were inactivated in heat-denatured lysozyme solution. In this study, we investigated whether or not heat-denatured lysozyme is effective in inactivating HAV and whether it could be an effective disinfectant for berries contaminated with HAV or MNV-1. We examined the inactivating effect of heat-denatured lysozyme on three strains of HAV and found that it reduced the infectivity of all three strains. We then immersed blueberries and mixed berries into solutions of HAV or MNV-1, and disinfected them by soaking them in 1% heat-denatured lysozyme for 1min. Consequently, the infectious HAV and MNV-1 contaminating the berries were decreased by >3.1 log units in all samples. Our results demonstrate that heat-denatured lysozyme effectively inactivates HAV and suggest that heat-denatured lysozyme may be an effective disinfectant for berry fruit, which is a potential source of HAV food poisoning. Copyright © 2017 Elsevier B.V. All rights reserved.

What is Proof of Concept Research and how does it Generate Epistemic and Ethical Categories for Future Scientific Practice?

PubMed

Kendig, Catherine Elizabeth

2016-06-01

"Proof of concept" is a phrase frequently used in descriptions of research sought in program announcements, in experimental studies, and in the marketing of new technologies. It is often coupled with either a short definition or none at all, its meaning assumed to be fully understood. This is problematic. As a phrase with potential implications for research and technology, its assumed meaning requires some analysis to avoid it becoming a descriptive category that refers to all things scientifically exciting. I provide a short analysis of proof of concept research and offer an example of it within synthetic biology. I suggest that not only are there activities that circumscribe new epistemological categories but there are also associated normative ethical categories or principles linked to the research. I examine these and provide an outline for an alternative ethical account to describe these activities that I refer to as "extended agency ethics". This view is used to explain how the type of research described as proof of concept also provides an attendant proof of principle that is the result of decision-making that extends across practitioners, their tools, techniques, and the problem solving activities of other research groups.

9 CFR 325.13 - Denaturing procedures.

Code of Federal Regulations, 2011 CFR

2011-01-01

...) Tripe may be denatured by dipping it in a 6 percent solution of tannic acid for 1 minute followed by... coloring; (4) Meat may be denatured by dipping it in a solution of 0.0625 percent tannic acid, followed by... carbolic acid; cresylic disinfectant; a formula consisting of 1 part FD&C green No. 3 coloring, 40 parts...

9 CFR 325.13 - Denaturing procedures.

Code of Federal Regulations, 2010 CFR

2010-01-01

...) Tripe may be denatured by dipping it in a 6 percent solution of tannic acid for 1 minute followed by... coloring; (4) Meat may be denatured by dipping it in a solution of 0.0625 percent tannic acid, followed by... carbolic acid; cresylic disinfectant; a formula consisting of 1 part FD&C green No. 3 coloring, 40 parts...

27 CFR 19.385 - Making alcohol or water solutions of denaturants.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2013-04-01 2013-04-01 false Making alcohol or water solutions of denaturants. 19.385 Section 19.385 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY ALCOHOL DISTILLED SPIRITS PLANTS Denaturing Operations and Manufacture of Articles Rules for...

Toward a Proof of Concept Cloud Framework for Physics Applications on Blue Gene Supercomputers

NASA Astrophysics Data System (ADS)

Dreher, Patrick; Scullin, William; Vouk, Mladen

2015-09-01

Traditional high performance supercomputers are capable of delivering large sustained state-of-the-art computational resources to physics applications over extended periods of time using batch processing mode operating environments. However, today there is an increasing demand for more complex workflows that involve large fluctuations in the levels of HPC physics computational requirements during the simulations. Some of the workflow components may also require a richer set of operating system features and schedulers than normally found in a batch oriented HPC environment. This paper reports on progress toward a proof of concept design that implements a cloud framework onto BG/P and BG/Q platforms at the Argonne Leadership Computing Facility. The BG/P implementation utilizes the Kittyhawk utility and the BG/Q platform uses an experimental heterogeneous FusedOS operating system environment. Both platforms use the Virtual Computing Laboratory as the cloud computing system embedded within the supercomputer. This proof of concept design allows a cloud to be configured so that it can capitalize on the specialized infrastructure capabilities of a supercomputer and the flexible cloud configurations without resorting to virtualization. Initial testing of the proof of concept system is done using the lattice QCD MILC code. These types of user reconfigurable environments have the potential to deliver experimental schedulers and operating systems within a working HPC environment for physics computations that may be different from the native OS and schedulers on production HPC supercomputers.

From statistical proofs of the Kochen-Specker theorem to noise-robust noncontextuality inequalities

NASA Astrophysics Data System (ADS)

Kunjwal, Ravi; Spekkens, Robert W.

2018-05-01

The Kochen-Specker theorem rules out models of quantum theory wherein projective measurements are assigned outcomes deterministically and independently of context. This notion of noncontextuality is not applicable to experimental measurements because these are never free of noise and thus never truly projective. For nonprojective measurements, therefore, one must drop the requirement that an outcome be assigned deterministically in the model and merely require that it be assigned a distribution over outcomes in a manner that is context-independent. By demanding context independence in the representation of preparations as well, one obtains a generalized principle of noncontextuality that also supports a quantum no-go theorem. Several recent works have shown how to derive inequalities on experimental data which, if violated, demonstrate the impossibility of finding a generalized-noncontextual model of this data. That is, these inequalities do not presume quantum theory and, in particular, they make sense without requiring an operational analog of the quantum notion of projectiveness. We here describe a technique for deriving such inequalities starting from arbitrary proofs of the Kochen-Specker theorem. It extends significantly previous techniques that worked only for logical proofs, which are based on sets of projective measurements that fail to admit of any deterministic noncontextual assignment, to the case of statistical proofs, which are based on sets of projective measurements that d o admit of some deterministic noncontextual assignments, but not enough to explain the quantum statistics.

Protein denaturants at aqueous-hydrophobic interfaces: self-consistent correlation between induced interfacial fluctuations and denaturant stability at the interface.

PubMed

Cui, Di; Ou, Shu-Ching; Patel, Sandeep

2015-01-08

The notion of direct interaction between denaturing cosolvent and protein residues has been proposed in dialogue relevant to molecular mechanisms of protein denaturation. Here we consider the correlation between free energetic stability and induced fluctuations of an aqueous-hydrophobic interface between a model hydrophobically associating protein, HFBII, and two common protein denaturants, guanidinium cation (Gdm(+)) and urea. We compute potentials of mean force along an order parameter that brings the solute molecule close to the known hydrophobic region of the protein. We assess potentials of mean force for different relative orientations between the protein and denaturant molecule. We find that in both cases of guanidinium cation and urea relative orientations of the denaturant molecule that are parallel to the local protein-water interface exhibit greater stability compared to edge-on or perpendicular orientations. This behavior has been observed for guanidinium/methylguanidinium cations at the liquid-vapor interface of water, and thus the present results further corroborate earlier findings. Further analysis of the induced fluctuations of the aqueous-hydrophobic interface upon approach of the denaturant molecule indicates that the parallel orientation, displaying a greater stability at the interface, also induces larger fluctuations of the interface compared to the perpendicular orientations. The correlation of interfacial stability and induced interface fluctuation is a recurring theme for interface-stable solutes at hydrophobic interfaces. Moreover, observed correlations between interface stability and induced fluctuations recapitulate connections to local hydration structure and patterns around solutes as evidenced by experiment (Cooper et al., J. Phys. Chem. A 2014, 118, 5657.) and high-level ab initio/DFT calculations (Baer et al., Faraday Discuss 2013, 160, 89).

Protein Denaturants at Aqueous–Hydrophobic Interfaces: Self-Consistent Correlation between Induced Interfacial Fluctuations and Denaturant Stability at the Interface

PubMed Central

2015-01-01

The notion of direct interaction between denaturing cosolvent and protein residues has been proposed in dialogue relevant to molecular mechanisms of protein denaturation. Here we consider the correlation between free energetic stability and induced fluctuations of an aqueous–hydrophobic interface between a model hydrophobically associating protein, HFBII, and two common protein denaturants, guanidinium cation (Gdm+) and urea. We compute potentials of mean force along an order parameter that brings the solute molecule close to the known hydrophobic region of the protein. We assess potentials of mean force for different relative orientations between the protein and denaturant molecule. We find that in both cases of guanidinium cation and urea relative orientations of the denaturant molecule that are parallel to the local protein–water interface exhibit greater stability compared to edge-on or perpendicular orientations. This behavior has been observed for guanidinium/methylguanidinium cations at the liquid–vapor interface of water, and thus the present results further corroborate earlier findings. Further analysis of the induced fluctuations of the aqueous–hydrophobic interface upon approach of the denaturant molecule indicates that the parallel orientation, displaying a greater stability at the interface, also induces larger fluctuations of the interface compared to the perpendicular orientations. The correlation of interfacial stability and induced interface fluctuation is a recurring theme for interface-stable solutes at hydrophobic interfaces. Moreover, observed correlations between interface stability and induced fluctuations recapitulate connections to local hydration structure and patterns around solutes as evidenced by experiment (Cooper et al., J. Phys. Chem. A2014, 118, 5657.) and high-level ab initio/DFT calculations (Baer et al., Faraday Discuss2013, 160, 89). PMID:25536388

Interplay of secondary structures and side-chain contacts in the denatured state of BBA1

NASA Astrophysics Data System (ADS)

Wen, Edward Z.; Luo, Ray

2004-08-01

The denatured state of a miniprotein BBA1 is studied under the native condition with the AMBER/Poisson-Boltzmann energy model and with the self-guided enhanced sampling technique. Forty independent trajectories are collected to sample the highly diversified denatured structures. Our simulation data show that the denatured BBA1 contains high percentage of native helix and native turn, but low percentage of native hairpin. Conditional population analysis indicates that the native helix formation and the native hairpin formation are not cooperative in the denatured state. Side-chain analysis shows that the native hydrophobic contacts are more preferred than the non-native hydrophobic contacts in the denatured BBA1. In contrast, the salt-bridge contacts are more or less nonspecific even if their populations are higher than those of hydrophobic contacts. Analysis of the trajectories shows that the native helix mostly initiates near the N terminus and propagates to the C terminus, and mostly forms from 310-helix/turn to α helix. The same analysis shows that the native turn is important but not necessary in its formation in the denatured BBA1. In addition, the formations of the two strands in the native hairpin are rather asymmetric, demonstrating the likely influence of the protein environment. Energetic analysis shows that the native helix formation is largely driven by electrostatic interactions in denatured BBA1. Further, the native helix formation is associated with the breakup of non-native salt-bridge contacts and the accumulation of native salt-bridge contacts. However, the native hydrophobic contacts only show a small increase upon the native helix formation while the non-native hydrophobic contacts stay essentially the same, different from the evolution of hydrophobic contacts observed in an isolated helix folding.

Effect of heating strategies on whey protein denaturation--Revisited by liquid chromatography quadrupole time-of-flight mass spectrometry.

PubMed

Akkerman, M; Rauh, V M; Christensen, M; Johansen, L B; Hammershøj, M; Larsen, L B

2016-01-01

Previous standards in the area of effect of heat treatment processes on milk protein denaturation were based primarily on laboratory-scale analysis and determination of denaturation degrees by, for example, electrophoresis. In this study, whey protein denaturation was revisited by pilot-scale heating strategies and liquid chromatography quadrupole time-of-flight mass spectrometer (LC/MC Q-TOF) analysis. Skim milk was heat treated by the use of 3 heating strategies, namely plate heat exchanger (PHE), tubular heat exchanger (THE), and direct steam injection (DSI), under various heating temperatures (T) and holding times. The effect of heating strategy on the degree of denaturation of β-lactoglobulin and α-lactalbumin was determined using LC/MC Q-TOF of pH 4.5-soluble whey proteins. Furthermore, effect of heating strategy on the rennet-induced coagulation properties was studied by oscillatory rheometry. In addition, rennet-induced coagulation of heat-treated micellar casein concentrate subjected to PHE was studied. For skim milk, the whey protein denaturation increased significantly as T and holding time increased, regardless of heating method. High denaturation degrees were obtained for T >100°C using PHE and THE, whereas DSI resulted in significantly lower denaturation degrees, compared with PHE and THE. Rennet coagulation properties were impaired by increased T and holding time regardless of heating method, although DSI resulted in less impairment compared with PHE and THE. No significant difference was found between THE and PHE for effect on rennet coagulation time, whereas the curd firming rate was significantly larger for THE compared with PHE. Micellar casein concentrate possessed improved rennet coagulation properties compared with skim milk receiving equal heat treatment. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Candidate proof mass actuator control laws for the vibration suppression of a frame

NASA Technical Reports Server (NTRS)

Umland, Jeffrey W.; Inman, Daniel J.

1991-01-01

The vibration of an experimental flexible space truss is controlled with internal control forces produced by several proof mass actuators. Four candidate control law strategies are evaluated in terms of performance and robustness. These control laws are experimentally implemented on a quasi free-free planar truss. Sensor and actuator dynamics are included in the model such that the final closed loop is self-equilibrated. The first two control laws considered are based on direct output feedback and consist of tuning the actuator feedback gains to the lowest mode intended to receive damping. The first method feeds back only the position and velocity of the proof mass relative to the structure; this results in a traditional vibration absorber. The second method includes the same feedback paths as the first plus feedback of the local structural velocity. The third law is designed with robust H infinity control theory. The fourth strategy is an active implementation of a viscous damper, where the actuator is configured to provide a bending moment at two points on the structure. The vibration control system is then evaluated in terms of how it would benefit the space structure's position control system.

Imaging Prostate Cancer Microenvironment by Collagen Hybridization

DTIC Science & Technology

2016-10-01

affinity to denatured collagens and collagens undergoing remodeling which simulate the microenvironment of metastatic tumors. We will focus on previously...specifically target digested collagens with unfolded and partially denatured collagen triple helices. 2. Demonstration of ex vivo and in vivo targeting...invasive prostate cancer due to the absence of non-specific affinity and high propensity to hybridize with denatured collagen strand (Aim 1). We

Effects of thermally induced denaturation on technological-functional properties of whey protein isolate-based films.

PubMed

Schmid, M; Krimmel, B; Grupa, U; Noller, K

2014-09-01

This study examined how and to what extent the degree of denaturation affected the technological-functional properties of whey protein isolate (WPI)-based coatings. It was observed that denaturation affected the material properties of WPI-coated films significantly. Surface energy decreased by approximately 20% compared with native coatings. Because the surface energy of a coating should be lower than that of the substrate, this might result in enhanced wettability characteristics between WPI-based solution and substrate surface. Water vapor barrier properties increased by about 35% and oxygen barrier properties increased by approximately 33%. However, significant differences were mainly observed between coatings made of fully native WPI and ones with a degree of denaturation of 25%. Higher degrees of denaturation did not lead to further improvement of material properties. This observation offers cost-saving potential: a major share of denatured whey proteins may be replaced by fully native ones that are not exposed to energy-intensive heat treatment. Furthermore, native WPI solutions can be produced with higher dry matter content without gelatinizing. Hence, less moisture has to be removed through drying, resulting in reduced energy consumption. Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Cold denaturation and 2H2O stabilization of a staphylococcal nuclease mutant.

PubMed Central

Antonino, L C; Kautz, R A; Nakano, T; Fox, R O; Fink, A L

1991-01-01

Cold denaturation is now recognized as a general property of proteins but has been observed only under destabilizing conditions, such as moderate denaturant concentration or low pH. By destabilizing the protein using site-directed mutagenesis, we have observed cold denaturation at pH 7.0 in the absence of denaturants in a mutant of staphylococcal nuclease, which we call NCA S28G for a hybrid protein between staphylococcal nuclease and concanavalin A in which there is the point mutation Ser-28----Gly. The temperature of maximum stability (tmax) as determined by circular dichroism (CD) was 18.1 degrees C, and the midpoints of the thermal unfolding transitions (tm) were 0.6 degrees C and 30.0 degrees C. These values may be compared with the tm of 52.5 degrees C for wild-type staphylococcal nuclease, for which no cold denaturation was observed under these conditions. When the stability of the mutant was examined in 2H2O by NMR, CD, or fluorescence, a substantial increase in the amount of folded protein at the tmax was noted as well as a decrease in tmax, reflecting increased stability. PMID:1652762

Anti-myeloperoxidase autoantibodies react with native but not denatured myeloperoxidase.

PubMed Central

Falk, R J; Becker, M; Terrell, R; Jennette, J C

1992-01-01

We wondered whether anti-myeloperoxidase (MPO) autoantibodies (MPO-ANCA) found in patients with systemic vasculitis react with a conformational epitope or epitopes on the MPO molecule. Sera from 15 human MPO-ANCA, and a polyclonal and a monoclonal anti-MPO antibodies were reacted with MPO in native and denatured states. Human MPO-ANCA and mouse monoclonal anti-MPO reacted with native MPO, and a 120-kD band representing the MPO hologenzyme, but not with denatured MPO fragments; however, MPO-ANCA and mouse anti-MPO did not demonstrate competitive inhibition of binding to MPO. Polyclonal rabbit anti-MPO reacted with both native and denatured MPO. All MPO-ANCA tested showed the same patterns of reactivity with native and denatured MPO in dot blot and Western blot analyses. Both polyclonal and monoclonal anti-MPO antibodies inhibited MPO's protein iodination by over 90%, whereas MPO-ANCA IgGs, normal IgGs and disease control IgGs did not. These data suggest that (i) MPO-ANCA interact with a conformational epitope on the MPO molecule; and (ii) MPO-ANCA from different patients have similar reactivity with native versus denatured MPO. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:1379133

Reversible and non-reversible thermal denaturation of lysozyme with varying pH at low ionic strength.

PubMed

Blumlein, Alice; McManus, Jennifer J

2013-10-01

DSC analysis has been used to quantify the reversibility of unfolding following thermal denaturation of lysozyme. Since the temperature at which protein unfolding occurs, Tm, varies with different solution conditions, the effect on the melting temperature and the degree of refolding after thermal denaturation in low ionic strength sodium phosphate buffers (5-1000mM) over a range of pH (5-9) in the presence/absence of disaccharides is examined. This study compares the enthalpies of unfolding during successive heating cycles to quantify reversibility following thermal denaturation. The disaccharides, trehalose and maltose were used to assess if the disaccharide induced increase in Tm is reflected in the reversibility of thermally induced denaturation. There was extensive overlap between the Tm values where non-reversible and reversible thermal denaturation occurred. Indeed, for pH6, at the highest and lowest Tm, no refolding was observed whereas refolding was observed for intermediate values, but with similar Tm values having different proportions of refolded protein. We established a method to measure the degree of reversible unfolding following thermal denaturation and hence indirectly, the degree to which protein is lost to irreversible aggregation, and show that solution conditions which increase melt transition temperatures do not automatically confer an increase in reversibility. This type of analysis may prove useful in assessing the stability of proteins in both the biopharmaceutical and food industries. Copyright © 2013 Elsevier B.V. All rights reserved.

Effect of mechanical denaturation on surface free energy of protein powders.

PubMed

Mohammad, Mohammad Amin; Grimsey, Ian M; Forbes, Robert T; Blagbrough, Ian S; Conway, Barbara R

2016-10-01

Globular proteins are important both as therapeutic agents and excipients. However, their fragile native conformations can be denatured during pharmaceutical processing, which leads to modification of the surface energy of their powders and hence their performance. Lyophilized powders of hen egg-white lysozyme and β-galactosidase from Aspergillus oryzae were used as models to study the effects of mechanical denaturation on the surface energies of basic and acidic protein powders, respectively. Their mechanical denaturation upon milling was confirmed by the absence of their thermal unfolding transition phases and by the changes in their secondary and tertiary structures. Inverse gas chromatography detected differences between both unprocessed protein powders and the changes induced by their mechanical denaturation. The surfaces of the acidic and basic protein powders were relatively basic, however the surface acidity of β-galactosidase was higher than that of lysozyme. Also, the surface of β-galactosidase powder had a higher dispersive energy compared to lysozyme. The mechanical denaturation decreased the dispersive energy and the basicity of the surfaces of both protein powders. The amino acid composition and molecular conformation of the proteins explained the surface energy data measured by inverse gas chromatography. The biological activity of mechanically denatured protein powders can either be reversible (lysozyme) or irreversible (β-galactosidase) upon hydration. Our surface data can be exploited to understand and predict the performance of protein powders within pharmaceutical dosage forms. Copyright © 2016 Elsevier B.V. All rights reserved.

Starkey experimental forest and range.

Treesearch

Valerie. Rapp

2004-01-01

The Starkey Experimental Forest and Range. (Starkey) is a one-of-a-kind, world class research facility, located in the Blue Mountains of northeastern Oregon. Starkey is the primary field location for scientific study of the effects of deer, elk, and cattle on ecosystems. Most of the 28,000-acre forest and range is enclosed by a game-proof fence.The research...

Thermal analysis of the vortex tube based thermocycler for fast DNA amplification: Experimental and two-dimensional numerical results

NASA Astrophysics Data System (ADS)

Raghavan, V.; Whitney, Scott E.; Ebmeier, Ryan J.; Padhye, Nisha V.; Nelson, Michael; Viljoen, Hendrik J.; Gogos, George

2006-09-01

In this article, experimental and numerical analyses to investigate the thermal control of an innovative vortex tube based polymerase chain reaction (VT-PCR) thermocycler are described. VT-PCR is capable of rapid DNA amplification and real-time optical detection. The device rapidly cycles six 20μl 96bp λ-DNA samples between the PCR stages (denaturation, annealing, and elongation) for 30cycles in approximately 6min. Two-dimensional numerical simulations have been carried out using computational fluid dynamics (CFD) software FLUENT v.6.2.16. Experiments and CFD simulations have been carried out to measure/predict the temperature variation between the samples and within each sample. Heat transfer rate (primarily dictated by the temperature differences between the samples and the external air heating or cooling them) governs the temperature distribution between and within the samples. Temperature variation between and within the samples during the denaturation stage has been quite uniform (maximum variation around ±0.5 and 1.6°C, respectively). During cooling, by adjusting the cold release valves in the VT-PCR during some stage of cooling, the heat transfer rate has been controlled. Improved thermal control, which increases the efficiency of the PCR process, has been obtained both experimentally and numerically by slightly decreasing the rate of cooling. Thus, almost uniform temperature distribution between and within the samples (within 1°C) has been attained for the annealing stage as well. It is shown that the VT-PCR is a fully functional PCR machine capable of amplifying specific DNA target sequences in less time than conventional PCR devices.

Decoupling of size and shape fluctuations in heteropolymeric sequences reconciles discrepancies in SAXS vs. FRET measurements

PubMed Central

Fuertes, Gustavo; Banterle, Niccolò; Ruff, Kiersten M.; Chowdhury, Aritra; Mercadante, Davide; Koehler, Christine; Kachala, Michael; Estrada Girona, Gemma; Milles, Sigrid; Mishra, Ankur; Onck, Patrick R.; Gräter, Frauke; Esteban-Martín, Santiago; Pappu, Rohit V.; Svergun, Dmitri I.; Lemke, Edward A.

2017-01-01

Unfolded states of proteins and native states of intrinsically disordered proteins (IDPs) populate heterogeneous conformational ensembles in solution. The average sizes of these heterogeneous systems, quantified by the radius of gyration (RG), can be measured by small-angle X-ray scattering (SAXS). Another parameter, the mean dye-to-dye distance (RE) for proteins with fluorescently labeled termini, can be estimated using single-molecule Förster resonance energy transfer (smFRET). A number of studies have reported inconsistencies in inferences drawn from the two sets of measurements for the dimensions of unfolded proteins and IDPs in the absence of chemical denaturants. These differences are typically attributed to the influence of fluorescent labels used in smFRET and to the impact of high concentrations and averaging features of SAXS. By measuring the dimensions of a collection of labeled and unlabeled polypeptides using smFRET and SAXS, we directly assessed the contributions of dyes to the experimental values RG and RE. For chemically denatured proteins we obtain mutual consistency in our inferences based on RG and RE, whereas for IDPs under native conditions, we find substantial deviations. Using computations, we show that discrepant inferences are neither due to methodological shortcomings of specific measurements nor due to artifacts of dyes. Instead, our analysis suggests that chemical heterogeneity in heteropolymeric systems leads to a decoupling between RE and RG that is amplified in the absence of denaturants. Therefore, joint assessments of RG and RE combined with measurements of polymer shapes should provide a consistent and complete picture of the underlying ensembles. PMID:28716919

Collapse kinetics and chevron plots from simulations of denaturant-dependent folding of globular proteins

PubMed Central

Liu, Zhenxing; Reddy, Govardhan; O’Brien, Edward P.; Thirumalai, D.

2011-01-01

Quantitative description of how proteins fold under experimental conditions remains a challenging problem. Experiments often use urea and guanidinium chloride to study folding whereas the natural variable in simulations is temperature. To bridge the gap, we use the molecular transfer model that combines measured denaturant-dependent transfer free energies for the peptide group and amino acid residues, and a coarse-grained Cα-side chain model for polypeptide chains to simulate the folding of src SH3 domain. Stability of the native state decreases linearly as [C] (the concentration of guanidinium chloride) increases with the slope, m, that is in excellent agreement with experiments. Remarkably, the calculated folding rate at [C] = 0 is only 16-fold larger than the measured value. Most importantly ln kobs (kobs is the sum of folding and unfolding rates) as a function of [C] has the characteristic V (chevron) shape. In every folding trajectory, the times for reaching the native state, interactions stabilizing all the substructures, and global collapse coincide. The value of (mf is the slope of the folding arm of the chevron plot) is identical to the fraction of buried solvent accessible surface area in the structures of the transition state ensemble. In the dominant transition state, which does not vary significantly at low [C], the core of the protein and certain loops are structured. Besides solving the long-standing problem of computing the chevron plot, our work lays the foundation for incorporating denaturant effects in a physically transparent manner either in all-atom or coarse-grained simulations. PMID:21512127

Collapse kinetics and chevron plots from simulations of denaturant-dependent folding of globular proteins.

PubMed

Liu, Zhenxing; Reddy, Govardhan; O'Brien, Edward P; Thirumalai, D

2011-05-10

Quantitative description of how proteins fold under experimental conditions remains a challenging problem. Experiments often use urea and guanidinium chloride to study folding whereas the natural variable in simulations is temperature. To bridge the gap, we use the molecular transfer model that combines measured denaturant-dependent transfer free energies for the peptide group and amino acid residues, and a coarse-grained C(α)-side chain model for polypeptide chains to simulate the folding of src SH(3) domain. Stability of the native state decreases linearly as [C] (the concentration of guanidinium chloride) increases with the slope, m, that is in excellent agreement with experiments. Remarkably, the calculated folding rate at [C] = 0 is only 16-fold larger than the measured value. Most importantly ln k(obs) (k(obs) is the sum of folding and unfolding rates) as a function of [C] has the characteristic V (chevron) shape. In every folding trajectory, the times for reaching the native state, interactions stabilizing all the substructures, and global collapse coincide. The value of (m(f) is the slope of the folding arm of the chevron plot) is identical to the fraction of buried solvent accessible surface area in the structures of the transition state ensemble. In the dominant transition state, which does not vary significantly at low [C], the core of the protein and certain loops are structured. Besides solving the long-standing problem of computing the chevron plot, our work lays the foundation for incorporating denaturant effects in a physically transparent manner either in all-atom or coarse-grained simulations.

Role of Urea-Aromatic Stacking Interactions in Stabilizing the Aromatic Residues of the Protein in Urea-Induced Denatured State.

PubMed

Goyal, Siddharth; Chattopadhyay, Aditya; Kasavajhala, Koushik; Priyakumar, U Deva

2017-10-25

A delicate balance of different types of intramolecular interactions makes the folded states of proteins marginally more stable than the unfolded states. Experiments use thermal, chemical, or mechanical stress to perturb the folding equilibrium for examining protein stability and the protein folding process. Elucidation of the mechanism by which chemical denaturants unfold proteins is crucial; this study explores the nature of urea-aromatic interactions relevant in urea-assisted protein denaturation. Free energy profiles corresponding to the unfolding of Trp-cage miniprotein in the presence and absence of urea at three different temperatures demonstrate the distortion of the hydrophobic core to be a crucial step. Exposure of the Trp6 residue to the solvent is found to be favored in the presence of urea. Previous experiments showed that urea has a high affinity for aromatic groups of proteins. We show here that this is due to the remarkable ability of urea to form stacking and NH-π interactions with aromatic groups of proteins. Urea-nucleobase stacking interactions have been shown to be crucial in urea-assisted RNA unfolding. Examination of these interactions using microsecond-long unrestrained simulations shows that urea-aromatic stacking interactions are stabilizing and long lasting. Further MD simulations, thermodynamic integration, and quantum mechanical calculations on aromatic model systems reveal that such interactions are possible for all the aromatic amino acid side-chains. Finally, we validate the ubiquitous nature of urea-aromatic stacking interactions by analyzing experimental structures of urea transporters and proteins crystallized in the presence of urea or urea derivatives.

Proceedings of the European ISTVS Conference (6th), OVK Symposium (4th), on "Off Road Vehicles in Theory and Practice". Held at Vienna, Austria on 28-30 September 1994. Volume 2.

DTIC Science & Technology

1994-09-30

experimental proof-of-concept series hybrid thermo-mechano-elec- tromechanical and/or electromechanical All-Wheel-Driven (AWD) pro- pulsion, All-Wheel...caterpillar-tracks’ motorized sprocket-, road- and tensioner- wheels form a complete proof-of-concept series hybrid thermo- mechano-electromechanical and/or...tromechanical AWA supension spheres and skid-steering conversion spheres used as integral spheres together with future new concept hybrid thermo

Dynamics of Ionic Liquid-Assisted Refolding of Denatured Cytochrome c: A Study of Preferential Interactions toward Renaturation.

PubMed

Singh, Upendra Kumar; Patel, Rajan

2018-05-25

In vitro refolding of denatured protein and the influence of the alkyl chain on the refolding of a protein were tested using long chain imidazolium chloride salts, 1-methyl-3-octylimidazolium chloride [C 8 mim][Cl], and 1-decyl-3-methylimidazolium chloride [C 10 mim][Cl]. The horse heart cytochrome c (h-cyt c) was denatured by urea and guanidinium hydrochloride (GdnHCl), as well as by base-induced denaturation at pH 13, to provide a broad overview of the overall refolding behavior. The variation in the alkyl chain of the ionic liquids (ILs) showed a profound effect on the refolding of denatured h-cyt c. The ligand-induced refolding was correlated to understand the mechanism of the conformational stability of proteins in aqueous solutions of ILs. The results showed that the long chain ILs having the [C 8 mim] + and [C 10 mim] + cations promote the refolding of alkali-denatured h-cyt c. The IL having the [C 10 mim] + cation efficiently refolded the alkali-denatured h-cyt c with the formation of the MG state, whereas the IL having the [C 8 mim] + cation, which is known to be compatible for protein stability, shows slight refolding and forms a different transition state. The lifetime results show successful refolding of alkaline-denatured h-cyt c by both of the ILs, however, more refolding was observed in the case of [C 10 mim][Cl], and this was correlated with the fast and medium lifetimes (τ 1 and τ 2 ) obtained, which show an increase accompanied by an increase in secondary structure. The hydrophobic interactions plays an important role in the refolding of chemically and alkali-denatured h-cyt c by long chain imidazolium ILs. The formation of the MG state by [C 10 mim][Cl] was also confirmed, as some regular structure exists far below the CMC of IL. The overall results suggested that the [C 10 mim] + cation bound to the unfolded h-cyt c triggers its refolding by electrostatic and hydrophobic interactions that stabilize the MG state.

Transurethral radiofrequency collagen denaturation for the treatment of women with urinary incontinence.

PubMed

Kang, Diana; Han, Julia; Neuberger, Molly M; Moy, M Louis; Wallace, Sheila A; Alonso-Coello, Pablo; Dahm, Philipp

2015-03-18

Transurethral radiofrequency collagen denaturation is a relatively novel, minimally invasive device-based intervention used to treat individuals with urinary incontinence (UI). No systematic review of the evidence supporting its use has been published to date. To evaluate the efficacy of transurethral radiofrequency collagen denaturation, compared with other interventions, in the treatment of women with UI.Review authors sought to compare the following.• Transurethral radiofrequency collagen denaturation versus no treatment/sham treatment.• Transurethral radiofrequency collagen denaturation versus conservative physical treatment.• Transurethral radiofrequency collagen denaturation versus mechanical devices (pessaries for UI).• Transurethral radiofrequency collagen denaturation versus drug treatment.• Transurethral radiofrequency collagen denaturation versus injectable treatment for UI.• Transurethral radiofrequency collagen denaturation versus other surgery for UI. We conducted a systematic search of the Cochrane Incontinence Group Specialised Register (searched 19 December 2014), EMBASE and EMBASE Classic (January 1947 to 2014 Week 50), Google Scholar and three trials registries in December 2014, along with reference checking. We sought to identify unpublished studies by handsearching abstracts of major gynaecology and urology meetings, and by contacting experts in the field and the device manufacturer. Randomised and quasi-randomised trials of transurethral radiofrequency collagen denaturation versus no treatment/sham treatment, conservative physical treatment, mechanical devices, drug treatment, injectable treatment for UI or other surgery for UI in women were eligible. We screened search results and selected eligible studies for inclusion. We assessed risk of bias and analysed dichotomous variables as risk ratios (RRs) with 95% confidence intervals (CIs) and continuous variables as mean differences (MDs) with 95% CIs. We rated the quality of evidence using the GRADE (Grading of Recommendations Assessment, Development and Evaluation) approach. We included in the analysis one small sham-controlled randomised trial of 173 women performed in the United States. Participants enrolled in this study had been diagnosed with stress UI and were randomly assigned to transurethral radiofrequency collagen denaturation (treatment) or a sham surgery using a non-functioning catheter (no treatment). Mean age of participants in the 12-month multi-centre trial was 50 years (range 22 to 76 years).Of three patient-important primary outcomes selected for this systematic review, the number of women reporting UI symptoms after intervention was not reported. No serious adverse events were reported for the transurethral radiofrequency collagen denaturation arm or the sham treatment arm during the 12-month trial. Owing to high risk of bias and imprecision, we downgraded the quality of evidence for this outcome to low. The effect of transurethral radiofrequency collagen denaturation on the number of women with an incontinence quality of life (I-QOL) score improvement ≥ 10 points at 12 months was as follows: RR 1.11, 95% CI 0.77 to 1.62; participants = 142, but the confidence interval was wide. For this outcome, the quality of evidence was also low as the result of high risk of bias and imprecision.We found no evidence on the number of women undergoing repeat continence surgery. The risk of other adverse events (pain/dysuria (RR 5.73, 95% CI 0.75 to 43.70; participants = 173); new detrusor overactivity (RR 1.36, 95% CI 0.63 to 2.93; participants = 173); and urinary tract infection (RR 0.95, 95% CI 0.24 to 3.86; participants = 173) could not be established reliably as the trial was small. Evidence was insufficient for assessment of whether use of transurethral radiofrequency collagen denaturation was associated with an increased rate of urinary retention, haematuria and hesitancy compared with sham treatment in 173 participants. The GRADE quality of evidence for all other adverse events with available evidence was low as the result of high risk of bias and imprecision.We found no evidence to inform comparisons of transurethral radiofrequency collagen denaturation with conservative physical treatment, mechanical devices, drug treatment, injectable treatment for UI or other surgery for UI. It is not known whether transurethral radiofrequency collagen denaturation, as compared with sham treatment, improves patient-reported symptoms of UI. Evidence is insufficient to show whether the procedure improves disease-specific quality of life. Evidence is also insufficient to show whether the procedure causes serious adverse events or other adverse events in comparison with sham treatment, and no evidence was found for comparison with any other method of treatment for UI.

Nanomolar concentration of blood-soluble drag-reducing polymer inhibits experimental metastasis of human breast cancer cells

PubMed Central

Ding, Zhijie; Joy, Marion; Kameneva, Marina V; Roy, Partha

2017-01-01

Metastasis is the leading cause of cancer mortality. Extravasation of cancer cells is a critical step of metastasis. We report a novel proof-of-concept study that investigated whether non-toxic blood-soluble chemical agents capable of rheological modification of the near-vessel-wall blood flow can reduce extravasation of tumor cells and subsequent development of metastasis. Using an experimental metastasis model, we demonstrated that systemic administration of nanomolar concentrations of so-called drag-reducing polymer dramatically impeded extravasation and development of pulmonary metastasis of breast cancer cells in mice. This is the first proof-of-principle study to directly demonstrate physical/rheological, as opposed to chemical, way to prevent cancer cells from extravasation and developing metastasis and, thus, it opens the possibility of a new direction of adjuvant interventional approach in cancer. PMID:28280386

AfterMath: the work of proof in the age of human-machine collaboration.

PubMed

Dick, Stephanie

2011-09-01

During the 1970s and 1980s, a team of Automated Theorem Proving researchers at the Argonne National Laboratory near Chicago developed the Automated Reasoning Assistant, or AURA, to assist human users in the search for mathematical proofs. The resulting hybrid humans+AURA system developed the capacity to make novel contributions to pure mathematics by very untraditional means. This essay traces how these unconventional contributions were made and made possible through negotiations between the humans and the AURA at Argonne and the transformation in mathematical intuition they produced. At play in these negotiations were experimental practices, nonhumans, and nonmathematical modes of knowing. This story invites an earnest engagement between historians of mathematics and scholars in the history of science and science studies interested in experimental practice, material culture, and the roles of nonhumans in knowledge making.

On the Effect of Sodium Chloride and Sodium Sulfate on Cold Denaturation

PubMed Central

Pica, Andrea; Graziano, Giuseppe

2015-01-01

Both sodium chloride and sodium sulfate are able to stabilize yeast frataxin, causing an overall increase of its thermodynamic stability curve, with a decrease in the cold denaturation temperature and an increase in the hot denaturation one. The influence of low concentrations of these two salts on yeast frataxin stability can be assessed by the application of a theoretical model based on scaled particle theory. First developed to figure out the mechanism underlying cold denaturation in water, this model is able to predict the stabilization of globular proteins provided by these two salts. The densities of the salt solutions and their temperature dependence play a fundamental role. PMID:26197394

Substrate-permeable encapsulation of enzymes maintains effective activity, stabilizes against denaturation, and protects against proteolytic degradation.

PubMed

Nasseau, M; Boublik, Y; Meier, W; Winterhalter, M; Fournier, D

2001-12-05

How can enzymes be protected against denaturation and proteolysis while keeping them in a fully functional state? One solution is to encapsulate the enzymes into liposomes, which enhances their stability against denaturation and proteases. However, the permeability barrier of the lipid membrane drastically reduces the activity of enzyme entrapped in the liposome by reducing the internal concentration of the substrate. To overcome this problem, we permeabilized the wall of the liposome by reconstitution of a porin from Escherichia coli. In this way, we recovered the full functionality of the enzyme while retaining the protection against denaturation and proteolytic enzymes. Copyright 2001 John Wiley & Sons, Inc.

Using a second‐order differential model to fit data without baselines in protein isothermal chemical denaturation

PubMed Central

Tang, Chuanning; Lew, Scott

2016-01-01

Abstract In vitro protein stability studies are commonly conducted via thermal or chemical denaturation/renaturation of protein. Conventional data analyses on the protein unfolding/(re)folding require well‐defined pre‐ and post‐transition baselines to evaluate Gibbs free‐energy change associated with the protein unfolding/(re)folding. This evaluation becomes problematic when there is insufficient data for determining the pre‐ or post‐transition baselines. In this study, fitting on such partial data obtained in protein chemical denaturation is established by introducing second‐order differential (SOD) analysis to overcome the limitations that the conventional fitting method has. By reducing numbers of the baseline‐related fitting parameters, the SOD analysis can successfully fit incomplete chemical denaturation data sets with high agreement to the conventional evaluation on the equivalent completed data, where the conventional fitting fails in analyzing them. This SOD fitting for the abbreviated isothermal chemical denaturation further fulfills data analysis methods on the insufficient data sets conducted in the two prevalent protein stability studies. PMID:26757366

Heat-induced Irreversible Denaturation of the Camelid Single Domain VHH Antibody Is Governed by Chemical Modifications

PubMed Central

Akazawa-Ogawa, Yoko; Takashima, Mizuki; Lee, Young-Ho; Ikegami, Takahisa; Goto, Yuji; Uegaki, Koichi; Hagihara, Yoshihisa

2014-01-01

The variable domain of camelid heavy chain antibody (VHH) is highly heat-resistant and is therefore ideal for many applications. Although understanding the process of heat-induced irreversible denaturation is essential to improve the efficacy of VHH, its inactivation mechanism remains unclear. Here, we showed that chemical modifications predominantly governed the irreversible denaturation of VHH at high temperatures. After heat treatment, the activity of VHH was dependent only on the incubation time at 90 °C and was insensitive to the number of heating (90 °C)-cooling (20 °C) cycles, indicating a negligible role for folding/unfolding intermediates on permanent denaturation. The residual activity was independent of concentration; therefore, VHH lost its activity in a unimolecular manner, not by aggregation. A VHH mutant lacking Asn, which is susceptible to chemical modifications, had significantly higher heat resistance than did the wild-type protein, indicating the importance of chemical modifications to VHH denaturation. PMID:24739391

A differential scanning calorimetric study of the effects of metal ions, substrate/product, substrate analogues and chaotropic anions on the thermal denaturation of yeast enolase 1.

PubMed

Brewer, J M; Wampler, J E

2001-03-14

The thermal denaturation of yeast enolase 1 was studied by differential scanning calorimetry (DSC) under conditions of subunit association/dissociation, enzymatic activity or substrate binding without turnover and substrate analogue binding. Subunit association stabilizes the enzyme, that is, the enzyme dissociates before denaturing. The conformational change produced by conformational metal ion binding increases thermal stability by reducing subunit dissociation. 'Substrate' or analogue binding additionally stabilizes the enzyme, irrespective of whether turnover is occurring, perhaps in part by the same mechanism. More strongly bound metal ions also stabilize the enzyme more, which we interpret as consistent with metal ion loss before denaturation, though possibly the denaturation pathway is different in the absence of metal ion. We suggest that some of the stabilization by 'substrate' and analogue binding is owing to the closure of moveable polypeptide loops about the active site, producing a more 'closed' and hence thermostable conformation.

A restricted proof that the weak equivalence principle implies the Einstein equivalence principle

NASA Technical Reports Server (NTRS)

Lightman, A. P.; Lee, D. L.

1973-01-01

Schiff has conjectured that the weak equivalence principle (WEP) implies the Einstein equivalence principle (EEP). A proof is presented of Schiff's conjecture, restricted to: (1) test bodies made of electromagnetically interacting point particles, that fall from rest in a static, spherically symmetric gravitational field; (2) theories of gravity within a certain broad class - a class that includes almost all complete relativistic theories that have been found in the literature, but with each theory truncated to contain only point particles plus electromagnetic and gravitational fields. The proof shows that every nonmentric theory in the class (every theory that violates EEP) must violate WEP. A formula is derived for the magnitude of the violation. It is shown that WEP is a powerful theoretical and experimental tool for constraining the manner in which gravity couples to electromagnetism in gravitation theories.

Imaging Prostate Cancer Microenvironment by Collagen Hybridization

DTIC Science & Technology

2015-10-01

expected to exhibit selective affinity to metastatic PCa tumors known to contain processed and denatured collagens. The motivating hypothesis is that the...CMP’s ability to bind to collagen/ denatured collagen can be used to image PCa in vivo as well as to determine the level of PCa malignancy. 15...targeted by antibodies (monoclonal antibody raised against denatured collagen); however antibodies have poor pharmacokinetics for in vivo imaging2. Recently

Salicylic Acid and Ethylene Pathways Are Differentially Activated in Melon Cotyledons by Active or Heat-Denatured Cellulase from Trichoderma longibrachiatum

PubMed Central

Martinez, Christelle; Blanc, Frédéric; Le Claire, Emilie; Besnard, Olivier; Nicole, Michel; Baccou, Jean-Claude

2001-01-01

Infiltration of cellulase (EC 3.2.1.4) from Trichoderma longibrachiatum into melon (Cucumis melo) cotyledons induced several key defense mechanisms and hypersensitive reaction-like symptoms. An oxidative burst was observed 3 hours after treatment and was followed by activation of ethylene and salicylic acid (SA) signaling pathways leading to marked induction of peroxidase and chitinase activities. The treatment of cotyledons by heat-denatured cellulase also led to some induction of peroxidase and chitinase activities, but the oxidative burst and SA production were not observed. Co-infiltration of aminoethoxyvinil-glycine (an ethylene inhibitor) with the active cellulase did not affect the high increase of peroxidase and chitinase activities. In contrast, co-infiltration of aminoethoxyvinil-glycine with the denatured enzyme blocked peroxidase and chitinase activities. Our data suggest that the SA pathway (induced by the cellulase activity) and ethylene pathway (induced by heat-denatured and active protein) together coordinate the activation of defense mechanisms. We found a partial interaction between both signaling pathways since SA caused an inhibition of the ethylene production and a decrease in peroxidase activity when co-infiltrated with denatured cellulase. Treatments with active or denatured cellulase caused a reduction in powdery mildew (Sphaerotheca fuliginea) disease. PMID:11553761

Kinetic and thermodynamic parameters for heat denaturation of human recombinant lactoferrin from rice.

PubMed

Castillo, Eduardo; Pérez, María Dolores; Franco, Indira; Calvo, Miguel; Sánchez, Lourdes

2012-06-01

Heat denaturation of recombinant human lactoferrin (rhLf) from rice with 3 different iron-saturation degrees, holo rhLf (iron-saturated), AsIs rhLf (60% iron saturation), and apo rhLf (iron-depleted), was studied. The 3 forms of rhLf were subjected to heat treatment, and the kinetic and thermodynamic parameters of the denaturation process were determined. Thermal denaturation of rhLf was assessed by measuring the loss of reactivity against specific antibodies. D(t) values (time to reduce 90% of immunoreactivity) decreased with increasing temperature of treatment for apo and holo rhLf, those values being higher for the iron-saturated form, which indicates that iron confers thermal stability to rhLf. However, AsIs rhLf showed a different behaviour with an increase in resistance to heat between 79 °C and 84 °C, so that the kinetic parameters could not be calculated. The heat denaturation process for apo and holo rhLf was best described assuming a reaction order of 1.5. The activation energy of the denaturation process was 648.20 kJ/mol for holo rhLf and 406.94 kJ/mol for apo rhLf, confirming that iron-depleted rhLf is more sensitive to heat treatment than iron-saturated rhLf.

MECHANISMS INVOLVED IN FIBRIN FORMATION

PubMed Central

Boyles, Paul W.; Ferguson, John H.; Muehlke, Paul H.

1951-01-01

That the role of thrombin in the conversion of fibrinogen to fibrin is essentially enzymatic, is established not only by the minute amounts of thrombin which are effective but also by the complete independence of fibrin yields and thrombin concentrations over a very wide range of thrombin dilutions and clotting times. The thrombin-fibrinogen reaction, in the phase beyond the "latent period" at least, seems fundamentally "first order." Technical requirements of the experiments leading to these conclusions include: (1) a highly purified (e.g. 97 per cent "clottable") fibrinogen, (2) absence of traces of thrombic impurities in the fibrinogen, (3) absence of fibrinolytic protease contaminant of the thrombin and the fibrinogen, and (4) sufficient stability of the thrombin even at very high dilutions. Four conditions affecting thrombin stability have been investigated. Fibrin yields are not significantly modified by numerous experimental circumstances that influence the clotting time, such as (1) temperature, (2) pH, (3) non-specific salt action due to electrical (ionic) charges, which alter the Coulomb forces involved in the fibrillar aggregation, (4) specific ion effects, whether clot-accelerating (e.g. Ca++) or clot-inhibitory (e.g. Fe(CN)6''''), (5) occluding (adsorptive) colloids, which have a "fibrinoplastic" action, e.g. (a) acacia and probably (b) fibrinogen which has been mildly "denatured" by salt-heating, acidification, etc. The data with which several European workers have attempted to substantiate the idea of a two-stage thrombin-fibrinogen reaction with an intermediary "profibrin" (allegedly partly "denatured") have been reanalyzed with controls which lead us to very different conclusions, viz. (1) denaturation and fibrin formation are independent; (2) partial denaturation is "fibrinoplastic" (see above); and (3) conditions of strong salinity and acid pH (5.1) usually do not completely prevent the thrombin-fibrinogen reaction but merely prolong the "latent" phase and lessen the time required for completion of essentially the same reaction (fibrin polymerization) when more favorable clotting conditions are restored. Thus, our experiments advance the modern concepts concerning the coagulation mechanisms along lines that, for the most part, agree with those of the Harvard physical chemists, and we oppose the European views concerning a two-stage reaction, "profibrin," and "the denaturase theory" of clotting. PMID:14832433

Slow Proton Transfer Coupled to Unfolding Explains the Puzzling Results of Single-Molecule Experiments on BBL, a Paradigmatic Downhill Folding Protein

PubMed Central

Cerminara, Michele; Campos, Luis A.; Ramanathan, Ravishankar; Muñoz, Victor

2013-01-01

A battery of thermodynamic, kinetic, and structural approaches has indicated that the small α-helical protein BBL folds-unfolds via the one-state downhill scenario. Yet, single-molecule fluorescence spectroscopy offers a more conflicting view. Single-molecule experiments at pH 6 show a unique half-unfolded conformational ensemble at mid denaturation, whereas other experiments performed at higher pH show a bimodal distribution, as expected for two-state folding. Here we use thermodynamic and laser T-jump kinetic experiments combined with theoretical modeling to investigate the pH dependence of BBL stability, folding kinetics and mechanism within the pH 6–11 range. We find that BBL unfolding is tightly coupled to the protonation of one of its residues with an apparent pKa of ∼7. Therefore, in chemical denaturation experiments around neutral pH BBL unfolds gradually, and also converts in binary fashion to the protonated species. Moreover, under the single-molecule experimental conditions (denaturant midpoint and 279 K), we observe that proton transfer is much slower than the ∼15 microseconds folding-unfolding kinetics of BBL. The relaxation kinetics is distinctly biphasic, and the overall relaxation time (i.e. 0.2–0.5 ms) becomes controlled by the proton transfer step. We then show that a simple theoretical model of protein folding coupled to proton transfer explains quantitatively all these results as well as the two sets of single-molecule experiments, including their more puzzling features. Interestingly, this analysis suggests that BBL unfolds following a one-state downhill folding mechanism at all conditions. Accordingly, the source of the bimodal distributions observed during denaturation at pH 7–8 is the splitting of the unique conformational ensemble of BBL onto two slowly inter-converting protonation species. Both, the unprotonated and protonated species unfold gradually (one-state downhill), but they exhibit different degree of unfolding at any given condition because the native structure is less stable for the protonated form. PMID:24205082

Relative Stability of the Scleroglucan Triple-Helix and Single Strand: an Insight from Computational and Experimental Techniques

NASA Astrophysics Data System (ADS)

Bocchinfuso, Gianfranco; Mazzuca, Claudia; Conflitti, Paolo; Cori, Davide; Coviello, Tommasina; Palleschi, Antonio

2016-09-01

Scleroglucan (Sclg) is a polysaccharide that exhibits a triple helix conformation (triplex), both in aqueous solution and in the solid state, which is lost in DMSO solution, at high temperature and at high pH values. The triplex conformation is characterized by a high rigidity, responsible of Sclg peculiar properties. Although the relative stability of triplex and single strand has already been investigated, different structural details are still missing. In the present study, we analyse the structural properties and the factors stabilizing the single chain and the triple helix of Sclg in different conditions. To this end, we simulated both systems in water and in DMSO. The triple helix has been also simulated in the presence of chemical damages on one of the three strands (to reproduce in silico the effect of sonication) or by inducing a partial unfolding of the triplex structure. The computational results have been compared with experimental evidences in which the triplex denaturation, at alkaline pH values, has been followed by monitoring the UV and CD spectra of Congo red, used as a probe molecule. Our results indicate that sonication breaks the Sclg chains without appreciably changing the stability of the other tracts of triple helix. The simulated perturbed or partially unfolded triplexes show a clear tendency to form less ordered aggregates. Finally, our simulations put in evidence an important role of the hydrophobic interactions both in the triplex stability and in the aggregation processes observed after induced denaturation.

Physics of base-pairing dynamics in DNA

NASA Astrophysics Data System (ADS)

Manghi, Manoel; Destainville, Nicolas

2016-05-01

As a key molecule of life, Deoxyribo-Nucleic Acid (DNA) is the focus of numbers of investigations with the help of biological, chemical and physical techniques. From a physical point of view, both experimental and theoretical works have brought quantitative insights into DNA base-pairing dynamics that we review in this Report, putting emphasis on theoretical developments. We discuss the dynamics at the base-pair scale and its pivotal coupling with the polymer one, with a polymerization index running from a few nucleotides to tens of kilo-bases. This includes opening and closure of short hairpins and oligomers as well as zipping and unwinding of long macromolecules. We review how different physical mechanisms are either used by Nature or utilized in biotechnological processes to separate the two intertwined DNA strands, by insisting on quantitative results. They go from thermally-assisted denaturation bubble nucleation to force- or torque-driven mechanisms. We show that the helical character of the molecule, possibly supercoiled, can play a key role in many denaturation and renaturation processes. We categorize the mechanisms according to the relative timescales associated with base-pairing and chain orientational degrees of freedom such as bending and torsional elastic ones. In some specific situations, these chain orientational degrees of freedom can be integrated out, and the quasi-static approximation is valid. The complex dynamics then reduces to the diffusion in a low-dimensional free-energy landscape. In contrast, some important cases of experimental interest necessarily appeal to far-from-equilibrium statistical mechanics and hydrodynamics.

Determination of gas phase protein ion densities via ion mobility analysis with charge reduction.

PubMed

Maisser, Anne; Premnath, Vinay; Ghosh, Abhimanyu; Nguyen, Tuan Anh; Attoui, Michel; Hogan, Christopher J

2011-12-28

We use a charge reduction electrospray (ESI) source and subsequent ion mobility analysis with a differential mobility analyzer (DMA, with detection via both a Faraday cage electrometer and a condensation particle counter) to infer the densities of single and multiprotein ions of cytochrome C, lysozyme, myoglobin, ovalbumin, and bovine serum albumin produced from non-denaturing (20 mM aqueous ammonium acetate) and denaturing (1 : 49.5 : 49.5, formic acid : methanol : water) ESI. Charge reduction is achieved through use of a Po-210 radioactive source, which generates roughly equal concentrations of positive and negative ions. Ions produced by the source collide with and reduce the charge on ESI generated drops, preventing Coulombic fissions, and unlike typical protein ESI, leading to gas-phase protein ions with +1 to +3 excess charges. Therefore, charge reduction serves to effectively mitigate any role that Coulombic stretching may play on the structure of the gas phase ions. Density inference is made via determination of the mobility diameter, and correspondingly the spherical equivalent protein volume. Through this approach it is found that for both non-denaturing and denaturing ESI-generated ions, gas-phase protein ions are relatively compact, with average densities of 0.97 g cm(-3) and 0.86 g cm(-3), respectively. Ions from non-denaturing ESI are found to be slightly more compact than predicted from the protein crystal structures, suggesting that low charge state protein ions in the gas phase are slightly denser than their solution conformations. While a slight difference is detected between the ions produced with non-denaturing and denaturing ESI, the denatured ions are found to be much more dense than those examined previously by drift tube mobility analysis, in which charge reduction was not employed. This indicates that Coulombic stretching is typically what leads to non-compact ions in the gas-phase, and suggests that for gas phase measurements to be correlated to biomolecular structures in solution, low charge state ions should be analyzed. Further, to determine if different solution conditions give rise to ions of different structure, ions of similar charge state should be compared. Non-denatured protein ion densities are found to be in excellent agreement with non-denatured protein ion densities inferred from prior DMA and drift tube measurements made without charge reduction (all ions with densities in the 0.85-1.10 g cm(-3) range), showing that these ions are not strongly influenced by Coulombic stretching nor by analysis method.

Periodontal bacterial invasion and infection: contribution to atherosclerotic pathology.

PubMed

Reyes, Leticia; Herrera, David; Kozarov, Emil; Roldán, Silvia; Progulske-Fox, Ann

2013-04-01

The objective of this review was to perform a systematic evaluation of the literature reporting current scientific evidence for periodontal bacteria as contributors to atherosclerosis. Literature from epidemiological, clinical and experimental studies concerning periodontal bacteria and atherosclerosis were reviewed. Gathered data were categorized into seven "proofs" of evidence that periodontal bacteria: 1) disseminate from the oral cavity and reach systemic vascular tissues; 2) can be found in the affected tissues; 3) live within the affected site; 4) invade affected cell types in vitro; 5) induce atherosclerosis in animal models of disease; 6) non-invasive mutants of periodontal bacteria cause significantly reduced pathology in vitro and in vivo; and 7) periodontal isolates from human atheromas can cause disease in animal models of infection. Substantial evidence for proofs 1 to 6 was found. However, proof 7 has not yet been fulfilled. Despite the lack of evidence that periodontal bacteria obtained from human atheromas can cause atherosclerosis in animal models of infection, attainment of proofs 1 to 6 provides support that periodontal pathogens can contribute to atherosclerosis. © 2013 European Federation of Periodontology and American Academy of Periodontology.

Reprogramming of the Ovarian Tumor Stroma by Activation of a Biomechanical ECM Switch

DTIC Science & Technology

2016-09-01

Denatured collagen was detec- ted with anticollagen antibody (1:1000). For integrin-blocking enzyme -linked immunosorbent assay, wells were coated with...migration on denatured collagen; it failed to reduce cell adhesion. Moreover a peptide antagonist of alpha 10 beta 1 may inhibit ovarian tumor growth in...stromal cell adhesion, migration and proliferation on distinct ECM substrates including native and denatured collagen. 4   D). As outlined in aim 2

High-resolution nuclear magnetic resonance studies of proteins.

PubMed

Jonas, Jiri

2002-03-25

The combination of advanced high-resolution nuclear magnetic resonance (NMR) techniques with high-pressure capability represents a powerful experimental tool in studies of protein folding. This review is organized as follows: after a general introduction of high-pressure, high-resolution NMR spectroscopy of proteins, the experimental part deals with instrumentation. The main section of the review is devoted to NMR studies of reversible pressure unfolding of proteins with special emphasis on pressure-assisted cold denaturation and the detection of folding intermediates. Recent studies investigating local perturbations in proteins and the experiments following the effects of point mutations on pressure stability of proteins are also discussed. Ribonuclease A, lysozyme, ubiquitin, apomyoglobin, alpha-lactalbumin and troponin C were the model proteins investigated.

Experimental preparation and verification of quantum money

NASA Astrophysics Data System (ADS)

Guan, Jian-Yu; Arrazola, Juan Miguel; Amiri, Ryan; Zhang, Weijun; Li, Hao; You, Lixing; Wang, Zhen; Zhang, Qiang; Pan, Jian-Wei

2018-03-01

A quantum money scheme enables a trusted bank to provide untrusted users with verifiable quantum banknotes that cannot be forged. In this work, we report a proof-of-principle experimental demonstration of the preparation and verification of unforgeable quantum banknotes. We employ a security analysis that takes experimental imperfections fully into account. We measure a total of 3.6 ×106 states in one verification round, limiting the forging probability to 10-7 based on the security analysis. Our results demonstrate the feasibility of preparing and verifying quantum banknotes using currently available experimental techniques.

The effect of pyrite on E. coli in water: Proof-of-concept for the elimination of waterborne bacteria by reactive minerals

PubMed Central

Friedlander, Lonia R.; Puri, Neha; Schoonen, Martin A.A.; Karzai, A. Wali

2015-01-01

We present proof-of-concept results for the elimination of waterborne bacteria by reactive minerals. We exposed E.coli MG1655 suspended in water to the reactive mineral pyrite (FeS2) at room temperature and ambient light. This slurry eliminates 99.9% of bacteria in fewer than 4 hours. We also exposed E. coli to pyrite leachate (supernatant liquid from slurry after 24-hours), which eliminates 99.99% of bacteria over the same time-scale. Unlike SOlar water DISinfection (SODIS) our results do not depend on the presence of ultraviolet (UV) light. We confirmed this by testing proposed SODIS additive and known photo-catalyst anatase (TiO2) for antibacterial properties and found that, in contrast to pyrite, it does not eliminate E. coli under our experimental conditions. Previous investigations of naturally antibiotic minerals have focused on the medical applications of antibiotic clays, and thus have not been conducted under experimental conditions resembling those found in water purification. In our examination of the relevant literature, we have not found previously reported evidence for the use of reactive minerals in water sanitization. The results from this proof-of-concept experiment may have important implications for future directions in household water purification research. PMID:25719464

Continuous-variable protocol for oblivious transfer in the noisy-storage model.

PubMed

Furrer, Fabian; Gehring, Tobias; Schaffner, Christian; Pacher, Christoph; Schnabel, Roman; Wehner, Stephanie

2018-04-13

Cryptographic protocols are the backbone of our information society. This includes two-party protocols which offer protection against distrustful players. Such protocols can be built from a basic primitive called oblivious transfer. We present and experimentally demonstrate here a quantum protocol for oblivious transfer for optical continuous-variable systems, and prove its security in the noisy-storage model. This model allows us to establish security by sending more quantum signals than an attacker can reliably store during the protocol. The security proof is based on uncertainty relations which we derive for continuous-variable systems, that differ from the ones used in quantum key distribution. We experimentally demonstrate in a proof-of-principle experiment the proposed oblivious transfer protocol for various channel losses by using entangled two-mode squeezed states measured with balanced homodyne detection. Our work enables the implementation of arbitrary two-party quantum cryptographic protocols with continuous-variable communication systems.

Oxidatively denatured proteins are degraded by an ATP-independent proteolytic pathway in Escherichia coli.

PubMed

Davies, K J; Lin, S W

1988-01-01

E. coli contains a soluble proteolytic pathway which can recognize and degrade oxidatively denatured proteins and protein fragments, and which may act as a "secondary antioxidant defense." We now provide evidence that this proteolytic pathway is distinct from the previously described ATP-dependent, and protease "La"-dependent, pathway which may degrade other abnormal proteins. Cells (K12) which were depleted of ATP, by arsenate treatment or anaerobic incubation (after growth on succinate), exhibited proteolytic responses to oxidative stress which were indistinguishable from those observed in cells with normal ATP levels. Furthermore, the proteolytic responses to oxidative damage by menadione or H2O2 were almost identical in the isogenic strains RM312 (a K12 derivative) and RM1385 (a lon deletion mutant of RM312). Since the lon (or capR) gene codes for the ATP-dependent protease "La," these results indicate that neither ATP nor protease "La" are required for the degradation of oxidatively denatured proteins. We next prepared cell-free extracts of K12, RM312, and RM1385 and tested the activity of their soluble proteases against proteins (albumin, hemoglobin, superoxide dismutase, catalase) which had been oxidatively denatured (in vitro) by exposure to .OH, .OH + O2- (+O2), H2O2, or ascorbate plus iron. The breakdown of oxidatively denatured proteins was several-fold higher than that of untreated proteins in extracts from all three strains, and ATP did not stimulate degradation. Incubation of extracts at 45 degrees C, which inactivates protease "La," actually stimulated the degradation of oxidatively denatured proteins. Although Ca2+ had little effect on proteolysis, serine reagents, transition metal chelators, and hemin effectively inhibited the degradation of oxidatively denatured proteins in both intact cells and cell-free extracts. Degradation of oxidatively denatured proteins in cell-free extracts was maximal at pH 7.8, and was unaffected by dialysis of the extracts against membranes with molecular weight cutoffs as high as 50,000. Our results indicate the presence of a neutral, ATP- and calcium- independent proteolytic pathway in the E. coli cytosol, which contains serine- and metallo- proteases (with molecular weights greater than 50,000), and which preferentially degrades oxidatively denatured proteins.

Raman spectral markers of collagen denaturation and hydration in human cortical bone tissue are affected by radiation sterilization and high cycle fatigue damage.

PubMed

Flanagan, Christopher D; Unal, Mustafa; Akkus, Ozan; Rimnac, Clare M

2017-11-01

Thermal denaturation and monotonic mechanical damage alter the organic and water-related compartments of cortical bone. These changes can be detected using Raman spectroscopy. However, less is known regarding Raman sensitivity to detect the effects of cyclic fatigue damage and allograft sterilization doses of gamma radiation. To determine if Raman spectroscopic biomarkers of collagen denaturation and hydration are sensitive to the effects of (a) high cycle fatigue damage and (b) 25kGy irradiation. Unirradiated and gamma-radiation sterilized human cortical bone specimens previously tested in vitro under high-cycle (> 100,000 cycles) fatigue conditions at 15MPa, 25MPa, 35MPa, 45MPa, and 55MPa cyclic stress levels were studied. Cortical bone Raman spectral profiles from wavenumber ranges of 800-1750cm -1 and 2700-3800cm -1 were obtained and compared from: a) non-fatigue vs fatigue fracture sites and b) radiated vs. unirradiated states. Raman biomarker ratios 1670/1640 and 3220/2949, which reflect collagen denaturation and organic matrix (mainly collagen)-bound water, respectively, were assessed. One- and two-way ANOVA analyses were utilized to identify differences between groups along with interaction effects between cyclic fatigue and radiation-induced damage. Cyclic fatigue damage resulted in increases in collagen denaturation (1670/1640: 1.517 ± 0.043 vs 1.579 ± 0.021, p < 0.001) and organic matrix-bound water (3220/2949: 0.109 ± 0.012 vs 0.131 ± 0.008, p < 0.001). Organic matrix-bound water increased secondary to 25kGy irradiation (3220/2949: 0.105 ± 0.010 vs 0.1161 ± 0.009, p = 0.003). Organic matrix-bound water was correlated positively with collagen denaturation (r = 0.514, p < 0.001). Raman spectroscopy can detect the effects of cyclic fatigue damage and 25kGy irradiation via increases in organic matrix (mainly collagen)-bound water. A Raman measure of collagen denaturation was sensitive to cyclic fatigue damage but not 25kGy irradiation. Collagen denaturation was correlated with organic matrix-bound water, suggesting that denaturation of collagen to gelatinous form may expose more binding sites to water by unwinding the triple alpha chains. This research may eventually be useful to help identify allograft quality and more appropriately match donors to recipients. Copyright © 2017 Elsevier Ltd. All rights reserved.

Kinetic Aspects of Surfactant-Induced Structural Changes of Proteins - Unsolved Problems of Two-State Model for Protein Denaturation -.

PubMed

Takeda, Kunio; Moriyama, Yoshiko

2015-01-01

The kinetic mechanism of surfactant-induced protein denaturation is discussed on the basis of not only stopped-flow kinetic data but also the changes of protein helicities caused by the surfactants and the discontinuous mobility changes of surfactant-protein complexes. For example, the α-helical structures of bovine serum albumin (BSA) are partially disrupted due to the addition of sodium dodecyl sulfate (SDS). Formation of SDS-BSA complex can lead to only four complex types with specific mobilities depending on the surfactant concentration. On the other hand, the apparent rate constant of the structural change of BSA increases with an increase of SDS concentration, indicating that the rate of the structural change becomes fast as the degree of the change increases. When a certain amount of surfactant ions bind to proteins, their native structures transform directly to particular structures without passing through intermediate stages that might be induced due to the binding of fewer amounts of the surfactant ions. Furthermore, this review brings up a question about two-state and three-state models, N⇌D and N⇌D'⇌D (N: native state, D: denatured sate, D': intermediate between N and D), which have been often adopted without hesitation in discussion on general denaturations of proteins. First of all, doubtful is whether any equilibrium relationship exists in such denaturation reactions. It cannot be disregarded that the D states in these models differ depending on the changes of intensities of the denaturing factors. The authors emphasize that the denaturations or the structural changes of proteins should be discussed assuming one-way reaction models with no backward processes rather than assuming the reversible two-state reaction models or similar modified reaction models.

Urea-mediated protein denaturation: a consensus view.

PubMed

Das, Atanu; Mukhopadhyay, Chaitali

2009-09-24

We have performed all-atom molecular dynamics simulations of three structurally similar small globular proteins in 8 M urea and compared the results with pure aqueous simulations. Protein denaturation is preceded by an initial loss of water from the first solvation shell and consequent in-flow of urea toward the protein. Urea reaches the first solvation shell of the protein mainly due to electrostatic interaction with a considerable contribution coming from the dispersion interaction. Urea shifts the equilibrium from the native to denatured ensemble by making the protein-protein contact less stable than protein-urea contact, which is just the reverse of the condition in pure water, where protein-protein contact is more stable than protein-water contact. We have also seen that water follows urea and reaches the protein interior at later stages of denaturation, while urea preferentially and efficiently solvates different parts of the protein. Solvation of the protein backbone via hydrogen bonding, favorable electrostatic interaction with hydrophilic residues, and dispersion interaction with hydrophobic residues are the key steps through which urea intrudes the core of the protein and denatures it. Why urea is preferred over water for binding to the protein backbone and how urea orients itself toward the protein backbone have been identified comprehensively. All the key components of intermolecular forces are found to play a significant part in urea-induced protein denaturation and also toward the stability of the denatured state ensemble. Changes in water network/structure and dynamical properties and higher degree of solvation of the hydrophobic residues validate the presence of "indirect mechanism" along with the "direct mechanism" and reinforce the effect of urea on protein.

Development of a proof of concept low temperature 4He Superfluid Magnetic Pump

NASA Astrophysics Data System (ADS)

Jahromi, Amir E.; Miller, Franklin K.

2017-03-01

We describe the development and experimental results of a proof of concept Superfluid Magnetic Pump in this work. This novel low temperature, no moving part pump can replace the existing bellows-piston driven 4He or 3He-4He mixture compressor/circulators used in various sub Kelvin refrigeration systems such as dilution, Superfluid pulse tube, Stirling, or active magnetic regenerative refrigerators. Due to the superior thermal transport properties of sub-Lambda 4He this pump can also be used as a simple circulator to distribute cooling over large surface areas. Our pump was experimentally shown to produce a maximum flow rate of 440 mg/s (averaged over cycle), 665 mg/s (peak) and produced a maximum pressure difference of 2323 Pa using only the more common isotope of helium, 4He. This pump worked in an ;ideal; thermodynamic state: The experimental results matched with the theoretical values predicted by a computer model. Pump curves were developed to map the performance of this pump. This successful demonstration will enable this novel pump to be implemented in suitable sub Kelvin refrigeration systems.

Multiplexed microfluidic approach for nucleic acid enrichment

DOEpatents

VanderNoot, Victoria A.; Langevin, Stanley Alan; Bent, Zachary; Renzi, Ronald F.; Ferko, Scott M.; Van De Vreugde, James L.; Lane, Todd; Patel, Kamlesh; Branda, Steven

2016-04-26

A system for enhancing a nucleic acid sample may include a one pump, a denaturing chamber; a microfluidic hydroxyapatite chromatography device configured for performing hydroxyapatite chromatography on the nucleic acid sample, a sample collector, and tubing connecting the pump with the denaturing chamber, the hydroxyapatite chromatography device and the sample collector such that the pump may be used to move the nucleic acid sample from the denaturing chamber to the hydroxyapatite chromatography device and then to the sample collector.

Electrophoretic mobility patterns of collagen following laser welding

NASA Astrophysics Data System (ADS)

Bass, Lawrence S.; Moazami, Nader; Pocsidio, Joanne O.; Oz, Mehmet C.; LoGerfo, Paul; Treat, Michael R.

1991-06-01

Clinical application of laser vascular anastomosis in inhibited by a lack of understanding of its mechanism. Whether tissue fusion results from covalent or non-covalent bonding of collagen and other structural proteins is unknown. We compared electrophoretic mobility of collagen in laser treated and untreated specimens of rat tail tendon (>90% type I collagen) and rabbit aorta. Welding was performed, using tissue shrinkage as the clinical endpoint, using the 808 nm diode laser (power density 14 watts/cm2) and topical indocyanine green dye (max absorption 805 nm). Collagen was extracted with 8 M urea (denaturing), 0.5 M acetic acid (non-denaturing) and acetic acid/pepsin (cleaves non- helical protein). Mobility patterns on gel electrophoresis (SDS-PAGE) after urea or acetic acid extraction were identical in the lasered and control tendon and vessel (confirmed by optical densitometry), revealing no evidence of formation of novel covalent bonds. Alpha and beta band intensity was diminished in pepsin incubated lasered specimens compared with controls (optical density ratio 0.00 +/- 9 tendon, 0.65 +/- 0.12 aorta), indicating the presence of denatured collagen. With the laser parameters used, collagen is denatured without formation of covalent bonds, suggesting that non-covalent interaction between denatured collagen molecules may be responsible for the weld. Based on this mechanism, welding parameters can be chosen which produce collagen denaturation without cell death.

Identification of intracellular degradation intermediates of aldolase B by antiserum to the denatured enzyme.

PubMed Central

Reznick, A Z; Rosenfelder, L; Shpund, S; Gershon, D

1985-01-01

A method has been developed that enables us to identify intracellular degradation intermediates of fructose-bisphosphate aldolase B (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13). This method is based on the use of antibody against thoroughly denatured purified aldolase. This antibody has been shown to recognize only denatured molecules, and it did not interact with "native" enzyme. supernatants (24,000 X g for 30 min) of liver and kidney homogenates were incubated with antiserum to denatured enzyme. The antigen-antibody precipitates thus formed were subjected to NaDodSO4/PAGE, followed by electrotransfer to nitrocellulose paper and immunodecoration with antiserum to denatured enzyme and 125I-labeled protein A. Seven peptides with molecular weights ranging from 38,000 (that of the intact subunit) to 18,000, which cross-reacted antigenically with denatured fructose-bisphosphate aldolase, could be identified in liver. The longest three peptides were also present in kidney. The possibility that these peptides were artifacts of homogenization was ruled out as follows: 125I-labeled tagged purified native aldolase was added to the buffer prior to liver homogenization. The homogenates were than subjected to NaDodSO4/PAGE followed by autoradiography, and the labeled enzyme was shown to remain intact. This method is suggested for general use in the search for degradation products of other cellular proteins. Images PMID:3898080

The role of intra-domain disulfide bonds in heat-induced irreversible denaturation of camelid single domain VHH antibodies

PubMed Central

Akazawa-Ogawa, Yoko; Uegaki, Koichi; Hagihara, Yoshihisa

2016-01-01

Camelid-derived single domain VHH antibodies are highly heat resistant, and the mechanism of heat-induced VHH denaturation predominantly relies on the chemical modification of amino acids. Although chemical modification of disulfide bonds has been recognized as a cause for heat-induced denaturation of many proteins, there have been no mutagenesis studies, in which the number of disulfide bonds was controlled. In this article, we examined a series of mutants of two different VHHs with single, double or no disulfide bonds, and scrutinized the effects of these disulfide bond modifications on VHH denaturation. With the exception of one mutant, the heat resistance of VHHs decreased when the number of disulfide bonds increased. The effect of disulfide bonds on heat denaturation was more striking if the VHH had a second disulfide bond, suggesting that the contribution of disulfide shuffling is significant in proteins with multiple disulfide bonds. Furthermore, our results directly indicate that removal of a disulfide bond can indeed increase the heat resistance of a protein, irrespective of the negative impact on equilibrium thermodynamic stability. PMID:26289739

Joule Heating and Thermal Denaturation of Proteins in Nano-ESI Theta Tips

NASA Astrophysics Data System (ADS)

Zhao, Feifei; Matt, Sarah M.; Bu, Jiexun; Rehrauer, Owen G.; Ben-Amotz, Dor; McLuckey, Scott A.

2017-10-01

Electro-osmotically induced Joule heating in theta tips and its effect on protein denaturation were investigated. Myoglobin, equine cytochrome c, bovine cytochrome c, and carbonic anhydrase II solutions were subjected to electro-osmosis in a theta tip and all of the proteins were denatured during the process. The extent of protein denaturation was found to increase with the applied square wave voltage and electrolyte concentration. The solution temperature at the end of a theta tip was measured directly by Raman spectroscopy and shown to increase with the square wave voltage, thereby demonstrating the effect of Joule heating through an independent method. The electro-osmosis of a solution comprised of myoglobin, bovine cytochrome c, and ubiquitin demonstrated that the magnitude of Joule heating that causes protein denaturation is positively correlated with protein melting temperature. This allows for a quick determination of a protein's relative thermal stability. This work establishes a fast, novel method for protein conformation manipulation prior to MS analysis and provides a temperature-controllable platform for the study of processes that take place in solution with direct coupling to mass spectrometry. [Figure not available: see fulltext.

Thermal denaturation of β-glucosidase B from Paenibacillus polymyxa proceeds through a Lumry-Eyring mechanism.

PubMed

Camarillo-Cadena, Menandro; Garza-Ramos, Georgina; Peimbert, Mariana; Pérez-Hernández, Gerardo; Zubillaga, Rafael A

2011-06-01

β-glucosidase B (BglB), 1,4-β-D: -glucanohydrolase, is an enzyme with various technological applications for which some thermostable mutants have been obtained. Because BglB denatures irreversibly with heating, the stabilities of these mutants are assessed kinetically. It, therefore, becomes relevant to determine whether the measured rate constants reflect one or several elementary kinetic steps. We have analyzed the kinetics of heat denaturation of BglB from Paenibacillus polymyxa under various conditions by following the loss of secondary structure and enzymatic activity. The denaturation is accompanied by aggregation and an initial reversible step at low temperatures. At T ≥ T ( m ), the process follows a two-state irreversible mechanism for which the kinetics does not depend on the enzyme concentration. This behavior can be explained by a Lumry-Eyring model in which the difference between the rates of the irreversible and the renaturation steps increases with temperature. Accordingly, at high scan rates (≥1 °C min(-1)) or temperatures (T ≥ T ( m )), the measurable activation energy involves only the elementary step of denaturation.

Two-dimensional cross correlation analysis of protein unfolding: Portrayal of the thermal denaturation of CMP kinases in the absence and presence of substrates

NASA Astrophysics Data System (ADS)

Schultz, Christian P.; Bârzu, Octavian; Mantsch, Henry H.

2000-03-01

The functional role of CMP kinases is to regenerate mono-phosphate nucleotides in cells by transferring phosphate residues from tri-phosphorylated nucleotides to monophosphorylated nucleotides. These enzymes possess two binding sites and maintain a highly conserved secondary structure. They are essential for cell survival. Herein we compare the infrared spectra of two similar, but not identical enzymes, the CMP kinases from Escherichia coli and Bacillus subtilis. A two-dimensional cross correlation analysis of the infrared spectra reveals differences in the denaturation behavior of the two proteins. Different secondary structure elements show different time-delayed or advanced unfolding events in the two enzymes. When bound to the active sites, the two nucleotide-substrates CMP and ATP exert a stabilizing effect on the structure of both proteins. The changes observed upon thermal denaturation are different for the two enzymes. Model 2D correlations are used to simulate the different denaturation of the two enzymes. Thermal denaturation and aggregation can be distinguished as two processes separated in time.

[Inactivating Effect of Heat-Denatured Lysozyme on Murine Norovirus in Bread Fillings].

PubMed

Takahashi, Michiko; Yasuda, Yuka; Takahashi, Hajime; Takeuchi, Akira; Kuda, Takashi; Kimura, Bon

2018-01-01

In this study, we investigated the viability of murine norovirus strain 1 (MNV-1), a surrogate for human norovirus, in bread fillings used for making stuffed buns and pastries. The inactivating effect of heat-denatured lysozyme, which was recently reported to have an antiviral effect, on MNV-1 contaminating the bread fillings was also examined. MNV-1 was inoculated into two types of fillings (chocolate cream, marmalade jam) at 4.5 log PFU/g, and the bread fillings were stored at 4℃ for 5 days. MNV-1 remained viable in the bread fillings during storage. However, addition of 1% heat-denatured lysozyme to the fillings resulted in a decrease of MNV-1 infectivity immediately after inoculation, in both fillings. On the fifth day of storage, MNV-1 infectivity was decreased by 1.2 log PFU/g in chocolate cream and by 0.9 log PFU/g in marmalade jam. Although the mechanism underlying the anti-norovirus effect of heat-denatured lysozyme has not been clarified, our results suggest that heat-denatured lysozyme can be used as an inactivating agent against norovirus in bread fillings.

Removal of Chronic Intravascular Blood Clots using Liquid Plasma

NASA Astrophysics Data System (ADS)

Jung, Jae-Chul; Choi, Myeong; Koo, Il; Yu, Zengqi; Collins, George

2011-10-01

An electrical embolectomy device for removing chronic intravascular blood clots using liquid plasma under saline environment was demonstrated. We employed a proxy experimental blood clot model of deep vein thrombosis (DVT) and actual equine blood clot. Thermal damage to contiguous tissue and the collagen denaturing via the plasma irradiation were investigated by histological analysis using birefringence of the tissue and verified by FT-IR spectroscopic study, respectively, which showed the high removal rate up to 2 mm per minute at room temperature and small thermal damage less than 200 μm.

Cold and warm swelling of hydrophobic polymers

NASA Astrophysics Data System (ADS)

de Los Rios, Paolo; Caldarelli, Guido

2001-03-01

We introduce a polymer model where the transition from swollen to compact configurations is due to interactions between the monomers and the solvent. These interactions are the origin of the effective attractive interactions between hydrophobic amino acids in proteins. We find that in the low and high temperature phases polymers are swollen, and there is an intermediate phase where the most favorable configurations are compact. We argue that such a model captures in a single framework both the cold and the warm denaturation experimentally detected for thermosensitive polymers and for proteins.

Control of a flexible planar truss using proof mass actuators

NASA Technical Reports Server (NTRS)

Minas, Constantinos; Garcia, Ephrahim; Inman, Daniel J.

1989-01-01

A flexible structure was modeled and actively controlled by using a single space realizable linear proof mass actuator. The NASA/UVA/UB actuator was attached to a flexible planar truss structure at an optimal location and it was considered as both passive and active device. The placement of the actuator was specified by examining the eigenvalues of the modified model that included the actuator dynamics, and the frequency response functions of the modified system. The electronic stiffness of the actuator was specified, such that the proof mass actuator system was tuned to the fourth structural mode of the truss by using traditional vibration absorber design. The active control law was limited to velocity feedback by integrating of the signals of two accelerometers attached to the structure. The two lower modes of the closed-loop structure were placed further in the LHS of the complex plane. The theoretically predicted passive and active control law was experimentally verified.

Effect of Weight on the Resonant Tuning of Energy Harvesting Devices Using Giant Magnetostrictive Materials.

PubMed

Mori, Kotaro; Horibe, Tadashi; Ishikawa, Shigekazu

2018-04-10

This study deals with the numerical and experimental study of the effect of weight on the resonant tuning and energy harvesting characteristics of energy harvesting devices using giant magnetostrictive materials. The energy harvesting device is made in a cantilever shape using a thin Terfenol-D layer, stainless steel (SUS) layer and a movable proof mass, among other things. In this study, two types of movable proof mass were prepared, and the device was designed to adjust its own resonant frequency automatically to match external vibration frequency in real time. Three-dimensional finite element analysis (FEA) was performed, and the resonant frequency, tip displacement, and output voltage in the devices were predicted and measured, and the simulation and experiment results were compared. The effects of the weight of the proof mass on self-tuning ability and time-varying behavior were then considered in particular.

Proof of concept of a novel SMA cage actuator

NASA Astrophysics Data System (ADS)

Deyer, Christopher W.; Brei, Diann E.

2001-06-01

Numerous industrial applications that currently utilize expensive solenoids or slow wax motors are good candidates for smart material actuation. Many of these applications require millimeter-scale displacement and low cost; thereby, eliminating piezoelectric technologies. Fortunately, there is a subset of these applications that can tolerate the slower response of shape memory alloys. This paper details a proof-of-concept study of a novel SMA cage actuator intended for proportional braking in commercial appliances. The chosen actuator architecture consists of a SMA wire cage enclosing a return spring. To develop an understanding of the influences of key design parameters on the actuator response time and displacement amplitude, a half-factorial 25 Design of Experiment (DOE) study was conducted utilizing eight differently configured prototypes. The DOE results guided the selection of the design parameters for the final proof-of-concept actuator. This actuator was built and experimentally characterized for stroke, proportional control and response time.

Decoupling of size and shape fluctuations in heteropolymeric sequences reconciles discrepancies in SAXS vs. FRET measurements.

PubMed

Fuertes, Gustavo; Banterle, Niccolò; Ruff, Kiersten M; Chowdhury, Aritra; Mercadante, Davide; Koehler, Christine; Kachala, Michael; Estrada Girona, Gemma; Milles, Sigrid; Mishra, Ankur; Onck, Patrick R; Gräter, Frauke; Esteban-Martín, Santiago; Pappu, Rohit V; Svergun, Dmitri I; Lemke, Edward A

2017-08-01

Unfolded states of proteins and native states of intrinsically disordered proteins (IDPs) populate heterogeneous conformational ensembles in solution. The average sizes of these heterogeneous systems, quantified by the radius of gyration ( R G ), can be measured by small-angle X-ray scattering (SAXS). Another parameter, the mean dye-to-dye distance ( R E ) for proteins with fluorescently labeled termini, can be estimated using single-molecule Förster resonance energy transfer (smFRET). A number of studies have reported inconsistencies in inferences drawn from the two sets of measurements for the dimensions of unfolded proteins and IDPs in the absence of chemical denaturants. These differences are typically attributed to the influence of fluorescent labels used in smFRET and to the impact of high concentrations and averaging features of SAXS. By measuring the dimensions of a collection of labeled and unlabeled polypeptides using smFRET and SAXS, we directly assessed the contributions of dyes to the experimental values R G and R E For chemically denatured proteins we obtain mutual consistency in our inferences based on R G and R E , whereas for IDPs under native conditions, we find substantial deviations. Using computations, we show that discrepant inferences are neither due to methodological shortcomings of specific measurements nor due to artifacts of dyes. Instead, our analysis suggests that chemical heterogeneity in heteropolymeric systems leads to a decoupling between R E and R G that is amplified in the absence of denaturants. Therefore, joint assessments of R G and R E combined with measurements of polymer shapes should provide a consistent and complete picture of the underlying ensembles.

PARAMETERS OF TEXTURE CHANGE IN PROCESSED FISH: MYOSIN DENATURATION.

PubMed

Chu, George Hao; Sterling, Clarence

1970-03-01

The white muscle of the Sacramento blackfish (Orthodon microlepidotus) was processed by freezing, dehydration, and cooking. Myosin was extracted immediately afterwards or following a period of storage in order to examine evidence for denaturation. The tests used were the solubility of whole muscle protein and the intrinsic viscosity, isoelectric point, ATPase activity, ultra-violet absorption spectrum, and optical rotatory dispersion of purified myosin extract. Almost all measures used showed that denaturation increased in the order: fresh < frozen < frozen-stored < dehydrated < dehydrated-stored < cooked.

27 CFR 20.54 - Photocopying of permits.

Code of Federal Regulations, 2010 CFR

2010-04-01

..., DEPARTMENT OF THE TREASURY LIQUORS DISTRIBUTION AND USE OF DENATURED ALCOHOL AND RUM Qualification of Dealers... withdraw specially denatured spirits from a distilled spirits plant and other persons authorized under this...

Proline Can Have Opposite Effects on Fast and Slow Protein Folding Phases

PubMed Central

Osváth, Szabolcs; Gruebele, Martin

2003-01-01

Proline isomerization is well known to cause additional slow phases during protein refolding. We address a new question: does the presence of prolines significantly affect the very fast kinetics that lead to the formation of folding intermediates? We examined both the very slow (10–100 min) and very fast (4 μs–2.5 ms) folding kinetics of the two-domain enzyme yeast phosphoglycerate kinase by temperature-jump relaxation. Phosphoglycerate kinase contains a conserved cis-proline in position 204, in addition to several trans-prolines. Native cis-prolines have the largest effect on folding kinetics because the unfolded state favors trans isomerization, so we compared the kinetics of a P204H mutant with the wild-type as a proof of principle. The presence of Pro-204 causes an additional slow phase upon refolding from the cold denatured state, as reported in the literature. Contrary to this, the fast folding events are sped up in the presence of the cis-proline, probably by restriction of the conformational space accessible to the molecule. The wild-type and Pro204His mutant would be excellent models for off-lattice simulations probing the effects of conformational restriction on short timescales. PMID:12885665

Effect of urea on heat-induced gelation of bovine serum albumin (BSA) studied by rheology and small angle neutron scattering (SANS)

NASA Astrophysics Data System (ADS)

Nnyigide, Osita Sunday; Oh, Yuna; Song, Hyeong Yong; Park, Eun-kyoung; Choi, Soo-Hyung; Hyun, Kyu

2017-05-01

This paper reports the effects of urea on the heat-induced gelation of bovine serum albumin (BSA), which was studied by the tube inversion method, rheological measurements, and small-angle neutron scattering (SANS). An increase in the urea concentration accelerated the rate of gelation because the protein molecules have already been unfolded to some extent during sample preparation in the urea solution. In addition, the BSA solution in the presence of urea underwent a sol-gel-sol transition during the time sweep test at a constant temperature of 80oC. On the other hand, the BSA solution without urea turned into a hard and brittle gel that did not return to the solution state during isothermal heating at a constant temperature of 80oC. Aggregation and re-bonding of the denatured and unfolded protein chains led to gel formation. Urea added to the protein denatures its tertiary and secondary structures by simultaneously disrupting the hydrogen bonds, hydrophobic interactions, and altering the solvent properties. Furthermore, urea induces thermoreversible chemical interactions in BSA solutions leading to the formation of a gel with dynamic properties under these experimental conditions.

Thermal Destabilization of Collagen Matrix Hierarchical Structure by Freeze/Thaw

PubMed Central

Ozcelikkale, Altug; Han, Bumsoo

2016-01-01

This study aims to characterize and understand the effects of freezing on collagen structures and functionality. Specifically, thermodynamic destabilization of collagen at molecular- and fibril-levels by combination of low temperatures and freezing were experimentally characterized using modulated differential scanning calorimetry. In order to delineate the effects of sub-zero temperature and water-ice phase change, we hypothesized that the extent of destabilization can be determined based on post-thaw heat induced thermal denaturation of collagen. It is found that thermal denaturation temperature of collagen in hydrogel decreases by 1.4–1.6°C after freeze/thaw while no such decrease is observed in the case of molecular solution. The destabilization is predominantly due to ice formation. Exposure to low temperatures in the absence of ice has only minimal effect. Calorimetry measurements combined with morphological examination of collagen matrices by scanning electron microscopy suggest that freezing results in destabilization of collagen fibrils due to expansion of intrafibrillar space by ice formation. This fibril-level damage can be alleviated by use of cryoprotectant DMSO at concentrations as low as 0.5 M. A theoretical model explaining the change in collagen post-thaw thermal stability by freezing-induced fibril expansion is also proposed. PMID:26765741

Stability of spermine oxidase to thermal and chemical denaturation: comparison with bovine serum amine oxidase.

PubMed

Cervelli, Manuela; Leonetti, Alessia; Cervoni, Laura; Ohkubo, Shinji; Xhani, Marla; Stano, Pasquale; Federico, Rodolfo; Polticelli, Fabio; Mariottini, Paolo; Agostinelli, Enzo

2016-10-01

Spermine oxidase (SMOX) is a flavin-containing enzyme that specifically oxidizes spermine to produce spermidine, 3-aminopropanaldehyde and hydrogen peroxide. While no crystal structure is available for any mammalian SMOX, X-ray crystallography showed that the yeast Fms1 polyamine oxidase has a dimeric structure. Based on this scenario, we have investigated the quaternary structure of the SMOX protein by native gel electrophoresis, which revealed a composite gel band pattern, suggesting the formation of protein complexes. All high-order protein complexes are sensitive to reducing conditions, showing that disulfide bonds were responsible for protein complexes formation. The major gel band other than the SMOX monomer is the covalent SMOX homodimer, which was disassembled by increasing the reducing conditions, while being resistant to other denaturing conditions. Homodimeric and monomeric SMOXs are catalytically active, as revealed after gel staining for enzymatic activity. An engineered SMOX mutant deprived of all but two cysteine residues was prepared and characterized experimentally, resulting in a monomeric species. High-sensitivity differential scanning calorimetry of SMOX was compared with that of bovine serum amine oxidase, to analyse their thermal stability. Furthermore, enzymatic activity assays and fluorescence spectroscopy were used to gain insight into the unfolding process.

27 CFR 20.171 - Record of shipment.

Code of Federal Regulations, 2010 CFR

2010-04-01

... OF THE TREASURY LIQUORS DISTRIBUTION AND USE OF DENATURED ALCOHOL AND RUM Operations by Dealers and Users of Specially Denatured Spirits Inventory and Records § 20.171 Record of shipment. (a) Dealer. When...

Ion-ion interactions in the denatured state contribute to the stabilization of CutA1 proteins.

PubMed

Yutani, Katsuhide; Matsuura, Yoshinori; Naitow, Hisashi; Joti, Yasumasa

2018-05-16

In order to elucidate features of the denatured state ensembles that exist in equilibrium with the native state under physiological conditions, we performed 1.4-μs molecular dynamics (MD) simulations at 400 K and 450 K using the monomer subunits of three CutA1 mutants from Escherichia coli: an SH-free mutant (Ec0SH) with denaturation temperature (T d ) = 85.6 °C, a hydrophobic mutant (Ec0VV) with T d  = 113.3 °C, and an ionic mutant (Ec0VV_6) with T d  = 136.8 °C. The occupancy of salt bridges by the six substituted charged residues in Ec0VV_6 was 140.1% at 300 K and 89.5% at 450 K, indicating that even in the denatured state, salt bridge occupancy was high, approximately 60% of that at 300 K. From these results, we can infer that proteins from hyperthermophiles with a high ratio of charged residues are stabilized by a decrease in conformational entropy due to ion-ion interactions in the denatured state. The mechanism must be comparable to the stabilization conferred by disulfide bonds within a protein. This suggests that introduction of charged residues, to promote formation of salt bridges in the denatured state, would be a simple way to rationally design stability-enhanced mutants.

Chemically crosslinked protein dimers: stability and denaturation effects.

PubMed Central

Byrne, M. P.; Stites, W. E.

1995-01-01

Nine single substitution cysteine mutants of staphylococcal nuclease (nuclease) were preferentially crosslinked at the introduced cysteine residues using three different bifunctional crosslinking reagents; 1,6-bismaleimidohexane (BMH), 1,3-dibromo-2-propanol (DBP), and the chemical warfare agent, mustard gas (bis(2-chloroethyl)sulfide; mustard). BMH and mustard gas are highly specific reagents for cysteine residues, whereas DBP is not as specific. Guanidine hydrochloride (GuHCl) denaturations of the resulting dimeric proteins exhibited biphasic unfolding behavior that did not fit the two-state model of unfolding. The monofunctional reagent, epsilon-maleimidocaproic acid (MCA), was used as a control for the effects of alkylation. Proteins modified with MCA unfolded normally, showing that this unusual unfolding behavior is due to crosslinking. The data obtained from these crosslinked dimers was fitted to a three-state thermodynamic model of two successive transitions in which the individual subunits cooperatively unfold. These two unfolding transitions were very different from the unfolding of the monomeric protein. These differences in unfolding behavior can be attributed in large part to changes in the denatured state. In addition to GuHCl titrations, the crosslinked dimers were also thermally unfolded. In contrast to the GuHCl denaturations, analysis of this data fit a two-state model well, but with greatly elevated van't Hoff enthalpies in many cases. However, clear correlations between the thermal and GuHCl denaturations exist, and the differences in thermal unfolding can be rationalized by postulating interactions of the denatured crosslinked proteins. PMID:8580845

30 CFR 18.80 - Approval of machines assembled with certified or explosion-proof components.

Code of Federal Regulations, 2010 CFR

2010-07-01

... Modifications of Approved Machines, and Permits To Use Experimental Equipment § 18.80 Approval of machines... approval. A design of the approval plate will accompany the notification of approval. (Refer to §§ 18.10...

30 CFR 18.80 - Approval of machines assembled with certified or explosion-proof components.

Code of Federal Regulations, 2012 CFR

2012-07-01

... Modifications of Approved Machines, and Permits To Use Experimental Equipment § 18.80 Approval of machines... approval. A design of the approval plate will accompany the notification of approval. (Refer to §§ 18.10...

30 CFR 18.80 - Approval of machines assembled with certified or explosion-proof components.

Code of Federal Regulations, 2013 CFR

2013-07-01

... Modifications of Approved Machines, and Permits To Use Experimental Equipment § 18.80 Approval of machines... approval. A design of the approval plate will accompany the notification of approval. (Refer to §§ 18.10...

30 CFR 18.80 - Approval of machines assembled with certified or explosion-proof components.

Code of Federal Regulations, 2011 CFR

2011-07-01

... Modifications of Approved Machines, and Permits To Use Experimental Equipment § 18.80 Approval of machines... approval. A design of the approval plate will accompany the notification of approval. (Refer to §§ 18.10...

Experimental proof of the existence of water clusters in fullerene-like PrF3 nanoparticles

NASA Astrophysics Data System (ADS)

Alakshin, E. M.; Blokhin, D. S.; Sabitova, A. M.; Klochkov, A. V.; Klochkov, V. V.; Kono, K.; Korableva, S. L.; Tagirov, M. S.

2012-10-01

Synthesized fullerene-like nanoparticles of the Van Vleck paramagnet PrF3 have been studied by nuclear magnetic resonance cryoporometry. Water clusters have been discovered in the internal cavities of the nanoparticles. The analysis of the experimental data indicates that the cluster radius is 1-2.3 nm. The obtained data agree well with the high-resolution transmission electron microscopy data.

Coherent Experimental and Simulation Approach To Explore the Underlying Mechanism of Denaturation of Stem Bromelain in Osmolytes.

PubMed

Rani, Anjeeta; Taha, Mohamed; Venkatesu, Pannuru; Lee, Ming-Jer

2017-07-13

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. Herein, we employed florescence, UV-visible, circular dichroism (CD), dynamic light scattering (DLS) study, and MD simulations for comprehensive understanding of stem bromelain (BM) in the presence of betaine, sarcosine, arginine, and proline. The thermal stability of BM in the presence of 1 M of osmolytes is found to be in order: proline > betaine > buffer > arginine > sarcosine. BM gets more preferentially hydrated in the presence of betaine and proline than in sarcosine and arginine. Nonetheless, MD simulations suggest that betaine, sarcosine, and arginine at 1 M interact with the active site of BM through H-bonding except proline which are responsible for more disruption of active site. The distances between the catalytic site residues are 1.6, 1.9, 4.3, 5.0, and 6.2 Å for BM in proline, buffer, betaine, arginine, and sarcosine at 1 M, respectively. To the best of our knowledge, this is the first report on detailed unequivocal evidence of denaturation and deactivation of BM in the presence of methylamines and amino acids.

[Technical peculiarities of the argon-plasma welding of gastrointestinal walls wounds in experimental environment].

PubMed

Terekhov, G V; Furmanov, Iu A; Gvozdetskiĭ, V S; Savitskaia, I M

2008-06-01

A new method of the live biological tissues connection, using thermal energy of a high-temperature argon plasma, constituting perspective trend of application of a new nonsuture methods of the tissues connection, original for the world practice, was elaborated in the Department of Experimental Surgery together with the Institute of welding named after Academician E. O. Paton NAS of Ukraine. The argon-plasma welding application secure safe adhesion of the connecting surfaces formation due to the protein complexes temperature denaturation occurrence. The absence of foreign bodies in the connection zone as well as the presence of the plasma flow bacterocidal properties secure, while application of this new method, a significant lowering of a bacterial soiling of the formatted anastomoses, not interfering with the tissue natural regeneration process course.

GROMOS polarizable charge-on-spring models for liquid urea: COS/U and COS/U2

NASA Astrophysics Data System (ADS)

Lin, Zhixiong; Bachmann, Stephan J.; van Gunsteren, Wilfred F.

2015-03-01

Two one-site polarizable urea models, COS/U and COS/U2, based on the charge-on-spring model are proposed. The models are parametrized against thermodynamic properties of urea-water mixtures in combination with the polarizable COS/G2 and COS/D2 models for liquid water, respectively, and have the same functional form of the inter-atomic interaction function and are based on the same parameter calibration procedure and type of experimental data as used to develop the GROMOS biomolecular force field. Thermodynamic, dielectric, and dynamic properties of urea-water mixtures simulated using the polarizable models are closer to experimental data than using the non-polarizable models. The COS/U and COS/U2 models may be used in biomolecular simulations of protein denaturation.

Mechanistic insights into osmolyte action in protein stabilization under harsh conditions: N-methylacetamide in glycine betaine-urea mixture

NASA Astrophysics Data System (ADS)

Kumar, Narendra; Kishore, Nand

2014-10-01

Glycine betaine (GB), a small naturally occurring osmolyte, stabilizes proteins and counteracts harsh denaturing conditions such as extremes of temperature, cellular dehydration, and presence of high concentration of urea. In spite of several studies on understanding mechanism of protein stabilization and counteraction of these harsh conditions by osmolytes, studies centred on GB, one of the most important osmolyte, are scarce, hence, there is need for more investigations. To explore mechanism of protein stabilization and counteraction of denaturing property of urea by GB, molecular dynamics studies of N-methylacetamide (NMA), a model peptide representing denatured state of a protein, in the presence of GB, urea, and GB-urea mixture were carried out. The results show that GB and urea work such that the strength of GB as a protecting osmolyte is increased and the denaturing ability of urea is decreased in the GB-urea mixture. It can be inferred that GB counteracts urea by decreasing its hydrophobic interactions with proteins. The mutual interactions between GB and urea also play an important role in protein stabilization. This study provides insights on osmolyte induced counteraction of denaturing property of urea.

Conformational stability and thermodynamic characterization of homotetrameric Plasmodium falciparum beta-ketoacyl-ACP reductase.

PubMed

Karmodiya, Krishanpal; Sajad, Syed; Sinha, Sharmistha; Maity, Koustav; Suguna, Kaza; Surolia, Namita

2007-07-01

The conformational stability of the homotetrameric Plasmodium falciparum beta-ketoacyl-ACP reductase (FabG) was determined by guanidinium chloride-induced isothermal and thermal denaturation. The reversible unfolding transitions were monitored by intrinsic fluorescence, circular dichroism (CD) spectroscopy and by measuring the enzyme activity of FabG. The denaturation profiles were analyzed to obtain the thermodynamic parameters associated with unfolding of the protein. The data confirm the simple A(4) <--> 4A model of unfolding, based on the corroboration of CD data by fluorescence transition and similar Delta G estimation for denaturation curves obtained at four different concentration of the FabG. Denaturation is well described by the linear extrapolation model for denaturant-protein interactions. In addition, the conformational stability (Delta G(s)) as well as the Delta C(p) for the protein unfolding is quite high, 22.68 kcal/mole and 5.83 kcal/(mole K), respectively, which may be a reflection of the relatively large size of the tetrameric molecule (Mr 120, 000) and a large buried hydrophobic core in the folded protein. This study provides a prototype for determining conformational stability of other members of the short-chain alcohol dehydrogenase/reductase superfamily of proteins to which PfFabG belongs.

Corneal collagen denaturation in laser thermokeratoplasty

NASA Astrophysics Data System (ADS)

Brinkmann, Ralf; Kampmeier, Juergen; Grotehusmann, Ulf; Vogel, Alfred; Koop, Norbert; Asiyo-Vogel, Mary; Birngruber, Reginald

1996-05-01

In laserthermokeratoplasty (LTK) thermal denaturation and shrinkage of corneal collagen is used to correct hyperopia and astigmatism. In order to optimize dosimetry, the temperature at which maximal shrinkage of collagen fibrils occurs is of major interest. Since the exposure time in clinical LTK-treatment is limited to a few seconds, the kinetics of collagen denaturation as a rate process has to be considered, thus the time of exposure is of critical importance for threshold and shrinkage temperatures. We investigated the time-temperature correlation for corneal collagen denaturation within different time domains by turbidimetry of scattered HeNe laser probe light using a temperature controlled water bath and pulsed IR laser irradiation. In the temperature range of 60 degree(s)C to 95 degree(s)C we found an exponential relation between the denaturation time and temperature. For the typical LTK-treatment time of 2 s, a temperature of 95 degree(s)C is needed to induce thermal damage. Use of pulsed Holmium laser radiation gave significant scattering of HeNe laser probe light at calculated temperatures of around 100 degree(s)DC. Rate parameters according to the formalism of Arrhenius were fitted to these results. Force measurements showed the simultaneous onset of light scattering and collagen shrinkage.

The structural basis of urea-induced protein unfolding in β-catenin

PubMed Central

Wang, Chao; Chen, Zhongzhou; Hong, Xia; Ning, Fangkun; Liu, Haolin; Zang, Jianye; Yan, Xiaoxue; Kemp, Jennifer; Musselman, Catherine A.; Kutateladze, Tatinna G.; Zhao, Rui; Jiang, Chengyu; Zhang, Gongyi

2014-01-01

Although urea and guanidine hydrochloride are commonly used to denature proteins, the molecular underpinnings of this process have remained unclear for a century. To address this question, crystal structures of β-catenin were determined at various urea concentrations. These structures contained at least 105 unique positions that were occupied by urea molecules, each of which interacted with the protein primarily via hydrogen bonds. Hydrogen-bond competition experiments showed that the denaturing effects of urea were neutralized when polyethylene glycol was added to the solution. These data suggest that urea primarily causes proteins to unfold by competing and disrupting hydrogen bonds in proteins. Moreover, circular-dichroism spectra and nuclear magnetic resonance (NMR) analysis revealed that a similar mechanism caused protein denaturation in the absence of urea at pH levels greater than 12. Taken together, the results led to the conclusion that the disruption of hydrogen bonds is a general mechanism of unfolding induced by urea, high pH and potentially other denaturing agents such as guanidine hydrochloride. Traditionally, the disruption of hydrophobic inter­actions instead of hydrogen bonds has been thought to be the most important cause of protein denaturation. PMID:25372676

The structural basis of urea-induced protein unfolding in β-catenin.

PubMed

Wang, Chao; Chen, Zhongzhou; Hong, Xia; Ning, Fangkun; Liu, Haolin; Zang, Jianye; Yan, Xiaoxue; Kemp, Jennifer; Musselman, Catherine A; Kutateladze, Tatinna G; Zhao, Rui; Jiang, Chengyu; Zhang, Gongyi

2014-11-01

Although urea and guanidine hydrochloride are commonly used to denature proteins, the molecular underpinnings of this process have remained unclear for a century. To address this question, crystal structures of β-catenin were determined at various urea concentrations. These structures contained at least 105 unique positions that were occupied by urea molecules, each of which interacted with the protein primarily via hydrogen bonds. Hydrogen-bond competition experiments showed that the denaturing effects of urea were neutralized when polyethylene glycol was added to the solution. These data suggest that urea primarily causes proteins to unfold by competing and disrupting hydrogen bonds in proteins. Moreover, circular-dichroism spectra and nuclear magnetic resonance (NMR) analysis revealed that a similar mechanism caused protein denaturation in the absence of urea at pH levels greater than 12. Taken together, the results led to the conclusion that the disruption of hydrogen bonds is a general mechanism of unfolding induced by urea, high pH and potentially other denaturing agents such as guanidine hydrochloride. Traditionally, the disruption of hydrophobic interactions instead of hydrogen bonds has been thought to be the most important cause of protein denaturation.

Structural perturbation of proteins in low denaturant concentrations.

PubMed

Basak, S; Debnath, D; Haque, E; Ray, S; Chakrabarti, A

2001-01-01

The presence of very low concentrations of the widely used chemical denaturants, guanidinium chloride and urea, induce changes in the tertiary structure of proteins. We have presented results on such changes in four structurally unrelated proteins to show that such structural perturbations are common irrespective of their origin. Data representative of such structural changes are shown for the monomeric globular proteins such as horseradish peroxidase (HRP) from a plant, human serum albumin (HSA) and prothrombin from ovine blood serum, and for the membrane-associated, worm-like elongated protein, spectrin, from ovine erythrocytes. Structural alterations in these proteins were reflected in quenching studies of tryptophan fluorescence using the widely used quencher acrylamide. Stern-Volmer quenching constants measured in presence of the denaturants, even at concentrations below 100 mM, were higher than those measured in absence of the denaturants. Both steady-state and time-resolved fluorescence emission properties of tryptophan and of the extrinsic probe PRODAN were used for monitoring conformational changes in the proteins in presence of different low concentrations of the denaturants. These results are consistent with earlier studies from our laboratory indicating structural perturbations in proteins at the tertiary level, keeping their native-like secondary structure and their biological activity more or less intact.

Sequential events in the irreversible thermal denaturation of human brain-type creatine kinase by spectroscopic methods.

PubMed

Gao, Yan-Song; Su, Jing-Tan; Yan, Yong-Bin

2010-06-25

The non-cooperative or sequential events which occur during protein thermal denaturation are closely correlated with protein folding, stability, and physiological functions. In this research, the sequential events of human brain-type creatine kinase (hBBCK) thermal denaturation were studied by differential scanning calorimetry (DSC), CD, and intrinsic fluorescence spectroscopy. DSC experiments revealed that the thermal denaturation of hBBCK was calorimetrically irreversible. The existence of several endothermic peaks suggested that the denaturation involved stepwise conformational changes, which were further verified by the discrepancy in the transition curves obtained from various spectroscopic probes. During heating, the disruption of the active site structure occurred prior to the secondary and tertiary structural changes. The thermal unfolding and aggregation of hBBCK was found to occur through sequential events. This is quite different from that of muscle-type CK (MMCK). The results herein suggest that BBCK and MMCK undergo quite dissimilar thermal unfolding pathways, although they are highly conserved in the primary and tertiary structures. A minor difference in structure might endow the isoenzymes dissimilar local stabilities in structure, which further contribute to isoenzyme-specific thermal stabilities.

The role of intra-domain disulfide bonds in heat-induced irreversible denaturation of camelid single domain VHH antibodies.

PubMed

Akazawa-Ogawa, Yoko; Uegaki, Koichi; Hagihara, Yoshihisa

2016-01-01

Camelid-derived single domain VHH antibodies are highly heat resistant, and the mechanism of heat-induced VHH denaturation predominantly relies on the chemical modification of amino acids. Although chemical modification of disulfide bonds has been recognized as a cause for heat-induced denaturation of many proteins, there have been no mutagenesis studies, in which the number of disulfide bonds was controlled. In this article, we examined a series of mutants of two different VHHs with single, double or no disulfide bonds, and scrutinized the effects of these disulfide bond modifications on VHH denaturation. With the exception of one mutant, the heat resistance of VHHs decreased when the number of disulfide bonds increased. The effect of disulfide bonds on heat denaturation was more striking if the VHH had a second disulfide bond, suggesting that the contribution of disulfide shuffling is significant in proteins with multiple disulfide bonds. Furthermore, our results directly indicate that removal of a disulfide bond can indeed increase the heat resistance of a protein, irrespective of the negative impact on equilibrium thermodynamic stability. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Heat-Denatured Lysozyme Inactivates Murine Norovirus as a Surrogate Human Norovirus.

PubMed

Takahashi, Hajime; Nakazawa, Moemi; Ohshima, Chihiro; Sato, Miki; Tsuchiya, Tomoki; Takeuchi, Akira; Kunou, Masaaki; Kuda, Takashi; Kimura, Bon

2015-07-02

Human norovirus infects humans through the consumption of contaminated food, contact with the excrement or vomit of an infected person, and through airborne droplets that scatter the virus through the air. Being highly infectious and highly viable in the environment, inactivation of the norovirus requires a highly effective inactivating agent. In this study, we have discovered the thermal denaturing capacity of a lysozyme with known antimicrobial activity against gram-positive bacteria, as well as its inactivating effect on murine norovirus. This study is the first report on the norovirus-inactivating effects of a thermally denatured lysozyme. We observed that lysozymes heat-treated for 40 min at 100 °C caused a 4.5 log reduction in infectivity of norovirus. Transmission electron microscope analysis showed that virus particles exposed to thermally denatured lysozymes were expanded, compared to the virus before exposure. The amino acid sequence of the lysozyme was divided into three sections and the peptides of each artificially synthesised, in order to determine the region responsible for the inactivating effect. These results suggest that thermal denaturation of the lysozyme changes the protein structure, activating the region responsible for imparting an inactivating effect against the virus.

27 CFR 19.460 - Conversion of denatured alcohol formulas.

Code of Federal Regulations, 2010 CFR

2010-04-01

... (a), (b), and (c) of this section shall obtain approval from the appropriate TTB officer prior to... containing methanol or wood alcohol may be converted to any one of the completely denatured alcohol formulas...

On the cold denaturation of globular proteins

NASA Astrophysics Data System (ADS)

Ascolese, Eduardo; Graziano, Giuseppe

2008-12-01

The recent finding that yeast frataxin shows, at pH 7.0, cold denaturation at 274 K and hot denaturation at 303 K [A. Pastore, S.R. Martin, A. Politou, K.C. Kondapalli, T. Stemmler, P.A. Temussi, J. Am. Chem. Soc. 129 (2007) 5374] calls for a deeper rationalization of the molecular mechanisms stabilizing-destabilizing the native state of globular proteins. It is shown that the statistical thermodynamic model originally developed by Ikegami can reproduce in a more-than-qualitative manner the two conformational transitions of yeast frataxin, providing important clues on their molecular origin.

27 CFR 21.151 - List of denaturants authorized for denatured spirits.

Code of Federal Regulations, 2010 CFR

2010-04-01

... Denatured Rum (S.D.R.) Acetaldehyde S.D.A. 29. Acetone, U.S.P S.D.A. 23-A, 23-H. Acetaldol C.D.A. 18. Almond... alcohol S.D.A. 39, 39-A, 39-B, 40, 40-A, 40-B, 40-C. Camphor, U.S.P S.D.A. 27, 27-A, 38-B. Caustic soda.... Formaldehyde solution, U.S.P S.D.A. 22, 38-C, 38-D. Gasoline C.D.A. 18, 19; S.D.A. 28-A. Gasoline, unleaded C.D...

27 CFR 21.151 - List of denaturants authorized for denatured spirits.

Code of Federal Regulations, 2011 CFR

2011-04-01

... Denatured Rum (S.D.R.) Acetaldehyde S.D.A. 29. Acetone, U.S.P S.D.A. 23-A, 23-H. Acetaldol C.D.A. 18. Almond... alcohol S.D.A. 39, 39-A, 39-B, 40, 40-A, 40-B, 40-C. Camphor, U.S.P S.D.A. 27, 27-A, 38-B. Caustic soda.... Formaldehyde solution, U.S.P S.D.A. 22, 38-C, 38-D. Gasoline C.D.A. 18, 19; S.D.A. 28-A. Gasoline, unleaded C.D...

M1.3 - a small scaffold for DNA origami

NASA Astrophysics Data System (ADS)

Said, Hassan; Schüller, Verena J.; Eber, Fabian J.; Wege, Christina; Liedl, Tim; Richert, Clemens

2012-12-01

The DNA origami method produces programmable nanoscale objects that form when one long scaffold strand hybridizes to numerous oligonucleotide staple strands. One scaffold strand is dominating the field: M13mp18, a bacteriophage-derived vector 7249 nucleotides in length. The full-length M13 is typically folded by using over 200 staple oligonucleotides. Here we report the convenient preparation of a 704 nt fragment dubbed ``M1.3'' as a linear or cyclic scaffold and the assembly of small origami structures with just 15-24 staple strands. A typical M1.3 origami is large enough to be visualized by TEM, but small enough to show a cooperativity in its assembly and thermal denaturation that is reminiscent of oligonucleotide duplexes. Due to its medium size, M1.3 origami with globally modified staples is affordable. As a proof of principle, two origami structures with globally 5'-capped staples were prepared and were shown to give higher UV-melting points than the corresponding assembly with unmodified DNA. M1.3 has the size of a gene, not a genome, and may function as a model for gene-based nanostructures. Small origami with M1.3 as a scaffold may serve as a workbench for chemical, physical, and biological experiments.The DNA origami method produces programmable nanoscale objects that form when one long scaffold strand hybridizes to numerous oligonucleotide staple strands. One scaffold strand is dominating the field: M13mp18, a bacteriophage-derived vector 7249 nucleotides in length. The full-length M13 is typically folded by using over 200 staple oligonucleotides. Here we report the convenient preparation of a 704 nt fragment dubbed ``M1.3'' as a linear or cyclic scaffold and the assembly of small origami structures with just 15-24 staple strands. A typical M1.3 origami is large enough to be visualized by TEM, but small enough to show a cooperativity in its assembly and thermal denaturation that is reminiscent of oligonucleotide duplexes. Due to its medium size, M1.3 origami with globally modified staples is affordable. As a proof of principle, two origami structures with globally 5'-capped staples were prepared and were shown to give higher UV-melting points than the corresponding assembly with unmodified DNA. M1.3 has the size of a gene, not a genome, and may function as a model for gene-based nanostructures. Small origami with M1.3 as a scaffold may serve as a workbench for chemical, physical, and biological experiments. Electronic supplementary information (ESI) available: Materials, full sequence of M1.3, alternative restriction reactions, sequences and origami designs, ALEX data, estimated cost of producing M13, additional melting data for origami, and MALDI spectra of individual capped oligonucleotides. See DOI: 10.1039/c2nr32393a

DEVELOP A CONCENTRATED SOLAR POWER-BASED THERMAL COOLING SYSTEM VIA SIMULATION AND EXPERIMENTAL STUDIES

EPA Science Inventory

A small scale CSP-based cooling system prototype (300W cooling capacity) and the system performance simulation tool will be developed as a proof of concept. Practical issues will be identified to improve our design.

The efficacy of denaturing actinide elements as a means of decreasing materials attractiveness

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hase, K.R.; Bathke, C.G.; Ebbinghaus, B.B.

2013-07-01

This study considers the concept of denaturing as applied to the actinide elements present in spent fuel as a means to reduce materials attractiveness. Highly attractive materials generally have low values of bare critical mass, heat content, and dose. To denature an attractive element, its spent-fuel isotopic composition (isotopic vector) is intentionally modified by introducing sufficient quantities of a significantly less attractive isotope to dilute the concentration of a highly attractive isotope so that the overall attractiveness of the element is reduced. The authors used FOM (Figure of Merit) formula as the material attractiveness metric for their parametric determination ofmore » the attractiveness of the Pu and U. Materials attractiveness needs to be considered in three distinct phases in the process to construct a nuclear explosive device (NED): the acquisition phase, processing phase, and utilization phase. The results show that denaturing uranium with {sup 238}U is actually an effective means of reducing the attractiveness. For uranium with a large minority of {sup 235}U, a mixture of 80% {sup 238}U to 20% {sup 235}U is required to reduce the attractiveness to low. For uranium with a large concentration of {sup 233}U, a mixture of 88% {sup 238}U to 12% {sup 233}U is required to reduce the attractiveness to low. The results also show that denaturing plutonium with {sup 238}Pu is less effective than denaturing uranium with {sup 238}U. Using {sup 238}Pu as the denaturing agent would require 80% or more by mass in order to reduce the attractiveness to low. No amount of {sup 240}Pu is enough to reduce the plutonium attractiveness below medium. The combination of {sup 238}Pu and {sup 240}Pu would require approximately 70% {sup 238}Pu and 25% {sup 240}Pu by mass to reduce the plutonium attractiveness to low.« less

Denaturant-Dependent Conformational Changes in a [beta]-Trefoil Protein: Global and Residue-Specific Aspects of an Equilibrium Denaturation Process

DOE Office of Scientific and Technical Information (OSTI.GOV)

Latypov, Ramil F.; Liu, Dingjiang; Jacob, Jaby

2010-01-12

Conformational properties of the folded and unfolded ensembles of human interleukin-1 receptor antagonist (IL-1ra) are strongly denaturant-dependent as evidenced by high-resolution two-dimensional nuclear magnetic resonance (NMR), limited proteolysis, and small-angle X-ray scattering (SAXS). The folded ensemble was characterized in detail in the presence of different urea concentrations by 1H-15N HSQC NMR. The {beta}-trefoil fold characteristic of native IL-1ra was preserved until the unfolding transition region beginning at 4 M urea. At the same time, a subset of native resonances disappeared gradually starting at low denaturant concentrations, indicating noncooperative changes in the folded state. Additional evidence of structural perturbations came frommore » the chemical shift analysis, nonuniform and bell-shaped peak intensity profiles, and limited proteolysis. In particular, the following nearby regions of the tertiary structure became progressively destabilized with increasing urea concentrations: the {beta}-hairpin interface of trefoils 1 and 2 and the H2a-H2 helical region. These regions underwent small-scale perturbations within the native baseline region in the absence of populated molten globule-like states. Similar regions were affected by elevated temperatures known to induce irreversible aggregation of IL-1ra. Further evidence of structural transitions invoking near-native conformations came from an optical spectroscopy analysis of its single-tryptophan variant W17A. The increase in the radius of gyration was associated with a single equilibrium unfolding transition in the case of two different denaturants, urea and guanidine hydrochloride (GuHCl). However, the compactness of urea- and GuHCl-unfolded molecules was comparable only at high denaturant concentrations and deviated under less denaturing conditions. Our results identified the role of conformational flexibility in IL-1ra aggregation and shed light on the nature of structural transitions within the folded ensembles of other {beta}-trefoil proteins, such as IL-1{beta} and hFGF-1.« less

Efficient renaturation of inclusion body proteins denatured by SDS.

PubMed

He, Chuan; Ohnishi, Kouhei

2017-09-02

Inclusion bodies are often formed when the foreign protein is over expressed in Escherichia coli. Since proteins in inclusion bodies are inactive, denaturing and refolding of inclusion body proteins are necessary to obtain the active form. Instead of the conventional denaturants, urea and guanidine hydrochloride, a strong anionic detergent SDS was used to solubilize C-terminal His-tag form of ulvan lyase in the inclusion bodies. Solution containing SDS-solubilized enzyme were kept on ice to precipitate SDS, followed by SDS-KCl insoluble crystal formation to remove SDS completely. After removing the precipitate by centrifugation, the supernatant was applied to Ni-NTA column to purify His-tagged ulvan lyase. The purified protein showed a dimeric form and ulvan lyase activity, demonstrating that SDS-denatured protein was renatured and recovered enzyme activity. This simple method could be useful for refolding other inclusion body proteins. Copyright © 2017 Elsevier Inc. All rights reserved.

Natural hidden autoantibodies to tissue transglutaminase cross-react with fibrinogen.

PubMed

Zöller-Utz, Ingrid M; Esslinger, Birgit; Schulze-Krebs, Anja; Dieterich, Walburga

2010-03-01

Patients with celiac disease display autoantibodies against tissue transglutaminase (TG2), and the high sensitivity and specificity of these autoantibodies render them a reliable tool for diagnosis. However, we found that denatured sera from healthy persons also showed reactivity against TG2. To further examine the specificity of this phenomenon, sera of healthy individuals and celiac patients were denatured by heat or pH shift. Denatured sera of all individuals showed autoantibodies against TG2 in ELISA that could be specifically inhibited by TG2, but the biological role of these autoantibodies remains unknown. The alpha fibrinogen precursor could be isolated as serum protein that reacts with TG2 antibodies and treated sera reacted with fibrinogen in Western blotting. Cross-reactivity of TG2 antibodies with fibrinogen and vice versa was observed. We hypothesise that denaturation of sera reveals hidden autoantibodies against TG2, which might be normally masked by fibrinogen.

Conformational stability and thermodynamic characterization of the lipoic acid bearing domain of human mitochondrial branched chain α-ketoacid dehydrogenase

PubMed Central

Naik, Mandar T.; Huang, Tai-Huang

2004-01-01

The lipoic acid bearing domain (hbLBD) of human mitochondrial branched chain α-ketoacid dehydrogenase (BCKD) plays important role of substrate channeling in oxidative decarboxylation of the branched chain α-ketoacids. Recently hbLBD has been found to follow two-step folding mechanism without detectable presence of stable or kinetic intermediates. The present study describes the conformational stability underlying the folding of this small β-barrel domain. Thermal denaturation in presence of urea and isothermal urea denaturation titrations are used to evaluate various thermodynamic parameters defining the equilibrium unfolding. The linear extrapolation model successfully describes the two-step; native state ↔denatured state unfolding transition of hbLBD. The average temperature of maximum stability of hbLBD is estimated as 295.6 ± 0.9 K. Cold denaturation of hbLBD is also predicted and discussed. PMID:15322287

Effective non-denaturing purification method for improving the solubility of recombinant actin-binding proteins produced by bacterial expression.

PubMed

Chung, Jeong Min; Lee, Sangmin; Jung, Hyun Suk

2017-05-01

Bacterial expression is commonly used to produce recombinant and truncated mutant eukaryotic proteins. However, heterologous protein expression may render synthesized proteins insoluble. The conventional method used to express a poorly soluble protein, which involves denaturation and refolding, is time-consuming and inefficient. There are several non-denaturing approaches that can increase the solubility of recombinant proteins that include using different bacterial cell strains, altering the time of induction, lowering the incubation temperature, and employing different detergents for purification. In this study, we compared several non-denaturing protocols to express and purify two insoluble 34 kDa actin-bundling protein mutants. The solubility of the mutant proteins was not affected by any of the approaches except for treatment with the detergent sarkosyl. These results indicate that sarkosyl can effectively improve the solubility of insoluble proteins during bacterial expression. Copyright © 2016. Published by Elsevier Inc.

The Unfolding and Refolding Reactions of Triosephosphate Isomerase from Trypanosoma Cruzi Follow Similar Pathways. Guanidinium Hydrochloride Studies

NASA Astrophysics Data System (ADS)

Vázquez-Contreras, Edgar; Pérez Hernández, Gerardo; Sánchez-Rebollar, Brenda Guadalupe; Chánez-Cárdenas, María Elena

2005-04-01

The unfolding and refolding reactions of Trypanosoma cruzi triosephosphate isomerase (TcTIM) was studied under equilibrium conditions at increasing guanidinium hydrochloride concentrations. The changes in activity intrinsic fluorescence and far-ultraviolet circular dichroism as a function of denaturant were used as a quaternary, tertiary and secondary structural probes respectively. The change in extrinsic ANS fluorescence intensity was also investigated. The results show that the transition between the homodimeric native enzyme to the unfolded monomers (unfolding), and its inverse reaction (refolding) are described by similar pathways and two equilibrium intermediates were detected in both reactions. The mild denaturant concentrations intermediate is active and contains significant amount of secondary and tertiary structures. The medium denaturant concentrations intermediate is inactive and able to bind the fluorescent dye. This intermediates are maybe related with those observed in the denaturation pattern of TIMs from other species; the results are discussed in this context.

Coherent microscopic picture for urea-induced denaturation of proteins.

PubMed

Yang, Zaixing; Xiu, Peng; Shi, Biyun; Hua, Lan; Zhou, Ruhong

2012-08-02

In a previous study, we explored the mechanism of urea-induced denaturation of proteins by performing molecular dynamics (MD) simulations of hen lysozyme in 8 M urea and supported the "direct interaction mechanism" whereby urea denatures protein via dispersion interaction (Hua, L.; Zhou, R. H.; Thirumalai, D.; Berne, B. J. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 16928). Here we perform large scale MD simulations of five representative protein/peptide systems in aqueous urea to investigate if the above mechanism is common to other proteins. In all cases, accumulations of urea around proteins/peptide are observed, suggesting that urea denatures proteins by directly attacking protein backbones and side chains rather than indirectly disrupting water structure as a "water breaker". Consistent with our previous case study of lysozyme, the current energetic analyses with five protein/peptide systems reveal that urea's preferential binding to proteins mainly comes from urea's stronger dispersion interactions with proteins than with bulk solution, whereas the electrostatic (hydrogen-bonded) interactions only play a relatively minor (even negative) role during this denaturation process. Furthermore, the simulations of the peptide system at different urea concentrations (8 and 4.5 M), and with different force fields (CHARMM and OPLSAA) suggest that the above mechanism is robust, independent of the urea concentration and force field used. Last, we emphasize the importance of periodic boundary conditions in pairwise energetic analyses. This article provides a comprehensive study on the physical mechanism of urea-induced protein denaturation and suggests that the "dispersion-interaction-driven" mechanism should be general.

Statistical Characterization of the Mechanical Parameters of Intact Rock Under Triaxial Compression: An Experimental Proof of the Jinping Marble

NASA Astrophysics Data System (ADS)

Jiang, Quan; Zhong, Shan; Cui, Jie; Feng, Xia-Ting; Song, Leibo

2016-12-01

We investigated the statistical characteristics and probability distribution of the mechanical parameters of natural rock using triaxial compression tests. Twenty cores of Jinping marble were tested under each different levels of confining stress (i.e., 5, 10, 20, 30, and 40 MPa). From these full stress-strain data, we summarized the numerical characteristics and determined the probability distribution form of several important mechanical parameters, including deformational parameters, characteristic strength, characteristic strains, and failure angle. The statistical proofs relating to the mechanical parameters of rock presented new information about the marble's probabilistic distribution characteristics. The normal and log-normal distributions were appropriate for describing random strengths of rock; the coefficients of variation of the peak strengths had no relationship to the confining stress; the only acceptable random distribution for both Young's elastic modulus and Poisson's ratio was the log-normal function; and the cohesive strength had a different probability distribution pattern than the frictional angle. The triaxial tests and statistical analysis also provided experimental evidence for deciding the minimum reliable number of experimental sample and for picking appropriate parameter distributions to use in reliability calculations for rock engineering.

Molecular assessment of collagen denaturation in decellularized tissues using a collagen hybridizing peptide.

PubMed

Hwang, Jeongmin; San, Boi Hoa; Turner, Neill J; White, Lisa J; Faulk, Denver M; Badylak, Stephen F; Li, Yang; Yu, S Michael

2017-04-15

Decellularized extracellular matrix (ECM) derived from tissues and organs are emerging as important scaffold materials for regenerative medicine. Many believe that preservation of the native ECM structure during decellularization is highly desirable. However, because effective techniques to assess the structural damage in ECM are lacking, the disruptive effects of a decellularization method and the impact of the associated structural damage upon the scaffold's regenerative capacity are often debated. Using a novel collagen hybridizing peptide (CHP) that specifically binds to unfolded collagen chains, we investigated the molecular denaturation of collagen in the ECM decellularized by four commonly used cell-removing detergents: sodium dodecyl sulfate (SDS), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), sodium deoxycholate (SD), and Triton X-100. Staining of the detergent-treated porcine ligament and urinary bladder matrix with carboxyfluorescein-labeled CHP demonstrated that SDS and Triton X-100 denature the triple helical collagen molecule while CHAPS and SD do not, although second harmonic generation imaging and transmission electron microscopy (TEM) revealed that all four detergents disrupt collagen fibrils. Our findings from the CHP staining were further confirmed by the circular dichroism spectra of intact triple helical collagen molecules in CHAPS and SD solutions, and the TEM images of CHP-conjugated gold nanoparticles binding only to the SDS and Triton X-100 treated collagen fibrils. CHP is a powerful new tool for direct and reliable measurement of denatured collagen molecules in decellularized tissues. It is expected to have wide applications in the development and standardization of the tissue/organ decellularization technology. Preservation of the native ECM structure in decellularized tissues is highly desirable, since denaturation of ECM molecules (e.g., collagen) during decellularization can strongly influence the cellular response. Unfortunately, conventional techniques (SEM, SHG) are not conducive to identifying denatured collagen molecules in tissues. We demonstrate the first investigation into the molecular denaturation of collagen in decellularized ECM enabled by a novel Collagen Hybridizing Peptide (CHP) that specifically binds to unfolded collagen chains. We show that SDS and Triton X-100 denature collagen molecules while CHAPS and SD cannot. Such detection has been nearly impossible with other existing techniques. The CHP technique will advance our understanding about the effect of the cell-removing process on ECM, and lead to development of the decellularization technology. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Molecular assessment of collagen denaturation in decellularized tissues using a collagen hybridizing peptide

PubMed Central

Hwang, Jeongmin; San, Boi Hoa; Turner, Neill J.; White, Lisa J.; Faulk, Denver M.; Badylak, Stephen F.; Li, Yang; Yu, S. Michael

2017-01-01

Decellularized extracellular matrix (ECM) derived from tissues and organs are emerging as important scaffold materials for regenerative medicine. Many believe that preservation of the native ECM structure during decellularization is highly desirable. However, because effective techniques to assess the structural damage in ECM are lacking, the disruptive effects of a decellularization method and the impact of the associated structural damage upon the scaffold’s regenerative capacity are often debated. Using a novel collagen hybridizing peptide (CHP) that specifically binds to unfolded collagen chains, we investigated the molecular denaturation of collagen in the ECM decellularized by four commonly used cellremoving detergents: sodium dodecyl sulfate (SDS), 3-[(3-cholamidopropyl)dimethylammonio]-1-propa nesulfonate (CHAPS), sodium deoxycholate (SD), and Triton X-100. Staining of the detergent-treated porcine ligament and urinary bladder matrix with carboxyfluorescein-labeled CHP demonstrated that SDS and Triton X-100 denature the triple helical collagen molecule while CHAPS and SD do not, although second harmonic generation imaging and transmission electron microscopy (TEM) revealed that all four detergents disrupt collagen fibrils. Our findings from the CHP staining were further confirmed by the circular dichroism spectra of intact triple helical collagen molecules in CHAPS and SD solutions, and the TEM images of CHP-conjugated gold nanoparticles binding only to the SDS and Triton X-100 treated collagen fibrils. CHP is a powerful new tool for direct and reliable measurement of denatured collagen molecules in decellularized tissues. It is expected to have wide applications in the development and standardization of the tissue/organ decellularization technology. Statement of Significance Preservation of the native ECM structure in decellularized tissues is highly desirable, since denaturation of ECM molecules (e.g., collagen) during decellularization can strongly influence the cellular response. Unfortunately, conventional techniques (SEM, SHG) are not conducive to identifying denatured collagen molecules in tissues. We demonstrate the first investigation into the molecular denaturation of collagen in decellularized ECM enabled by a novel Collagen Hybridizing Peptide (CHP) that specifically binds to unfolded collagen chains. We show that SDS and Triton X-100 denature collagen molecules while CHAPS and SD cannot. Such detection has been nearly impossible with other existing techniques. The CHP technique will advance our understanding about the effect of the cell-removing process on ECM, and lead to development of the decellularization technology. PMID:28161576

Quantification of the Thermodynamically Linked Quaternary and Tertiary Structural Stabilities of Transthyretin and its Disease-Associated Variants–the Relationship between Stability and Amyloidosis†

PubMed Central

Hurshman Babbes, Amy R.; Powers, Evan T.; Kelly, Jeffery W.

2009-01-01

Urea denaturation studies were carried out as a function of transthyretin (TTR) concentration to quantify the thermodynamically linked quaternary and tertiary structural stability and to better understand the relationship between mutant folding energetics and amyloid disease phenotype. Urea denaturation of TTR involves at least two equilibria—dissociation of tetramers into folded monomers, and monomer unfolding. To deal with the thermodynamic linkage of these equilibria, we analyzed concentration-dependent denaturation data by global fitting to an equation that simultaneously accounts for the two-step denaturation process. Using this method, the quaternary and tertiary structural stabilities of well-behaved TTR sequences, wild type (WT) TTR and the disease-associated variant V122I, were scrutinized. The V122I variant is linked to late onset familial amyloid cardiomyopathy, the most common familial TTR amyloid disease. V122I TTR exhibits a destabilized quaternary structure and a stable tertiary structure relative to WT TTR. Three other variants of TTR were also examined, L55P, V30M, and A25T TTR. The L55P mutation is associated with the most aggressive familial TTR amyloid disease. L55P TTR has a complicated denaturation pathway that includes dimers and trimers, and so globally fitting its concentration-dependent urea denaturation data yielded error-laden estimates of stability parameters. Nevertheless, it is clear that L55P TTR is substantially less stable than WT TTR, primarily because its tertiary structure is unstable, although its quaternary structure is destabilized as well. V30M is the most common mutation associated with neuropathic forms of TTR amyloid disease. V30M TTR is certainly destabilized relative to WT TTR, but like L55P TTR it has a complex denaturation pathway that cannot be fit to the aforementioned two-step denaturation model. Literature data suggest that V30M TTR has stable quaternary structure but unstable tertiary structure. The A25T mutant, associated with central nervous system amyloidosis, is highly aggregation-prone and exhibits drastically reduced quaternary and tertiary structural stability. The observed differences in stability amongst the disease-associated TTR variants highlight the complexity and the heterogeneity of TTR amyloid disease, an observation having important implications for the treatment of these diseases. PMID:18537267

Change in refractive index of muscle tissue during laser-induced interstitial thermotherapy.

PubMed

Chen, Na; Chen, Meimei; Liu, Shupeng; Guo, Qiang; Chen, Zhenyi; Wang, Tingyun

2014-01-01

This paper presents a long-period fiber-grating (LPG) based Michelson interferometric refractometry to monitor the change in refractive index of porcine muscle during laser-induced interstitial thermotherapy (LITT). As the wavelength of RI interferometer alters with the change in refractive index around the probe, the LPG based refractometry is combined with LITT system to measure the change in refractive index of porcine muscle when irradiated by laser. The experimental results show the denaturation of tissue alters the refractive index significantly and the LPG sensor can be applied to monitor the tissue state during the LITT.

Surface plasmon resonance-enabled antibacterial digital versatile discs

NASA Astrophysics Data System (ADS)

Dou, Xuan; Chung, Pei-Yu; Jiang, Peng; Dai, Jianli

2012-02-01

We report the achievement of effective sterilization of exemplary bacteria including Escherichia coli and Geobacillus stearothermophilus spores on a digital versatile disc (DVD). The spiral arrangement of aluminum-covered pits generates strong surface plasmon resonance (SPR) absorption of near-infrared light, leading to high surface temperature that could even damage the DVD plastics. Localized protein denaturation and high sterilization efficiency have been demonstrated by using a fluorescence microscope and cell cultures. Numerical simulations have also been conducted to model the SPR properties and the surface temperature distribution of DVDs under laser illumination. The theoretical predictions agree reasonably well with the experimental results.

Protein digestion in isolated lysosomes inhibited by intralysosomal trypan blue.

PubMed

Davies, M; Lloyd, J B; Beck, F

1969-03-28

Control rats and rats treated with subcutaneous trypan blue were injected intravenously with denatured albumin-I(125). Lysosome-rich fractions of their livers, when incubated at 22 degrees C in osmotically protected medium (pH 7.4), retained their capacit to digest albumin-I(125). The rate of digestion was lower in suspensions pre-pared from rats treated with trypan blue than in control suspensions, but rates of lysosome breakage were not different. T'hese results and other experimental evidence suggest that trypanblue concentrated within lysosomes can inhibit intralysosomal digestion, probably by inhibition of lysosomal proteinases.

Critical Consideration of the Multiplicity of Experimental and Organismic Determinants of Pyrethroid Neurotoxicity: A Proof of Concept

EPA Science Inventory

Pyrethroids (PYR) are pesticides with high insecticidal activity that may disrupt neuronal excitability in target and nontarget species. The accumulated evidence consistently showed that this neurophysiologic action is followed by alterations in motor, sensorimotor, neuromuscular...

On the Learning of Mathematics Through Conversation.

ERIC Educational Resources Information Center

Haroutunian-Gordan, Sophie; Tartakoff, David S.

1996-01-01

Describes an experimental mathematics course involving an interpretive discussion approach in which students and instructors work together to develop questions about the meaning of mathematical, philosophical, and musical texts. Appendix contains a cluster of questions for Euclid's proof of the infinity of prime numbers. (MKR)

Intracochlear pressure measurements to study bone conduction transmission: State-of-the art and proof of concept of the experimental Procedure

NASA Astrophysics Data System (ADS)

Borgers, Charlotte; van Wieringen, Astrid; D'hondt, Christiane; Verhaert, Nicolas

2018-05-01

The cochlea is the main contributor in bone conduction perception. Measurements of differential pressure in the cochlea give a good estimation of the cochlear input provided by bone conduction stimulation. Recent studies have proven the feasibility of intracochlear pressure measurements in chinchillas and in human temporal bones to study bone conduction. However, similar measurements in fresh-frozen whole human cadaveric heads could give a more realistic representation of the five different transmission pathways of bone conduction to the cochlea compared to human temporal bones. The aim of our study is to develop and validate a framework for intracochlear pressure measurements to evaluate different aspects of bone conduction in whole human cadaveric heads. A proof of concept describing our experimental setup is provided together with the procedure. Additionally, we also present a method to fix the stapes footplate in order to simulate otosclerosis in human temporal bones. The effectiveness of this method is verified by some preliminary results.

Combination of watermarking and joint watermarking-decryption for reliability control and traceability of medical images.

PubMed

Bouslimi, D; Coatrieux, G; Cozic, M; Roux, Ch

2014-01-01

In this paper, we propose a novel crypto-watermarking system for the purpose of verifying the reliability of images and tracing them, i.e. identifying the person at the origin of an illegal distribution. This system couples a common watermarking method, based on Quantization Index Modulation (QIM), and a joint watermarking-decryption (JWD) approach. At the emitter side, it allows the insertion of a watermark as a proof of reliability of the image before sending it encrypted; at the reception, another watermark, a proof of traceability, is embedded during the decryption process. The scheme we propose makes interoperate such a combination of watermarking approaches taking into account risks of interferences between embedded watermarks, allowing the access to both reliability and traceability proofs. Experimental results confirm the efficiency of our system, and demonstrate it can be used to identify the physician at the origin of a disclosure even if the image has been modified.

Effects of proof loads and combined mode loadings on fracture and flaw growth characteristics of aerospace alloys

NASA Technical Reports Server (NTRS)

Shah, R. C.

1974-01-01

This experimental program was undertaken to determine the effects of (1) combined tensile and bending loadings, (2) combined tensile and shear loadings, and (3) proof overloads on fracture and flaw growth characteristics of aerospace alloys. Tests were performed on four alloys: 2219-T87 aluminum, 5Al-2.5Sn (ELl) titanium, 6Al-4V beta STA titanium and high strength 4340 steel. Tests were conducted in room air, gaseous nitrogen at -200F (144K), liquid nitrogen and liquid hydrogen. Flat center cracked and surface flawed specimens, cracked tube specimens, circumferentially notched round bar and surface flawed cylindrical specimens were tested. The three-dimensional photoelastic technique of stress freezing and slicing was used to determine stress intensity factors for surface flawed cylindrical specimens subjected to tension or torsion. Results showed that proof load/temperature histories used in the tests have a small beneficial effect or no effect on subsequent fracture strength and flaw growth rates.

Epidemiology: second-rate science?

PubMed

Parascandola, M

1998-01-01

In recent years epidemiology has come under increasing criticism in regulatory and public arenas for being "unscientific." The tobacco industry has taken advantage of this, insisting for decades that evidence linking cigarettes and lung cancer falls short of proof. Moreover, many epidemiologists remain unduly skeptical and self-conscious about the status of their own causal claims. This situation persists in part because of a widespread belief that only the laboratory can provide evidence sufficient for scientific proof. Adherents of this view erroneously believe that there is no element of uncertainty or inductive inference in the "direct observation" of the laboratory researcher and that epidemiology provides mere "circumstantial" evidence. The historical roots of this attitude can be traced to philosopher John Stuart Mill and physiologist Claude Bernard and their influence on modern experimental thinking. The author uses the debate over cigarettes and lung cancer to examine ideas of proof in medical science and public health, concluding that inductive inference from a limited sample to a larger population is an element in all empirical science.

Investigation of thermal denaturation of solid oxytocin by terahertz dielectric spectroscopy

NASA Astrophysics Data System (ADS)

Li, Xiangjun; Yang, Xiaojie; Liu, Jianjun; Du, Yong; Hong, Zhi

2014-07-01

We investigate the thermal denaturation of solid oxytocin using terahertz time domain spectroscopy(THz-TDS). When the peptide is heated up from 25°C to 107°C and cooled down to 25°C again, an irreversible decrease in its THz absorption coefficient and refractive index is observed. The corresponding frequency-dependent permittivity during heating is fitted by the Debye model with single relaxation time. The relaxation times during temperature rising agree very well with Arrhenius equation with the activation energy of 3.12kJ/(K•mol) as an indicator for its thermal denaturation difficulty.

Isothermal assembly of DNA origami structures using denaturing agents.

PubMed

Jungmann, Ralf; Liedl, Tim; Sobey, Thomas L; Shih, William; Simmel, Friedrich C

2008-08-06

DNA origami is one of the most promising recent developments in DNA self-assembly. It allows for the construction of arbitrary nanoscale patterns and objects by folding a long viral scaffold strand using a large number of short "staple" strands. Assembly is usually accomplished by thermal annealing of the DNA molecules in buffer solution. We here demonstrate that both 2D and 3D origami structures can be assembled isothermally by annealing the DNA strands in denaturing buffer, followed by a controlled reduction of denaturant concentration. This opens up origami assembly for the integration of temperature-sensitive components.

Degradation of insulin by human fibroblasts: effects of inhibitors of pinocytosis and lysosomal activity.

PubMed

Kooistra, T; Lloyd, J B

1985-01-01

The role of the pinosome-lysosome pathway in the degradation of 125I-labelled bovine insulin by cultured human fibroblasts was examined by comparing the effects of various known inhibitors of pinocytosis and lysosomal degradation on the uptake and degradation of 125I-labelled polyvinylpyrrolidone, formaldehyde-denatured bovine serum albumin and bovine insulin by these cells. Fibroblasts incubated with polyvinylpyrrolidone steadily accumulate this substrate, whereas incubations with insulin or denatured albumin led to the progressive appearance in the culture medium of [125I]iodotyrosine. Inhibitors of pinocytosis (bacitracin, colchicine and monensin), metabolic inhibitors (2,4-dinitrophenol and NaF), lysosomotropic agents (chloroquine and NH4Cl) and an inhibitor of cysteine-proteinases (leupeptin) decreased the rate of uptake of polyvinylpyrrolidone and denatured albumin very similarly, but only bacitracin had an effect on the processing of insulin. Chloroquine, NH4Cl and leupeptin strongly inhibited the digestion of denatured albumin, but not of insulin. The different responses to the modifiers, with polyvinylpyrrolidone and denatured albumin on the one hand and insulin on the other, suggest that insulin degradation can occur by a non-lysosomal pathway. The very strong inhibitory effect of bacitracin on insulin processing by fibroblasts may point to an important role of plasma membrane proteinases in insulin degradation.

Towards a Model of Cold Denaturation of Proteins

NASA Astrophysics Data System (ADS)

Sanchez, Isaac

2010-10-01

Proteins/enzymes can undergo cold denaturation or cold deactivation. In the active or natured state, a protein exists in a unique folded/ordered state. In the deactivated (denatured) state, a protein unfolds and exists in a disordered expanded state. This protein folding/unfolding or order/disorder transition can be triggered by a temperature change. What seems paradoxical is that the active (ordered) state can be induced by heating, or equivalently, the disordered inactive state can be induced by cooling. This is equivalent to an Ising spin model passing from a disordered array of spins to an ordered array by increasing temperature! Hydrogels and their corresponding polyelectrolyte chains behave similarly, i.e., the swollen disordered state can be induced by cooling while the more ordered collapsed or globular state is induced by heating (an entropically driven phase transition). In a living cell at the physiological temperature of 37 C, activation and deactivation of proteins is triggered by local environmental changes in pH, salinity, etc. The important physics is that the denaturation temperature can be moved up or down relative to 37 C by these stimuli. Moving the transition temperature up can destabilize the active protein while moving it down leads to stabilization. An analytical polymer model will be described that exhibits cold denaturation behavior.

Experimental verification of rank 1 chaos in switch-controlled Chua circuit.

PubMed

Oksasoglu, Ali; Ozoguz, Serdar; Demirkol, Ahmet S; Akgul, Tayfun; Wang, Qiudong

2009-03-01

In this paper, we provide the first experimental proof for the existence of rank 1 chaos in the switch-controlled Chua circuit by following a step-by-step procedure given by the theory of rank 1 maps. At the center of this procedure is a periodically kicked limit cycle obtained from the unforced system. Then, this limit cycle is subjected to periodic kicks by adding externally controlled switches to the original circuit. Both the smooth nonlinearity and the piecewise linear cases are considered in this experimental investigation. Experimental results are found to be in concordance with the conclusions of the theory.

27 CFR 26.51 - Formulas for articles, eligible articles and products manufactured with denatured spirits.

Code of Federal Regulations, 2010 CFR

2010-04-01

... in the United States. (1) Products may be made with completely denatured alcohol for sale under brand... such a formula is not required to submit a new formula. (Approved by the Office of Management and...

27 CFR 20.172 - Records.

Code of Federal Regulations, 2010 CFR

2010-04-01

... TREASURY LIQUORS DISTRIBUTION AND USE OF DENATURED ALCOHOL AND RUM Operations by Dealers and Users of Specially Denatured Spirits Inventory and Records § 20.172 Records. In addition to the records required by... Office of Management and Budget under control number 1512-0337) Operations by Dealers ...

Stabilizing effect of biochar on soil extracellular enzymes after a denaturing stress

USDA-ARS?s Scientific Manuscript database

Stabilization of extracellular enzymes may maintain enzymatic activity for ecosystem services such as carbon sequestration, nutrient cycling, and bioremediation, while protecting enzymes from proteolysis and denaturation. A laboratory incubation study was conducted to determine whether a fast pyroly...

GROMOS polarizable charge-on-spring models for liquid urea: COS/U and COS/U2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin, Zhixiong; Bachmann, Stephan J.; Gunsteren, Wilfred F. van, E-mail: wfvgn@igc.phys.chem.ethz.ch

2015-03-07

Two one-site polarizable urea models, COS/U and COS/U2, based on the charge-on-spring model are proposed. The models are parametrized against thermodynamic properties of urea-water mixtures in combination with the polarizable COS/G2 and COS/D2 models for liquid water, respectively, and have the same functional form of the inter-atomic interaction function and are based on the same parameter calibration procedure and type of experimental data as used to develop the GROMOS biomolecular force field. Thermodynamic, dielectric, and dynamic properties of urea-water mixtures simulated using the polarizable models are closer to experimental data than using the non-polarizable models. The COS/U and COS/U2 modelsmore » may be used in biomolecular simulations of protein denaturation.« less

Operational evaluation of a proppeller test stand in the quiet flow facility at Langley Research Center

NASA Technical Reports Server (NTRS)

Block, P. J. W.

1982-01-01

Operational proof tests of a propeller test stand (PTS) in a quiet flow facility (QFF) are presented. The PTS is an experimental test bed for acoustic propeller research in the quiet flow environment of the QFF. These proof tests validate thrust and torque predictions, examine the repeatability of measurements on the PTS, and determine the effect of applying artificial roughness to the propeller blades. Since a thrusting propeller causes an open jet to contract, the potential flow core was surveyed to examine the magnitude of the contraction. These measurements are compared with predicted values. The predictions are used to determine operational limitations for testing a given propeller design in the QFF.

Crack Growth of D6 Steel in Air and High Pressure Oxygen

NASA Technical Reports Server (NTRS)

Bixler, W. D.; Engstrom, W. L.

1971-01-01

Fracture and subcritical flaw growth characteristics were experimentally deter­mined for electroless nickel plated D6 steel in dry air and high pressure oxygen environments as applicable to the Lunar Module/Environmental Control System (LM/ECS) descent gaseous oxygen (GOX) tank. The material tested included forgings, plate, and actual LM/ECS descent GOX tank material. Parent metal and TIG (tungsten inert gas) welds were tested. Tests indicate that proof testing the tanks at 4000 pounds per square inch or higher will insure safe operation at 3060 pounds per square inch. Although significant flaw growth can occur during proofing, subsequent growth of flaws during normal tank operation is negligible.

Low Temperature Shape Memory Alloys for Adaptive, Autonomous Systems Project

NASA Technical Reports Server (NTRS)

Falker, John; Zeitlin, Nancy; Williams, Martha; Benafan, Othmane; Fesmire, James

2015-01-01

The objective of this joint activity between Kennedy Space Center (KSC) and Glenn Research Center (GRC) is to develop and evaluate the applicability of 2-way SMAs in proof-of-concept, low-temperature adaptive autonomous systems. As part of this low technology readiness (TRL) activity, we will develop and train low-temperature novel, 2-way shape memory alloys (SMAs) with actuation temperatures ranging from 0 C to 150 C. These experimental alloys will also be preliminary tested to evaluate their performance parameters and transformation (actuation) temperatures in low- temperature or cryogenic adaptive proof-of-concept systems. The challenge will be in the development, design, and training of the alloys for 2-way actuation at those temperatures.

Evaluation of the flow properties of xanthan gum solution

DOE Office of Scientific and Technical Information (OSTI.GOV)

Duda, J.L.; Klaus, E.E.; Leung, W.C.

1981-02-01

In this study, the solution properties of two forms of xanthan gum, a powder and a broth, which are commercially available were evaluated. As previous studies have shown, the solutions prepared from the broth do exhibit better injectivity properties. However, this investigation also shows that other properties of these solutions are not equivalent. In its natural state, xanthane gum exists as a multistranded helix. This ordered confirmation can be destroyed and in a denatured state, the xanthan gum exhibits a more random configuration and consequently higher viscosity. One of the major conclusions of this study is that the xanthan powdermore » is partially denatured when compared to the xanthan molecules which exist in the broth. This denaturing may occur during the drying process in which the xanthan solids are removed from the broth. Solutions prepared from the broth in the absence of the added salt show a transition in the viscosity-temperature relationship at approximately 40 to 50/sup 0/C. This is consistent with the behavior of native xanthan gum solutions. At approximately 50/sup 0/C, the molecules in solution go into a more random state and consequently, an abrupt rise in the viscosity is observed. However, solutions prepared from the polymer powder do not show any evidence of such a transition. The solutions prepared from the broth can be thermally denatured, and this denaturing results in viscosities which are equivalent to the viscosities realized with the powdered polymer. Before denaturing, the broth solution showed a lower viscosity. Further, intrinsic viscosity measurements indicate that the hydrodynamic volume of the polymer solutions prepared from the borth are smaller than the hydrodynamic volumes of solutions prepared from the powder.« less

Reshaping the folding energy landscape by chloride salt: impact on molten-globule formation and aggregation behavior of carbonic anhydrase.

PubMed

Borén, Kristina; Grankvist, Hannah; Hammarström, Per; Carlsson, Uno

2004-05-21

During chemical denaturation different intermediate states are populated or suppressed due to the nature of the denaturant used. Chemical denaturation by guanidine-HCl (GuHCl) of human carbonic anhydrase II (HCA II) leads to a three-state unfolding process (Cm,NI=1.0 and Cm,IU=1.9 M GuHCl) with formation of an equilibrium molten-globule intermediate that is stable at moderate concentrations of the denaturant (1-2 M) with a maximum at 1.5 M GuHCl. On the contrary, urea denaturation gives rise to an apparent two-state unfolding transition (Cm=4.4 M urea). However, 8-anilino-1-naphthalene sulfonate (ANS) binding and decreased refolding capacity revealed the presence of the molten globule in the middle of the unfolding transition zone, although to a lesser extent than in GuHCl. Cross-linking studies showed the formation of moderate oligomer sized (300 kDa) and large soluble aggregates (>1000 kDa). Inclusion of 1.5 M NaCl to the urea denaturant to mimic the ionic character of GuHCl leads to a three-state unfolding behavior (Cm,NI=3.0 and Cm,IU=6.4 M urea) with a significantly stabilized molten-globule intermediate by the chloride salt. Comparisons between NaCl and LiCl of the impact on the stability of the various states of HCA II in urea showed that the effects followed what could be expected from the Hofmeister series, where Li+ is a chaotropic ion leading to decreased stability of the native state. Salt addition to the completely urea unfolded HCA II also led to an aggregation prone unfolded state, that has not been observed before for carbonic anhydrase. Refolding from this state only provided low recoveries of native enzyme.

The effects of hyaluronic acid incorporated as a wetting agent on lysozyme denaturation in model contact lens materials.

PubMed

Weeks, Andrea; Boone, Adrienne; Luensmann, Doerte; Jones, Lyndon; Sheardown, Heather

2013-09-01

Conventional and silicone hydrogels as models for contact lenses were prepared to determine the effect of the presence of hyaluronic acid on lysozyme sorption and denaturation. Hyaluronic acid was loaded into poly(2-hydroxyethyl methacrylate) and poly(2-hydroxyethyl methacrylate)/TRIS--methacryloxypropyltris (trimethylsiloxy silane) hydrogels, which served as models for conventional and silicone hydrogel contact lens materials. The hyaluronic acid was cross-linked using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide in the presence of dendrimers. Active lysozyme was quantified using a Micrococcus lysodeikticus assay while total lysozyme was determined using 125-I radiolabeled protein. To examine the location of hyaluronic acid in the gels, 6-aminofluorescein labeled hyaluronic acid was incorporated into the gels using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide chemistry and the gels were examined using confocal laser scanning microscopy. Hyaluronic acid incorporation significantly reduced lysozyme sorption in poly(2-hydroxyethyl methacrylate) (p < 0.00001) and poly(2-hydroxyethyl methacrylate)/TRIS--methacryloxypropyltris (trimethylsiloxy silane) (p < 0.001) hydrogels, with the modified materials sorbing only 20% and 16% that of the control, respectively. More importantly, hyaluronic acid also decreased lysozyme denaturation in poly(2-hydroxyethyl methacrylate) (p < 0.005) and poly(2-hydroxyethyl methacrylate)/TRIS--methacryloxypropyltris (trimethylsiloxy silane) (p < 0.02) hydrogels. The confocal laser scanning microscopy results showed that the hyaluronic acid distribution was dependent on both the material type and the molecular weight of hyaluronic acid. This study demonstrates that hyaluronic acid incorporated as a wetting agent has the potential to reduce lysozyme sorption and denaturation in contact lens applications. The distribution of hyaluronic acid within hydrogels appears to affect denaturation, with more surface mobile, lower molecular weight hyaluronic acid being more effective in preventing denaturation.

Physical interaction between bacterial heat shock protein (Hsp) 90 and Hsp70 chaperones mediates their cooperative action to refold denatured proteins.

PubMed

Nakamoto, Hitoshi; Fujita, Kensaku; Ohtaki, Aguru; Watanabe, Satoru; Narumi, Shoichi; Maruyama, Takahiro; Suenaga, Emi; Misono, Tomoko S; Kumar, Penmetcha K R; Goloubinoff, Pierre; Yoshikawa, Hirofumi

2014-02-28

In eukaryotes, heat shock protein 90 (Hsp90) is an essential ATP-dependent molecular chaperone that associates with numerous client proteins. HtpG, a prokaryotic homolog of Hsp90, is essential for thermotolerance in cyanobacteria, and in vitro it suppresses the aggregation of denatured proteins efficiently. Understanding how the non-native client proteins bound to HtpG refold is of central importance to comprehend the essential role of HtpG under stress. Here, we demonstrate by yeast two-hybrid method, immunoprecipitation assays, and surface plasmon resonance techniques that HtpG physically interacts with DnaJ2 and DnaK2. DnaJ2, which belongs to the type II J-protein family, bound DnaK2 or HtpG with submicromolar affinity, and HtpG bound DnaK2 with micromolar affinity. Not only DnaJ2 but also HtpG enhanced the ATP hydrolysis by DnaK2. Although assisted by the DnaK2 chaperone system, HtpG enhanced native refolding of urea-denatured lactate dehydrogenase and heat-denatured glucose-6-phosphate dehydrogenase. HtpG did not substitute for DnaJ2 or GrpE in the DnaK2-assisted refolding of the denatured substrates. The heat-denatured malate dehydrogenase that did not refold by the assistance of the DnaK2 chaperone system alone was trapped by HtpG first and then transferred to DnaK2 where it refolded. Dissociation of substrates from HtpG was either ATP-dependent or -independent depending on the substrate, indicating the presence of two mechanisms of cooperative action between the HtpG and the DnaK2 chaperone system.

Compact high-power microwave divider and combiner.

PubMed

Guo, L T; Chang, C; Huang, W H; Liu, Y S; Cao, Y B; Liu, C L; Sun, J

2016-02-01

A novel, compact, TM01-TE10 mode power divider and a novel, compact, four-way TE10-TM01 mode power combiner were theoretically designed and experimentally tested as a proof of principle. The theoretical and experimental S parameters are consistent with each other. High-power experiments show that their power capacities are no less than 1.5 GW and 3 GW, respectively. The devices have the merits of high power capacities and low insertion losses.

40 CFR 80.1650 - Registration.

Code of Federal Regulations, 2014 CFR

2014-07-01

... the first date that such person will blend oxygenate into RBOB, whichever is earlier. (4) Any ethanol... advance of the first date that such person will produce or import ethanol denaturant, whichever is earlier... inaccurate. (h) Certified ethanol denaturant producer registration. (1) Registration shall be on forms and...

40 CFR 80.1665 - Penalties.

Code of Federal Regulations, 2014 CFR

2014-07-01

... system. (2) Any person liable under § 80.1662(a)(6) for causing gasoline, oxygenate, or ethanol... the non-complying gasoline, oxygenate, or ethanol denaturant remains any place in the gasoline, oxygenate, or ethanol denaturant distribution system. (3) For purposes of this paragraph (c), the length of...

Protein sterilization method of firefly luciferase using reduced pressure and molecular sieves

NASA Technical Reports Server (NTRS)

Chappelle, E. W.; Rich, E., Jr. (Inventor)

1973-01-01

The sterilization of the protein fruitfly luciferase under conditions that prevent denaturation is examined. Denaturation is prevented by heating the protein in contact with molecular seives and under a reduced pressure of the order of 0.00005 millimeters of mercury.

Collective hydration dynamics of guanidinium chloride solutions and its possible role in protein denaturation: a terahertz spectroscopic study.

PubMed

Samanta, Nirnay; Mahanta, Debasish Das; Mitra, Rajib Kumar

2014-11-14

The remarkable ability of guanidinium chloride (GdmCl) to denature proteins is a well studied yet controversial phenomenon; the exact molecular mechanism is still debatable, especially the role of hydration dynamics, which has been paid less attention. In the present contribution, we have addressed the issue of whether the collective hydrogen bond dynamics of water gets perturbed in the presence of GdmCl and its possible impact on the denaturation of a globular protein human serum albumin (HSA), using terahertz (THz) time domain spectroscopy (TTDS) in the frequency range of 0.3-2.0 THz. The collective hydrogen bond dynamics is determined by fitting the obtained complex dielectric response in a multiple Debye relaxation model. To compare the results, the studies were extended to two more salts: tetramethylguanidinium chloride (TMGdmCl) and sodium chloride (NaCl). It was concluded that the change in hydration dynamics plays a definite role in the protein denaturation process.

Intrinsic alterations in the partial molar volume on the protein denaturation: surficial Kirkwood-Buff approach.

PubMed

Yu, Isseki; Takayanagi, Masayoshi; Nagaoka, Masataka

2009-03-19

The partial molar volume (PMV) of the protein chymotrypsin inhibitor 2 (CI2) was calculated by all-atom MD simulation. Denatured CI2 showed almost the same average PMV value as that of native CI2. This is consistent with the phenomenological question of the protein volume paradox. Furthermore, using the surficial Kirkwood-Buff approach, spatial distributions of PMV were analyzed as a function of the distance from the CI2 surface. The profiles of the new R-dependent PMV indicate that, in denatured CI2, the reduction in the solvent electrostatic interaction volume is canceled out mainly by an increment in thermal volume in the vicinity of its surface. In addition, the PMV of the denatured CI2 was found to increase in the region in which the number density of water atoms is minimum. These results provide a direct and detailed picture of the mechanism of the protein volume paradox suggested by Chalikian et al.

Interchange reaction of disulfides and denaturation of oxytocin by copper(II)/ascorbic acid/O2 system.

PubMed

Inoue, H; Hirobe, M

1987-05-29

The interchange reaction of disulfides was caused by the copper(II)/ascorbic acid/O2 system. The incubation of two symmetric disulfides, L-cystinyl-bis-L-phenylalanine (PP) and L-cystinyl-bis-L-tyrosine (TT), with L-ascorbic acid and CuSO4 in potassium phosphate buffer (pH 7.2, 50 mM) resulted in the formation of an asymmetric disulfide, L-cystinyl-L-phenylalanine-L-tyrosine (PT), and the final ratio of PP:PT:TT was 1:2:1. As the reaction was inhibited by catalase and DMSO only at the initial time, hydroxyl radical generated by the copper(II)/ascorbic acid/O2 system seemed to be responsible for the initiation of the reaction. Oxytocin and insulin were denatured by this system, and catalase and DMSO similarly inhibited these denaturations. As the composition of amino acids was unchanged after the reaction, hydroxyl radical was thought to cause the cleavage and/or interchange reaction of disulfides to denature the peptides.

COLD-PCR: improving the sensitivity of molecular diagnostics assays

PubMed Central

Milbury, Coren A; Li, Jin; Liu, Pingfang; Makrigiorgos, G Mike

2011-01-01

The detection of low-abundance DNA variants or mutations is of particular interest to medical diagnostics, individualized patient treatment and cancer prognosis; however, detection sensitivity for low-abundance variants is a pronounced limitation of most currently available molecular assays. We have recently developed coamplification at lower denaturation temperature-PCR (COLD-PCR) to resolve this limitation. This novel form of PCR selectively amplifies low-abundance DNA variants from mixtures of wild-type and mutant-containing (or variant-containing) sequences, irrespective of the mutation type or position on the amplicon, by using a critical denaturation temperature. The use of a lower denaturation temperature in COLD-PCR results in selective denaturation of amplicons with mutation-containing molecules within wild-type mutant heteroduplexes or with a lower melting temperature. COLD-PCR can be used in lieu of conventional PCR in several molecular applications, thus enriching the mutant fraction and improving the sensitivity of downstream mutation detection by up to 100-fold. PMID:21405967

Signal Peptide and Denaturing Temperature are Critical Factors for Efficient Mammalian Expression and Immunoblotting of Cannabinoid Receptors*

PubMed Central

WANG, Chenyun; WANG, Yingying; WANG, Miao; CHEN, Jiankui; YU, Nong; SONG, Shiping; KAMINSKI, Norbert E.; ZHANG, Wei

2013-01-01

Summary Many researchers employed mammalian expression system to artificially express cannabinoid receptors, but immunoblot data that directly prove efficient protein expression can hardly be seen in related research reports. In present study, we demonstrated cannabinoid receptor protein was not able to be properly expressed with routine mammalian expression system. This inefficient expression was rescued by endowing an exogenous signal peptide ahead of cannabinoid receptor peptide. In addition, the artificially synthesized cannabinoid receptor was found to aggregate under routine sample denaturing temperatures (i.e., ≥95°C), forming a large molecular weight band when analyzed by immunoblotting. Only denaturing temperatures ≤75°C yielded a clear band at the predicted molecular weight. Collectively, we showed that efficient mammalian expression of cannabinoid receptors need a signal peptide sequence, and described the requirement for a low sample denaturing temperature in immunoblot analysis. These findings provide very useful information for efficient mammalian expression and immunoblotting of membrane receptors. PMID:22528237

Stability, denaturation and refolding of Mycobacterium tuberculosis MfpA, a DNA mimicking protein that confers antibiotic resistance

PubMed Central

Khrapunov, Sergei; Brenowitz, Michael

2011-01-01

MfpA from Mycobacterium tuberculosis is a founding member of the pentapeptide repeat class of proteins (PRP) that is believed to confer bacterial resistance to the drug fluoroquinolone by mimicking the size, shape and surface charge of duplex DNA. We show that phenylalanine side chain stacking stabilizes the N-terminus of MfpA’s pentapeptide thus extending the DNA mimicry analogy. The Lumry-Eyring model was applied to multiple spectral measures of MfpA denaturation revealing that the MfpA dimer dissociates to monomers which undergo a structural transition that leads to aggregation. MfpA retains high secondary and tertiary structure content under denaturing conditions. Dimerization stabilizes MfpA’s pentapeptide repeat fold. The high Arrhenius activation energy of the barrier to aggregate formation rationalizes its stability. The mechanism of MfpA denaturation and refolding is a ‘double funnel’ energy landscape where the ‘native’ and ‘aggregate’ funnels are separated by the high barrier that is not overcome during in vitro refolding. PMID:21605934

Kinetic study of the thermal denaturation of a hyperthermostable extracellular α-amylase from Pyrococcus furiosus.

PubMed

Brown, I; Dafforn, T R; Fryer, P J; Cox, P W

2013-12-01

Hyperthermophilic enzymes are of industrial importance and interest, especially due to their denaturation kinetics at commercial sterilisation temperatures inside safety indicating time-temperature integrators (TTIs). The thermal stability and irreversible thermal inactivation of native extracellular Pyrococcus furiosus α-amylase were investigated using differential scanning calorimetry, circular dichroism and Fourier transform infrared spectroscopy. Denaturation of the amylase was irreversible above a Tm of approximately 106°C and could be described by a one-step irreversible model. The activation energy at 121°C was found to be 316kJ/mol. Using CD and FT-IR spectroscopy it was shown that folding and stability greatly increase with temperature. Under an isothermal holding temperature of 121°C, the structure of the PFA changes during denaturation from an α-helical structure, through a β-sheet structure to an aggregated protein. Such data reinforces the use of P. furiosus α-amylase as a labile species in TTIs. © 2013.

Protein renaturation by the liquid organic salt ethylammonium nitrate.

PubMed Central

Summers, C. A.; Flowers, R. A.

2000-01-01

The room-temperature liquid salt, ethylammonium nitrate (EAN), has been used to enhance the recovery of denatured-reduced hen egg white lysozyme (HEWL). Our results show that EAN has the ability to prevent aggregation of the denatured protein. The use of EAN as a refolding additive is advantageous because the renaturation is a one-step process. When HEWL was denatured reduced using routine procedures and renatured using EAN as an additive, HEWL was found to regain 75% of its activity. When HEWL was denatured and reduced in neat EAN, dilution resulted in over 90% recovery of active protein. An important aspect of this process is that renaturation of HEWL occurs at concentrations of 1.6 mg/mL, whereas other renaturation processes occur at significantly lower protein concentrations. Additionally, the refolded-active protein can be separated from the molten salt by simple desalting methods. Although the use of a low-temperature molten salt in protein renaturation is unconventional, the power of this approach lies in its simplicity and utility. PMID:11106174

AFM visualization at a single-molecule level of denaturated states of proteins on graphite.

PubMed

Barinov, Nikolay A; Prokhorov, Valery V; Dubrovin, Evgeniy V; Klinov, Dmitry V

2016-10-01

Different graphitic materials are either already used or believed to be advantageous in biomedical and biotechnological applications, e.g., as biomaterials or substrates for sensors. Most of these applications or associated important issues, such as biocompatibility, address the problem of adsorption of protein molecules and, in particular the conformational state of the adsorbed protein molecule on graphite. High-resolution AFM demonstrates highly oriented pyrolytic graphite (HOPG) induced denaturation of four proteins of blood plasma, such as ferritin, fibrinogen, human serum albumin (HSA) and immunoglobulin G (IgG), at a single molecule level. Protein denaturation is accompanied by the decrease of the heights of protein globules and spreading of the denatured protein fraction on the surface. In contrast, the modification of HOPG with the amphiphilic oligoglycine-hydrocarbon derivative monolayer preserves the native-like conformation and provides even more mild conditions for the protein adsorption than typically used mica. Protein unfolding on HOPG may have universal character for "soft" globular proteins. Copyright © 2016 Elsevier B.V. All rights reserved.

Effect of osmolytes on the thermal stability of proteins: replica exchange simulations of Trp-cage in urea and betaine solutions.

PubMed

Adamczak, Beata; Kogut, Mateusz; Czub, Jacek

2018-04-25

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. In contrast, betaine is sterically excluded from the protein surface thereby exerting entropic depletion forces that contribute to the stabilization of the native state. In fact, we find that while at low temperatures betaine slightly increases the folding free energy of Trp-cage by promoting another near-native conformation, it protects the protein against temperature-induced denaturation. This, in turn, can be attributed to enhanced exclusion of betaine at higher temperatures that arises from less attractive interactions with the protein surface.

Structural changes induced by high-pressure processing in micellar casein and milk protein concentrates.

PubMed

Cadesky, Lee; Walkling-Ribeiro, Markus; Kriner, Kyle T; Karwe, Mukund V; Moraru, Carmen I

2017-09-01

Reconstituted micellar casein concentrates and milk protein concentrates of 2.5 and 10% (wt/vol) protein concentration were subjected to high-pressure processing at pressures from 150 to 450 MPa, for 15 min, at ambient temperature. The structural changes induced in milk proteins by high-pressure processing were investigated using a range of physical, physicochemical, and chemical methods, including dynamic light scattering, rheology, mid-infrared spectroscopy, scanning electron microscopy, proteomics, and soluble mineral analyses. The experimental data clearly indicate pressure-induced changes of casein micelles, as well as denaturation of serum proteins. Calcium-binding α S1 - and α S2 -casein levels increased in the soluble phase after all pressure treatments. Pressurization up to 350 MPa also increased levels of soluble calcium and phosphorus, in all samples and concentrations, whereas treatment at 450 MPa reduced the levels of soluble Ca and P. Experimental data suggest dissociation of calcium phosphate and subsequent casein micelle destabilization as a result of pressure treatment. Treatment of 10% micellar casein concentrate and 10% milk protein concentrate samples at 450 MPa resulted in weak, physical gels, which featured aggregates of uniformly distributed, casein substructures of 15 to 20 nm in diameter. Serum proteins were significantly denatured by pressures above 250 MPa. These results provide information on pressure-induced changes in high-concentration protein systems, and may inform the development on new milk protein-based foods with novel textures and potentially high nutritional quality, of particular interest being the soft gel structures formed at high pressure levels. The Authors. Published by the Federation of Animal Science Societies and Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

Isothermal DNA origami folding: avoiding denaturing conditions for one-pot, hybrid-component annealing

NASA Astrophysics Data System (ADS)

Kopielski, Andreas; Schneider, Anne; Csáki, Andrea; Fritzsche, Wolfgang

2015-01-01

The DNA origami technique offers great potential for nanotechnology. Using biomolecular self-assembly, defined 2D and 3D nanoscale DNA structures can be realized. DNA origami allows the positioning of proteins, fluorophores or nanoparticles with an accuracy of a few nanometers and enables thereby novel nanoscale devices. Origami assembly usually includes a thermal denaturation step at 90 °C. Additional components used for nanoscale assembly (such as proteins) are often thermosensitive, and possibly damaged by such harsh conditions. They have therefore to be attached in an extra second step to avoid defects. To enable a streamlined one-step nanoscale synthesis - a so called one-pot folding - an adaptation of the folding procedures is required. Here we present a thermal optimization of this process for a 2D DNA rectangle-shaped origami resulting in an isothermal assembly protocol below 60 °C without thermal denaturation. Moreover, a room temperature protocol is presented using the chemical additive betaine, which is biocompatible in contrast to chemical denaturing approaches reported previously.The DNA origami technique offers great potential for nanotechnology. Using biomolecular self-assembly, defined 2D and 3D nanoscale DNA structures can be realized. DNA origami allows the positioning of proteins, fluorophores or nanoparticles with an accuracy of a few nanometers and enables thereby novel nanoscale devices. Origami assembly usually includes a thermal denaturation step at 90 °C. Additional components used for nanoscale assembly (such as proteins) are often thermosensitive, and possibly damaged by such harsh conditions. They have therefore to be attached in an extra second step to avoid defects. To enable a streamlined one-step nanoscale synthesis - a so called one-pot folding - an adaptation of the folding procedures is required. Here we present a thermal optimization of this process for a 2D DNA rectangle-shaped origami resulting in an isothermal assembly protocol below 60 °C without thermal denaturation. Moreover, a room temperature protocol is presented using the chemical additive betaine, which is biocompatible in contrast to chemical denaturing approaches reported previously. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr04176c

Thermal and Denaturation Studies of the Time-Resolved Fluorescence Decay of Human Superoxide Dismutase

NASA Astrophysics Data System (ADS)

Silva, Norberto De Jesus

Previous studies have shown that time-resolved fluorescence decay of various single tryptophan proteins is best described by a distribution of fluorescence lifetimes rather than one or two lifetimes. The thermal dependence of the lifetime distributions is consistent with the hypothesis that proteins fluctuate between a hierarchy of many conformational substates. With this scenario as a theoretical framework, the correlations between protein dynamic and structure are investigated by studying the time-resolved fluorescence and anisotropy decay of the single tryptophan (Trp) residue of human superoxide dismutase (HSOD) over a wide range of temperatures and at different denaturant concentrations. First, it is demonstrated that the center of the lifetime distribution can characterize the average deactivation environment of the excited Trp-protein system. A qualitative model is introduced to explain the time-resolved fluorescence decay of HSOD in 80% glycerol over a wide range of temperatures. The dynamical model features isoenergetic conformational substates separated by a hierarchy of energy barriers. The HSOD system is also investigated as a function of denaturant concentration in aqueous solution. As a function of guanidine hydrochloride (GdHCl), the width of the fluorescence lifetime distribution of HSOD displays a maximum which is not coincident with the fully denatured form of HSOD at 6.5M GdHCl. Furthermore, the width for the fully denatured form of HSOD is greater than that of the native form. This is consistent with the scenario that more conformational substates are being created upon denaturation of HSOD. HSOD is a dimeric protein and it was observed that the width of the lifetime distribution of HSOD at intermediate GdHCl concentrations increased with decreasing protein concentration. In addition, the secondary structure of HSOD at intermediate GdHCl concentration does not change with protein concentration. These results suggest that HSOD display structural microheterogeneity which is consistent with the hypothesis of conformational substates. Further analysis show that, during denaturation, the monomeric form of HSOD is an intermediate which displays native-like secondary structure and fluctuating tertiary structure; i.e., the monomeric form of HSOD is a molten globule.

Denaturation of Proteins by SDS and by Tetra-alkylammonium Dodecyl Sulfates

PubMed Central

Lee, Andrew; Tang, Sindy K. Y.; Mace, Charles R.

2011-01-01

This paper describes the use of capillary electrophoresis (CE) to examine the influence of different cations (C+; C+ = Na+ and tetra-n-alkylammonium, NR4 +, where R = Me, Et, Pr, Bu) on the rates of denaturation of bovine carbonic anhydrase II (BCA) in the presence of the anionic surfactant dodecylsulfate (DS−). Analysis of the denaturation of BCA in solutions of Na+DS− and NR4 +DS− (in Tris-Gly buffer) indicated that the rates of formation of complexes of denatured BCA with DS− (BCAD-DS−n,sat) are indistinguishable and independent of the cation below the critical micellar concentration (cmc), and independent of the total concentration of DS− above the cmc. At concentrations of C+DS− above the cmc, BCA denatured with rates that depended on the cation; the rates decreased by a factor > 104, in the order Na+ ~ NMe4 + > NEt4 + > NPr4 + > NBu4 + – the same order as the values of cmc (which decrease from 4.0 mM for Na+DS− to 0.9 mM for NBu4 +DS− in Tris-Gly buffer). The relationship between values of cmc and rates of formation of BCAD-DS−n,sat suggested that the kinetics of denaturation of BCA involve the association of this protein with monomeric DS−, rather than with micelles of (C+DS−)n. A less-detailed survey of seven other proteins (α-lactalbumin, β-lactoglobulin A, β-lactoglobulin B, carboxypeptidase B, creatine phosphokinase, myoglobin, and ubiquitin) showed that the difference between Na+DS− and NR4 +DS− observed with BCA was not general. Instead, the influence of NR4 + on the association of DS− with these proteins depended on the protein. The selection of cation contributed to the properties (including composition, electrophoretic mobility, and partitioning behavior in aqueous two-phase systems) of aggregates of denatured protein and DS−. These results suggest that variation in the behavior of NR4 +DS− with changes in R may be exploited in methods for analyzing and separating mixtures of proteins. PMID:21834533

Hemoglobin stability: observations on the denaturation of normal and abnormal hemoglobins by oxidant dyes, heat, and alkali

PubMed Central

Rieder, Ronald F.

1970-01-01

Several unstable mutant hemoglobins have alterations which affect areas of the molecule involved in the attachment of heme to globin. Loss of heme from globin has been demonstrated during the denaturation of some of these unstable mutants. The importance of heme ligands for the stability of hemoglobin was illustrated in the present experiments on the denaturation of several hemoglobins and hemoglobin derivatives by heat, oxidative dyes, and alkali. Heating of normal hemolysates diluted to 4 g of hemoglobin per 100 ml at 50°C for 20 hr in 0.05 M sodium phosphate, pH 7.4, caused precipitation of 23-54% of the hemoglobin. Dialysis against water or dilution of the sample decreased denaturation to 12-20%. Precipitation was decreased to less than 3.5% by the presence of 0.015 M potassium cyanide. Increasing the ionic strength of the medium increased precipitation. Cyanide prevented the formation of inclusion bodies when red cells containing unstable hemoglobin Philly, β35 tyr → phe, were incubated with the redox dye new methylene blue. Conversion to methemoglobin increased the rate of alkali denaturation of hemoglobin but the presence of potassium cyanide returned the denaturation rate to that of ferrohemoglobin. The ability of cyanide to decrease heat precipitation of hemoglobin may depend on a dimeric or tetrameric state of the hemoglobin molecule. Purified β-chains, which exist as tetramers, were stabilized but purified monomeric α-chains were not rendered more heat resistant by the ligand. Stabilization of hemoglobin by cyanide required binding of the ligand to only one heme of an αβ-dimer. Hemoglobin Gun Hill, an unstable molecule with heme groups present only on the α-chains was quite heat stable in the presence of cyanide. The binding of cyanide to the iron atom in methemoglobin is thought to be associated with increased planarity of the heme group and increased stability of the heme-globin complex. The stabilizing effect of cyanide in the above experiments suggests that Heinz body formation, heat precipitation of hemoglobin, and the increased alkali denaturation of methemoglobin depend on changes of heme-globin binding. Images PMID:5480860

Denaturation of proteins by SDS and tetraalkylammonium dodecyl sulfates.

PubMed

Lee, Andrew; Tang, Sindy K Y; Mace, Charles R; Whitesides, George M

2011-09-20

This article describes the use of capillary electrophoresis (CE) to examine the influence of different cations (C(+); C(+) = Na(+) and tetra-n-alkylammonium, NR(4)(+), where R = Me, Et, Pr, and Bu) on the rates of denaturation of bovine carbonic anhydrase II (BCA) in the presence of anionic surfactant dodecylsulfate (DS(-)). An analysis of the denaturation of BCA in solutions of Na(+)DS(-) and NR(4)(+)DS(-) (in Tris-Gly buffer) indicated that the rates of formation of complexes of denatured BCA with DS(-) (BCA(D)-DS(-)(n,sat)) are indistinguishable and independent of the cation below the critical micellar concentration (cmc) and independent of the total concentration of DS(-) above the cmc. At concentrations of C(+)DS(-) above the cmc, BCA denatured at rates that depended on the cation; the rates decreased by a factor >10(4) in the order of Na(+) ≈ NMe(4)(+) > NEt(4)(+) > NPr(4)(+) > NBu(4)(+), which is the same order as the values of the cmc (which decrease from 4.0 mM for Na(+)DS(-) to 0.9 mM for NBu(4)(+)DS(-) in Tris-Gly buffer). The relationship between the cmc values and the rates of formation of BCA(D)-DS(-)(n,sat()) suggested that the kinetics of denaturation of BCA involve the association of this protein with monomeric DS(-) rather than with micelles of (C(+)DS(-))(n). A less-detailed survey of seven other proteins (α-lactalbumin, β-lactoglobulin A, β-lactoglobulin B, carboxypeptidase B, creatine phosphokinase, myoglobin, and ubiquitin) showed that the difference between Na(+)DS(-) and NR(4)(+)DS(-) observed with BCA was not general. Instead, the influence of NR(4)(+) on the association of DS(-) with these proteins depended on the protein. The selection of the cation contributed to the properties (including the composition, electrophoretic mobility, and partitioning behavior in aqueous two-phase systems) of aggregates of denatured protein and DS(-). These results suggest that the variation in the behavior of NR(4)(+)DS(-) with changes in R may be exploited in methods used to analyze and separate mixtures of proteins. © 2011 American Chemical Society

Development of a multiphysics model to characterize the responsive behavior of urea-sensitive hydrogel as biosensor.

PubMed

Goh, K B; Li, Hua; Lam, K Y

2017-05-15

A remarkable feature of biomaterials is their ability to deform in response to certain external bio-stimuli. Here, a novel biochemo-electro-mechanical model is developed for the numerical characterization of the urea-sensitive hydrogel in response to the external stimulus of urea. The urea sensitivity of the hydrogel is usually characterized by the states of ionization and denaturation of the immobilized urease, as such the model includes the effect of the fixed charge groups and temperature coupled with pH on the activity of the urease. Therefore, a novel rate of reaction equation is proposed to characterize the hydrolysis of urea that accounts for both the ionization and denaturation states of the urease subject to the environmental conditions. After examination with the published experimental data, it is thus confirmed that the model can characterize well the responsive behavior of the urea-sensitive hydrogel subject to the urea stimulus, including the distribution patterns of the electrical potential and pH of the hydrogel. The results point to an innovative means for generating electrical power via the enzyme-induced pH and electrical potential gradients, when the hydrogel comes in contact with the urea-rich solution, such as human urine. Copyright © 2017 Elsevier B.V. All rights reserved.

Aqueous TMAO solutions as seen by theoretical THz spectroscopy: hydrophilic versus hydrophobic water.

PubMed

Imoto, Sho; Forbert, Harald; Marx, Dominik

2018-02-28

Solvation of trimethylamine-N-oxide (TMAO) by water is of great fundamental interest because this small molecule has both strongly hydrophilic and large hydrophobic groups at its opposite ends and, furthermore, stabilizes proteins against temperature and pressure denaturation. Since hydrophilic and hydrophobic groups affect the structural dynamics of the respective solvation water molecules in vastly different ways, we dissect their distinct influences on the THz spectrum of TMAO(aq) by using ab initio molecular dynamics simulations. In particular, we demonstrate that exclusively electronic polarization and charge transfer effects, being absent in the usual fixed-charge biomolecular force fields, are responsible for the significant enhancement of the effective molecular dipole moment of hydrophilic solvation water. This, in turn, leads to pronounced solute-solvent couplings and thus to specific THz modes that involve well-defined H-bond bending and stretching motion being characteristic to hydrophilic solvation. The THz response of individual H-bonded pairs of water molecules involving hydrophobic solvation water, in stark contrast, is nearly indistinguishable from such pairs in bulk water. Transcending the specific case, THz spectroscopy is suggested to be an ideal experimental approach to unravel the controversial piezolytic properties of TMAO including its counteracting effect on pressure-induced denaturation of proteins.

Interaction of proteins with ionic liquid, alcohol and DMSO and in situ generation of gold nano-clusters in a cell.

PubMed

Nandi, Somen; Parui, Sridip; Halder, Ritaban; Jana, Biman; Bhattacharyya, Kankan

2018-06-01

In this review, we give a brief overview on how the interaction of proteins with ionic liquids, alcohols and dimethyl sulfoxide (DMSO) influences the stability, conformational dynamics and function of proteins/enzymes. We present experimental results obtained from fluorescence correlation spectroscopy on the effect of ionic liquid or alcohol or DMSO on the size (more precisely, the diffusion constant) and conformational dynamics of lysozyme, cytochrome c and human serum albumin in aqueous solution. The interaction of ionic liquid with biomolecules (e.g. protein, DNA etc.) has emerged as a current frontier. We demonstrate that ionic liquids are excellent stabilizers of protein and DNA and, in some cases, cause refolding of a protein already denatured by chemical denaturing agents. We show that in ethanol-water binary mixture, proteins undergo non-monotonic changes in size and dynamics with increasing ethanol content. We also discuss the effect of water-DMSO mixture on the stability of proteins. We demonstrate how large-scale molecular dynamics simulations have revealed the molecular origin of this observed phenomenon and provide a microscopic picture of the immediate environment of the biomolecules. Finally, we describe how favorable interactions of ionic liquids may be utilized for in situ generation of fluorescent gold nano-clusters for imaging a live cell.

Folding free-energy landscape of villin headpiece subdomain from molecular dynamics simulations.

PubMed

Lei, Hongxing; Wu, Chun; Liu, Haiguang; Duan, Yong

2007-03-20

High-accuracy ab initio folding has remained an elusive objective despite decades of effort. To explore the folding landscape of villin headpiece subdomain HP35, we conducted two sets of replica exchange molecular dynamics for 200 ns each and three sets of conventional microsecond-long molecular dynamics simulations, using AMBER FF03 force field and a generalized-Born solvation model. The protein folded consistently to the native state; the lowest C(alpha)-rmsd from the x-ray structure was 0.46 A, and the C(alpha)- rmsd of the center of the most populated cluster was 1.78 A at 300 K. ab initio simulations have previously not reached this level. The folding landscape of HP35 can be partitioned into the native, denatured, and two intermediate-state regions. The native state is separated from the major folding intermediate state by a small barrier, whereas a large barrier exists between the major folding intermediate and the denatured states. The melting temperature T(m) = 339 K extracted from the heat-capacity profile was in close agreement with the experimentally derived T(m) = 342 K. A comprehensive picture of the kinetics and thermodynamics of HP35 folding emerges when the results from replica exchange and conventional molecular dynamics simulations are combined.

The Experimental Mathematician: The Pleasure of Discovery and the Role of Proof

ERIC Educational Resources Information Center

Borwein, Jonathan M.

2005-01-01

The emergence of powerful mathematical computing environments, the growing availability of correspondingly powerful (multi-processor) computers and the pervasive presence of the Internet allow for mathematicians, students and teachers, to proceed heuristically and "quasi-inductively." We may increasingly use symbolic and numeric computation,…

Raman imaging using fixed bandpass filter

NASA Astrophysics Data System (ADS)

Landström, L.; Kullander, F.; Lundén, H.; Wästerby, P.

2017-05-01

By using fixed narrow band pass optical filtering and scanning the laser excitation wavelength, hyperspectral Raman imaging could be achieved. Experimental, proof-of-principle results from the Chemical Warfare Agent (CWA) tabun (GA) as well as the common CWA simulant tributyl phosphate (TBP) on different surfaces/substrates are presented and discussed.

46 CFR 56.20-1 - General.

Code of Federal Regulations, 2013 CFR

2013-10-01

... accordance with § 56.60-1 of this part. (2) Valves must be designed so that the maximum allowable working... permits this; (ii) Subjecting a representative model to a proof test or experimental stress analysis... uncontrollable rise in pressure can result, means must be provided in the design, installation, and operation of...

Pupils' Cognitive Activity Stimulation by Means of Physical Training

ERIC Educational Resources Information Center

Nekhoroshkov, Anatolij V.

2016-01-01

The article presents the research results of the physical activity influence on the intellectual performance of high school students. The methods of experiments and standardized observation were used. The efficiency of the cognitive activity was assessed by "Proof test" technique of B. Burdon. Within the experimental class, the program…

Intrinsic Fluorescence as a Spectral Probe for Protein Denaturation Studies in the Presence of Honey

NASA Astrophysics Data System (ADS)

Wong, Y. H.; Kadir, H. A.; Tayyab, S.

2015-11-01

Honey was found to quench the intrinsic fluorescence of bovine serum albumin (BSA) in a concentration dependent manner, showing complete quenching in the presence of 5% (w/v) honey. Increasing the protein concentration up to 5.0 μM did not lead to the recovery of the protein fluorescence. Urea denaturation of BSA, which otherwise shows a two-step, three-state transition, using intrinsic fluorescence of the protein as the probe failed to produce any result in the presence of 5% (w/v) honey. Thus, intrinsic fluorescence cannot be used as a spectral probe for protein denaturation studies in the presence of honey.

Functional screening of pharmacological chaperones via restoration of enzyme activity upon denaturation.

PubMed

Shanmuganathan, Meera; Britz-McKibbin, Philip

2012-10-02

Pharmacological chaperones (PCs) are small molecules that stabilize and promote protein folding. Enzyme inhibition is widely used for PC selection; however, it does not accurately reflect chaperone activity. We introduce a functional assay for characterization of PCs based on their capacity to restore enzyme activity that is abolished upon chemical denaturation. Dose-dependent activity curves were performed as a function of urea to assess the chaperone potency of various ligands to β-glucocerebrosidase as a model system. Restoration of enzyme activity upon denaturation allows direct screening of PCs for treatment of genetic disorders associated with protein deficiency, such as Gaucher disease.

Magnetic Guarding: Experimental and Numerical Results

NASA Astrophysics Data System (ADS)

Heinrich, Jonathon; Font, Gabriel; Garrett, Michael; Rose, D.; Genoni, T.; Welch, D.; McGuire, Thomas

2017-10-01

The magnetic field topology of Lockheed Martin's Compact Fusion Reactor (CFR) concept requires internal magnetic field coils. Internal coils for similar devices have leveraged levitating coils or coils with magnetically guarded supports. Magnetic guarding of supports has been investigated for multipole devices (theoretically and experimentally) without conclusive results. One outstanding question regarding magnetic guarding of supports is the magnitude and behavior of secondary plasma drifts resulting from magnetic guard fields (grad-B drifts, etc). We present magnetic-implicit PIC modeling results and preliminary proof of concept experimental results on magnetic guarding of internal-supports and the subsequent reduction in total plasma losses.

Experimental implementation of non-Gaussian attacks on a continuous-variable quantum-key-distribution system.

PubMed

Lodewyck, Jérôme; Debuisschert, Thierry; García-Patrón, Raúl; Tualle-Brouri, Rosa; Cerf, Nicolas J; Grangier, Philippe

2007-01-19

An intercept-resend attack on a continuous-variable quantum-key-distribution protocol is investigated experimentally. By varying the interception fraction, one can implement a family of attacks where the eavesdropper totally controls the channel parameters. In general, such attacks add excess noise in the channel, and may also result in non-Gaussian output distributions. We implement and characterize the measurements needed to detect these attacks, and evaluate experimentally the information rates available to the legitimate users and the eavesdropper. The results are consistent with the optimality of Gaussian attacks resulting from the security proofs.

76 FR 60601 - Proposed Information Collections; Comment Request

Federal Register 2010, 2011, 2012, 2013, 2014

2011-09-29

... capital or start-up costs and costs of operation, maintenance, and purchase of services to provide the... of Spirits, Specially Denatured Spirits, or Wines for Exportation. OMB Number: 1513-0037. TTB Form..., denatured spirits, and wines from internal revenue bonded premises, without payment of tax for direct...

27 CFR 20.177 - Encased containers.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Encased containers. 20.177... Users of Specially Denatured Spirits Operations by Dealers § 20.177 Encased containers. (a) A dealer may package specially denatured spirits in unlabeled containers which are completely encased in wood...

27 CFR 20.145 - Encased containers.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Encased containers. 20.145... Denatured Alcohol § 20.145 Encased containers. Completely denatured alcohol may be packaged by distributors in unlabeled containers which are completely encased in wood, fiberboard, or similar material so that...

27 CFR 21.116 - Methyl alcohol.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Methyl alcohol. 21.116 Section 21.116 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY LIQUORS FORMULAS FOR DENATURED ALCOHOL AND RUM Specifications for Denaturants § 21...

27 CFR 21.113 - Isopropyl alcohol.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2011-04-01 2011-04-01 false Isopropyl alcohol. 21.113 Section 21.113 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY LIQUORS FORMULAS FOR DENATURED ALCOHOL AND RUM Specifications for Denaturants § 21...

27 CFR 21.116 - Methyl alcohol.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2011-04-01 2011-04-01 false Methyl alcohol. 21.116 Section 21.116 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY LIQUORS FORMULAS FOR DENATURED ALCOHOL AND RUM Specifications for Denaturants § 21...

27 CFR 21.113 - Isopropyl alcohol.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Isopropyl alcohol. 21.113 Section 21.113 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY LIQUORS FORMULAS FOR DENATURED ALCOHOL AND RUM Specifications for Denaturants § 21...

27 CFR 20.170 - Physical inventory.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2011-04-01 2011-04-01 false Physical inventory. 20.170... Users of Specially Denatured Spirits Inventory and Records § 20.170 Physical inventory. Once in each... physical inventory of each formula of new and recovered specially denatured spirits. (Approved by the...

27 CFR 20.170 - Physical inventory.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2012-04-01 2012-04-01 false Physical inventory. 20.170... Users of Specially Denatured Spirits Inventory and Records § 20.170 Physical inventory. Once in each... physical inventory of each formula of new and recovered specially denatured spirits. (Approved by the...

27 CFR 20.170 - Physical inventory.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2014-04-01 2014-04-01 false Physical inventory. 20.170... Users of Specially Denatured Spirits Inventory and Records § 20.170 Physical inventory. Once in each... physical inventory of each formula of new and recovered specially denatured spirits. (Approved by the...

27 CFR 20.170 - Physical inventory.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2013-04-01 2013-04-01 false Physical inventory. 20.170... Users of Specially Denatured Spirits Inventory and Records § 20.170 Physical inventory. Once in each... physical inventory of each formula of new and recovered specially denatured spirits. (Approved by the...

27 CFR 20.170 - Physical inventory.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Physical inventory. 20.170... Users of Specially Denatured Spirits Inventory and Records § 20.170 Physical inventory. Once in each... physical inventory of each formula of new and recovered specially denatured spirits. (Approved by the...

27 CFR 20.165 - Storage facilities.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2011-04-01 2011-04-01 false Storage facilities. 20.165... Users of Specially Denatured Spirits Premises and Equipment § 20.165 Storage facilities. (a) Storerooms... for locking. (b) Each stationary tank used for the storage of specially denatured spirits shall be...

27 CFR 20.165 - Storage facilities.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Storage facilities. 20.165... Users of Specially Denatured Spirits Premises and Equipment § 20.165 Storage facilities. (a) Storerooms... for locking. (b) Each stationary tank used for the storage of specially denatured spirits shall be...

27 CFR 20.165 - Storage facilities.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2012-04-01 2012-04-01 false Storage facilities. 20.165... Users of Specially Denatured Spirits Premises and Equipment § 20.165 Storage facilities. (a) Storerooms... for locking. (b) Each stationary tank used for the storage of specially denatured spirits shall be...

27 CFR 20.165 - Storage facilities.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2013-04-01 2013-04-01 false Storage facilities. 20.165... Users of Specially Denatured Spirits Premises and Equipment § 20.165 Storage facilities. (a) Storerooms... for locking. (b) Each stationary tank used for the storage of specially denatured spirits shall be...

27 CFR 20.165 - Storage facilities.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2014-04-01 2014-04-01 false Storage facilities. 20.165... Users of Specially Denatured Spirits Premises and Equipment § 20.165 Storage facilities. (a) Storerooms... for locking. (b) Each stationary tank used for the storage of specially denatured spirits shall be...

Lactoferrin denaturation induced by anionic surfactants: The role of the ferric ion in the protein stabilization.

PubMed

Ferreira, Gabriel Max Dias; Ferreira, Guilherme Max Dias; Agudelo, Álvaro Javier Patiño; Hudson, Eliara Acipreste; Dos Santos Pires, Ana Clarissa; da Silva, Luis Henrique Mendes

2018-05-11

Here, investigation was made of the interaction between Lactoferrin (Lf) and the anionic surfactants sodium dodecyl sulfate (SDS), sodium dodecylbenzene sulfonate (SDBS), and sodium decyl sulfate (DSS), using isothermal titration calorimetry, Nano differential scanning calorimetry (NanoDSC), and fluorescence spectroscopy. The Lf-surfactant interaction was enthalpically favorable (the integral enthalpy change ranged from -5.99 kJ mol -1 , for SDS at pH 3.0, to -0.61 kJ mol -1 , for DSS at pH 12.0) and promoted denaturation of the protein. The Lf denaturation efficiency followed the order DSS < SDS < SDBS. The extent of binding of the surfactants to Lf strongly depended on pH and the surfactant structure, reaching a maximum value of 505 SDBS molecules per gram of Lf at pH 3.0. The different efficiencies of the surfactants in denaturing Lf were attributed to the balance of hydrophobic and electrostatic interactions, which also depended on pH and the surfactant structure, highlighting the SDBS-tryptophan residue specific interaction, where SDBS acted as a quencher of fluorescence. Interestingly, the NanoDSC and fluorescence measurements showed that the ferric ion bound to Lf increased its stability against denaturation induced by the surfactants. The results have important implications for understanding the influence of surfactants on structural changes in metalloproteins. Copyright © 2017. Published by Elsevier B.V.

Designed to Fail: A Novel Mode of Collagen Fibril Disruption and Its Relevance to Tissue Toughness

PubMed Central

Veres, Samuel P.; Lee, J. Michael

2012-01-01

Collagen fibrils are nanostructured biological cables essential to the structural integrity of many of our tissues. Consequently, understanding the structural basis of their robust mechanical properties is of great interest. Here we present what to our knowledge is a novel mode of collagen fibril disruption that provides new insights into both the structure and mechanics of native collagen fibrils. Using enzyme probes for denatured collagen and scanning electron microscopy, we show that mechanically overloading collagen fibrils from bovine tail tendons causes them to undergo a sequential, two-stage, selective molecular failure process. Denatured collagen molecules—meaning molecules with a reduced degree of time-averaged helicity compared to those packed in undamaged fibrils—were first created within kinks that developed at discrete, repeating locations along the length of fibrils. There, collagen denaturation within the kinks was concentrated within certain subfibrils. Additional denatured molecules were then created along the surface of some disrupted fibrils. The heterogeneity of the disruption within fibrils suggests that either mechanical load is not carried equally by a fibril's subcomponents or that the subcomponents do not possess homogenous mechanical properties. Meanwhile, the creation of denatured collagen molecules, which necessarily involves the energy intensive breaking of intramolecular hydrogen bonds, provides a physical basis for the toughness of collagen fibrils. PMID:22735538

Counteraction of the deleterious effects of urea on structure and stability of mammalian kidney proteins by osmolytes.

PubMed

Dar, Mohammad Aasif; Wahiduzzaman; Islam, Asimul; Hassan, Md Imtaiyaz; Ahmad, Faizan

2018-02-01

Owing to the urine concentrating mechanism of kidney cells, urea concentration is very high (3.0-5.0M) in mammalian kidneys which may denature many kidney proteins. Methylamines are known to counteract the deleterious effects of urea on structure, stability and function of proteins at 2:1 molar ratio of urea to methylamines. It is known that mammalian kidney cells also contain stabilizing osmolytes, non-methylamines (myo-inositol and sorbitol). A question arises: Do these non-methylmine osmolytes have ability to counteract the deleterious effects of urea on kidney proteins? To answer this question, we took two kidney proteins, namely, sheep serum albumin and Human carbonic anhydrase II. We measured their thermodynamic stability (ΔG 0 N↔D , the Gibbs free energy change in absence of GdmCl (guanidinium chloride) associated with the equilibrium, native (N) state↔denatured (D) state) from the GdmCl-induced denaturation curves in the presence of different concentrations of urea and each kidney osmolyte individually and in combination. For both proteins, we observed that (i) glycine betaine and myo-inositol provide perfect counteraction at 2:1 molar ratio of urea to osmolyte, i.e., denaturing effect of 2M urea is 100% neutralized by 1M of glycine betaine (or myo-inositol), and (ii) sorbitol fails to refold urea denatured proteins. Copyright © 2017 Elsevier B.V. All rights reserved.

Guanidine hydrochloride-induced alkali molten globule model of horse ferrocytochrome c.

PubMed

Jain, Rishu; Kaur, Sandeep; Kumar, Rajesh

2013-02-01

This article compares structural, kinetic and thermodynamic properties of previously unknown guanidine hydrochloride (GdnHCl)-induced alkali molten globule (MG) state of horse 'ferrocytochrome c' (ferrocyt c) with the known NaCl-induced alkali-MG state of ferrocyt c. It is well known that Cl(-) arising from GdnHCl refolds and stabilizes the acid-denatured protein to MG state. We demonstrate that the GdnH(+) arising from GdnHCl (≤0.2 M) also transforms the base-denatured CO-liganded ferrocyt c (carbonmonoxycyt c) to MG state by making the electrostatic interactions to the negative charges of the protein. Structural and molecular properties extracted from the basic spectroscopic (circular dichroism (CD), fluorescence, FTIR and NMR) experiments suggest that the GdnH(+)- and Na(+)-induced MG states of base-denatured carbonmonoxycyt c are molecular compact states containing native-like secondary structures and disordered tertiary structures. Kinetic experiments involving the measurement of the CO association to the alkaline ferrocyt c in the presence of different GdnHCl and NaCl concentrations indicate that the Na(+)-induced MG state is more constrained relative to that of GdnH(+)-induced MG state. Analyses of thermal (near UV-CD) denaturation curves of the base-denatured protein in the presence of different GdnHCl and NaCl concentration also indicate that the Na(+)-induced MG state is thermally more stable than the GdnH(+)-induced MG state.

Effects of arginine on rabbit muscle creatine kinase and salt-induced molten globule-like state.

PubMed

Ou, Wen-bin; Wang, Ri-Sheng; Lu, Jie; Zhou, Hai-Meng

2003-11-03

The arginine (Arg)-induced unfolding and the salt-induced folding of creatine kinase (CK) have been studied by measuring enzyme activity, fluorescence emission spectra, native polyacrylamide gel electrophoresis and size exclusion chromatography (SEC). The results showed that Arg caused inactivation and unfolding of CK, but there was no aggregation during CK denaturation. The kinetics of CK unfolding followed a one-phase process. At higher concentrations of Arg (>160 mM), the CK dimers were fully dissociated, the alkali characteristic of Arg mainly led to the dissociation of dimers, but not denaturation effect of Arg's guanidine groups on CK. The inactivation of CK occurred before noticeable conformational changes of the whole molecules. KCl induced monomeric and dimeric molten globule-like states of CK denatured by Arg. These results suggest that as a protein denaturant, the effect of Arg on CK differed from that of guanidine and alkali, its denaturation for protein contains the double effects, which acts not only as guanidine hydrochloride but also as alkali. The active sites of CK have more flexibility than the whole enzyme conformation. Monomeric and dimeric molten globule-like states of CK were formed by the salt inducing in 160 and 500 mM Arg H(2)O solutions, respectively. The molten globule-like states indicate that monomeric and dimeric intermediates exist during CK folding. Furthermore, these results also proved the orderly folding model of CK.

Structural stability of E. coli transketolase to temperature and pH denaturation.

PubMed

Jahromi, Raha R F; Morris, Phattaraporn; Martinez-Torres, Ruben J; Dalby, Paul A

2011-09-10

We have previously shown that the denaturation of TK with urea follows a non-aggregating though irreversible denaturation pathway in which the cofactor binding appears to become altered but without dissociating, then followed at higher urea by partial denaturation of the homodimer prior to any further unfolding or dissociation of the two monomers. Urea is not typically present during biocatalysis, whereas access to TK enzymes that retain activity at increased temperature and extreme pH would be useful for operation under conditions that increase substrate and product stability or solubility. To provide further insight into the underlying causes of its deactivation in process conditions, we have characterised the effects of temperature and pH on the structure, stability, aggregation and activity of Escherichia coli transketolase. The activity of TK was initially found to progressively improve after pre-incubation at increasing temperatures. Loss of activity at higher temperature and low pH resulted primarily from protein denaturation and subsequent irreversible aggregation. By contrast, high pH resulted in the formation of a native-like state that was only partially inactive. The apo-TK enzyme structure content also increased at pH 9 to converge on that of the holo-TK. While cofactor dissociation was previously proposed for high pH deactivation, the observed structural changes in apo-TK but not holo-TK indicate a more complex mechanism. Copyright © 2011 Elsevier B.V. All rights reserved.

Prediction of protein mutant stability using classification and regression tool.

PubMed

Huang, Liang-Tsung; Saraboji, K; Ho, Shinn-Ying; Hwang, Shiow-Fen; Ponnuswamy, M N; Gromiha, M Michael

2007-02-01

Prediction of protein stability upon amino acid substitutions is an important problem in molecular biology and the solving of which would help for designing stable mutants. In this work, we have analyzed the stability of protein mutants using two different datasets of 1396 and 2204 mutants obtained from ProTherm database, respectively for free energy change due to thermal (DeltaDeltaG) and denaturant denaturations (DeltaDeltaG(H(2)O)). We have used a set of 48 physical, chemical energetic and conformational properties of amino acid residues and computed the difference of amino acid properties for each mutant in both sets of data. These differences in amino acid properties have been related to protein stability (DeltaDeltaG and DeltaDeltaG(H(2)O)) and are used to train with classification and regression tool for predicting the stability of protein mutants. Further, we have tested the method with 4 fold, 5 fold and 10 fold cross validation procedures. We found that the physical properties, shape and flexibility are important determinants of protein stability. The classification of mutants based on secondary structure (helix, strand, turn and coil) and solvent accessibility (buried, partially buried, partially exposed and exposed) distinguished the stabilizing/destabilizing mutants at an average accuracy of 81% and 80%, respectively for DeltaDeltaG and DeltaDeltaG(H(2)O). The correlation between the experimental and predicted stability change is 0.61 for DeltaDeltaG and 0.44 for DeltaDeltaG(H(2)O). Further, the free energy change due to the replacement of amino acid residue has been predicted within an average error of 1.08 kcal/mol and 1.37 kcal/mol for thermal and chemical denaturation, respectively. The relative importance of secondary structure and solvent accessibility, and the influence of the dataset on prediction of protein mutant stability have been discussed.

Rousseau's Philosophy of Transformative, "Denaturing" Education

ERIC Educational Resources Information Center

Riley, Patrick

2011-01-01

Rousseau's political philosophy presents the great legislator as a civic educator who must over time transform naturally self-loving egoists into citizens animated by a general will without destroying freedom. This is an educational process which is "denaturing" but which aims to produce autonomous adults who can ultimately say to their teacher…

27 CFR 26.36 - Products exempt from tax.

Code of Federal Regulations, 2012 CFR

2012-04-01

..., DEPARTMENT OF THE TREASURY LIQUORS LIQUORS AND ARTICLES FROM PUERTO RICO AND THE VIRGIN ISLANDS Products Coming Into the United States From Puerto Rico § 26.36 Products exempt from tax. (a) General. Industrial spirits, denatured spirits, and products made with denatured spirits in Puerto Rico may be brought into...

Step-wise refolding of recombinant proteins.

PubMed

Tsumoto, Kouhei; Arakawa, Tsutomu; Chen, Linda

2010-04-01

Protein refolding is still on trial-and-error basis. Here we describe step-wise dialysis refolding, in which denaturant concentration is altered in step-wise fashion. This technology controls the folding pathway by adjusting the concentrations of the denaturant and other solvent additives to induce sequential folding or disulfide formation.

40 CFR 80.40 - Fuel certification procedures.

Code of Federal Regulations, 2013 CFR

2013-07-01

... volume percent ethanol, or RBOB intended for blending with 10 to 15 volume percent ethanol, that is... contain denatured, anhydrous ethanol. The concentration of the ethanol, excluding the required denaturing agent, must be at least 9 percent and no more than 15 percent (by volume) of the gasoline. The ethanol...

40 CFR 80.40 - Fuel certification procedures.

Code of Federal Regulations, 2010 CFR

2010-07-01

... volume percent ethanol, or RBOB intended for blending with 10 volume percent ethanol, that is intended... contain denatured, anhydrous ethanol. The concentration of the ethanol, excluding the required denaturing agent, must be at least 9% and no more than 10% (by volume) of the gasoline. The ethanol content of the...

40 CFR 80.40 - Fuel certification procedures.

Code of Federal Regulations, 2012 CFR

2012-07-01

... volume percent ethanol, or RBOB intended for blending with 10 to 15 volume percent ethanol, that is... contain denatured, anhydrous ethanol. The concentration of the ethanol, excluding the required denaturing agent, must be at least 9 percent and no more than 15 percent (by volume) of the gasoline. The ethanol...

40 CFR 80.40 - Fuel certification procedures.

Code of Federal Regulations, 2011 CFR

2011-07-01

... volume percent ethanol, or RBOB intended for blending with 10 volume percent ethanol, that is intended... contain denatured, anhydrous ethanol. The concentration of the ethanol, excluding the required denaturing agent, must be at least 9% and no more than 10% (by volume) of the gasoline. The ethanol content of the...

40 CFR 80.40 - Fuel certification procedures.

Code of Federal Regulations, 2014 CFR

2014-07-01

... volume percent ethanol, or RBOB intended for blending with 10 to 15 volume percent ethanol, that is... contain denatured, anhydrous ethanol. The concentration of the ethanol, excluding the required denaturing agent, must be at least 9 percent and no more than 15 percent (by volume) of the gasoline. The ethanol...

27 CFR 19.726 - Authorized abbreviations to identify spirits.

Code of Federal Regulations, 2010 CFR

2010-04-01

... records: Kinds of spirits Abbreviations Alcohol A Brandy BR Bourbon Whisky BW Canadian Whisky CNW Completely Denatured Alcohol CDA Corn Whisky CW Grain Spirits GS Irish Whisky IW Light Whisky LW Malt Whisky MW Neutral Spirits NS Neutral Spirits Grain NSG Rye Whisky RW Scotch Whisky SW Specially Denatured...

48 CFR 829.202-70 - Tax exemptions for alcohol products.

Code of Federal Regulations, 2010 CFR

2010-10-01

...). The use of tax-free alcohol, whiskey, beer, wine, and denatured spirits for non-beverage purposes... authority may not be delegated. (b) Whiskey, alcohol, and denatured alcohol. (1) The contracting officer may... whiskey and alcohol only from qualified distillery plants or bonded dealers. The accountable officer must...

48 CFR 829.202-70 - Tax exemptions for alcohol products.

Code of Federal Regulations, 2011 CFR

2011-10-01

...). The use of tax-free alcohol, whiskey, beer, wine, and denatured spirits for non-beverage purposes... authority may not be delegated. (b) Whiskey, alcohol, and denatured alcohol. (1) The contracting officer may... whiskey and alcohol only from qualified distillery plants or bonded dealers. The accountable officer must...

76 FR 75836 - Revisions to Distilled Spirits Plant Operations Reports and Regulations

Federal Register 2010, 2011, 2012, 2013, 2014

2011-12-05

... relate to production, storage, denaturation, and processing activities, and may include other information..., Monthly Report of Production Operations; TTB F 5110.11, Monthly Report of Storage Operations; TTB F 5110.... These include a production report, up to four storage reports, a processing report, and a denaturing...

27 CFR 21.133 - Vinegar.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Vinegar. 21.133 Section 21.133 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY LIQUORS FORMULAS FOR DENATURED ALCOHOL AND RUM Specifications for Denaturants § 21.133 Vinegar. (a...

27 CFR 21.43 - Formula No. 18.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Formula No. 18. 21.43 Section 21.43 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY LIQUORS FORMULAS FOR DENATURED ALCOHOL AND RUM Specially Denatured Spirits Formulas...

27 CFR 21.62 - Formula No. 35-A.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Formula No. 35-A. 21.62 Section 21.62 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY LIQUORS FORMULAS FOR DENATURED ALCOHOL AND RUM Specially Denatured Spirits Formulas...

27 CFR 21.41 - Formula No. 13-A.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Formula No. 13-A. 21.41 Section 21.41 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY LIQUORS FORMULAS FOR DENATURED ALCOHOL AND RUM Specially Denatured Spirits Formulas...

27 CFR 21.133 - Vinegar.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2011-04-01 2011-04-01 false Vinegar. 21.133 Section 21.133 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY LIQUORS FORMULAS FOR DENATURED ALCOHOL AND RUM Specifications for Denaturants § 21.133 Vinegar. (a...

Using Myoglobin Denaturation to Help Biochemistry Students Understand Protein Structure

ERIC Educational Resources Information Center

Miao, Yilan; Thomas, Courtney L.

2017-01-01

Analyzing and understanding data directly from primary literature can be a daunting task for undergraduates. However, if information is put into context, students will be more successful when developing data analysis skills. A classroom activity is presented using protein denaturation to help undergraduate biochemistry students examine myoglobin…

27 CFR 21.21 - General.

Code of Federal Regulations, 2010 CFR

2010-04-01

....21 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE... subpart (or in accordance with § 21.5). (b) Denaturers may be authorized to add a small quantity of an... TTB officer. (c) Odorants or perfume materials may be added to denaturants authorized for completely...

27 CFR 21.21 - General.

Code of Federal Regulations, 2011 CFR

2011-04-01

....21 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE... subpart (or in accordance with § 21.5). (b) Denaturers may be authorized to add a small quantity of an... TTB officer. (c) Odorants or perfume materials may be added to denaturants authorized for completely...

27 CFR 21.21 - General.

Code of Federal Regulations, 2012 CFR

2012-04-01

....21 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE... subpart (or in accordance with § 21.5). (b) Denaturers may be authorized to add a small quantity of an... TTB officer. (c) Odorants or perfume materials may be added to denaturants authorized for completely...

27 CFR 21.21 - General.

Code of Federal Regulations, 2014 CFR

2014-04-01

....21 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE... subpart (or in accordance with § 21.5). (b) Denaturers may be authorized to add a small quantity of an... TTB officer. (c) Odorants or perfume materials may be added to denaturants authorized for completely...

27 CFR 21.21 - General.

Code of Federal Regulations, 2013 CFR

2013-04-01

....21 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE... subpart (or in accordance with § 21.5). (b) Denaturers may be authorized to add a small quantity of an... TTB officer. (c) Odorants or perfume materials may be added to denaturants authorized for completely...

27 CFR 21.44 - Formula No. 19.

Code of Federal Regulations, 2014 CFR

2014-04-01

... OF THE TREASURY ALCOHOL FORMULAS FOR DENATURED ALCOHOL AND RUM Specially Denatured Spirits Formulas and Authorized Uses § 21.44 Formula No. 19. (a) Formula. To every 100 gallons of alcohol add: One... 27 Alcohol, Tobacco Products and Firearms 1 2014-04-01 2014-04-01 false Formula No. 19. 21.44...

27 CFR 21.56 - Formula No. 29.

Code of Federal Regulations, 2013 CFR

2013-04-01

... OF THE TREASURY ALCOHOL FORMULAS FOR DENATURED ALCOHOL AND RUM Specially Denatured Spirits Formulas and Authorized Uses § 21.56 Formula No. 29. (a) Formula. To every 100 gallons of alcohol add: One... 27 Alcohol, Tobacco Products and Firearms 1 2013-04-01 2013-04-01 false Formula No. 29. 21.56...

27 CFR 21.44 - Formula No. 19.

Code of Federal Regulations, 2010 CFR

2010-04-01

... OF THE TREASURY LIQUORS FORMULAS FOR DENATURED ALCOHOL AND RUM Specially Denatured Spirits Formulas and Authorized Uses § 21.44 Formula No. 19. (a) Formula. To every 100 gallons of alcohol add: One... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Formula No. 19. 21.44...

27 CFR 21.52 - Formula No. 27.

Code of Federal Regulations, 2014 CFR

2014-04-01

... OF THE TREASURY ALCOHOL FORMULAS FOR DENATURED ALCOHOL AND RUM Specially Denatured Spirits Formulas and Authorized Uses § 21.52 Formula No. 27. (a) Formula. To every 100 gallons of alcohol add: One... 27 Alcohol, Tobacco Products and Firearms 1 2014-04-01 2014-04-01 false Formula No. 27. 21.52...

27 CFR 21.56 - Formula No. 29.

Code of Federal Regulations, 2014 CFR

2014-04-01

... OF THE TREASURY ALCOHOL FORMULAS FOR DENATURED ALCOHOL AND RUM Specially Denatured Spirits Formulas and Authorized Uses § 21.56 Formula No. 29. (a) Formula. To every 100 gallons of alcohol add: One... 27 Alcohol, Tobacco Products and Firearms 1 2014-04-01 2014-04-01 false Formula No. 29. 21.56...

27 CFR 21.44 - Formula No. 19.

Code of Federal Regulations, 2011 CFR

2011-04-01

... OF THE TREASURY LIQUORS FORMULAS FOR DENATURED ALCOHOL AND RUM Specially Denatured Spirits Formulas and Authorized Uses § 21.44 Formula No. 19. (a) Formula. To every 100 gallons of alcohol add: One... 27 Alcohol, Tobacco Products and Firearms 1 2011-04-01 2011-04-01 false Formula No. 19. 21.44...

27 CFR 21.44 - Formula No. 19.

Code of Federal Regulations, 2013 CFR

2013-04-01

... OF THE TREASURY ALCOHOL FORMULAS FOR DENATURED ALCOHOL AND RUM Specially Denatured Spirits Formulas and Authorized Uses § 21.44 Formula No. 19. (a) Formula. To every 100 gallons of alcohol add: One... 27 Alcohol, Tobacco Products and Firearms 1 2013-04-01 2013-04-01 false Formula No. 19. 21.44...

27 CFR 21.52 - Formula No. 27.

Code of Federal Regulations, 2010 CFR

2010-04-01

... OF THE TREASURY LIQUORS FORMULAS FOR DENATURED ALCOHOL AND RUM Specially Denatured Spirits Formulas and Authorized Uses § 21.52 Formula No. 27. (a) Formula. To every 100 gallons of alcohol add: One... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Formula No. 27. 21.52...

27 CFR 21.52 - Formula No. 27.

Code of Federal Regulations, 2012 CFR

2012-04-01

... OF THE TREASURY LIQUORS FORMULAS FOR DENATURED ALCOHOL AND RUM Specially Denatured Spirits Formulas and Authorized Uses § 21.52 Formula No. 27. (a) Formula. To every 100 gallons of alcohol add: One... 27 Alcohol, Tobacco Products and Firearms 1 2012-04-01 2012-04-01 false Formula No. 27. 21.52...

27 CFR 21.44 - Formula No. 19.

Code of Federal Regulations, 2012 CFR

2012-04-01

... OF THE TREASURY LIQUORS FORMULAS FOR DENATURED ALCOHOL AND RUM Specially Denatured Spirits Formulas and Authorized Uses § 21.44 Formula No. 19. (a) Formula. To every 100 gallons of alcohol add: One... 27 Alcohol, Tobacco Products and Firearms 1 2012-04-01 2012-04-01 false Formula No. 19. 21.44...

27 CFR 21.52 - Formula No. 27.

Code of Federal Regulations, 2011 CFR

2011-04-01

... OF THE TREASURY LIQUORS FORMULAS FOR DENATURED ALCOHOL AND RUM Specially Denatured Spirits Formulas and Authorized Uses § 21.52 Formula No. 27. (a) Formula. To every 100 gallons of alcohol add: One... 27 Alcohol, Tobacco Products and Firearms 1 2011-04-01 2011-04-01 false Formula No. 27. 21.52...

27 CFR 21.52 - Formula No. 27.

Code of Federal Regulations, 2013 CFR

2013-04-01

... OF THE TREASURY ALCOHOL FORMULAS FOR DENATURED ALCOHOL AND RUM Specially Denatured Spirits Formulas and Authorized Uses § 21.52 Formula No. 27. (a) Formula. To every 100 gallons of alcohol add: One... 27 Alcohol, Tobacco Products and Firearms 1 2013-04-01 2013-04-01 false Formula No. 27. 21.52...

27 CFR 21.56 - Formula No. 29.

Code of Federal Regulations, 2012 CFR

2012-04-01

... OF THE TREASURY LIQUORS FORMULAS FOR DENATURED ALCOHOL AND RUM Specially Denatured Spirits Formulas and Authorized Uses § 21.56 Formula No. 29. (a) Formula. To every 100 gallons of alcohol add: One... 27 Alcohol, Tobacco Products and Firearms 1 2012-04-01 2012-04-01 false Formula No. 29. 21.56...

Purification and Characterization of Recombinant Vaccinia L1R Protein from Escherichia coli

DTIC Science & Technology

2016-08-01

Solubilization .................................................2  2.4  Denaturing Chromatography (Purification Step 1...Concentration Determination ................................................................4  2.10  Enzyme -Linked Immunosorbent Assay (ELISA...the preparation of the recombinant VACV L1R protein fragment by denaturing , refolding, and purifying material expressed into inclusion bodies in

1H, 15N, 13C resonance assignment of folded and 8 M urea-denatured state of SUMO from Drosophila melanogaster.

PubMed

Kumar, Dinesh; Kumar, Ashutosh; Misra, Jyoti Ranjan; Chugh, Jeetender; Sharma, Shilpy; Hosur, Ramakrishna V

2008-06-01

SUMO, an important post-translational modifier of variety of substrate proteins, regulates different cellular functions. Here, we report the NMR resonance assignment of the folded and 8 M urea-denatured state of SUMO from Drosophila melanogaster (dsmt3).

Noncanonical self-assembly of multifunctional DNA nanoflowers for biomedical applications.

PubMed

Zhu, Guizhi; Hu, Rong; Zhao, Zilong; Chen, Zhuo; Zhang, Xiaobing; Tan, Weihong

2013-11-06

DNA nanotechnology has been extensively explored to assemble various functional nanostructures for versatile applications. Mediated by Watson-Crick base-pairing, these DNA nanostructures have been conventionally assembled through hybridization of many short DNA building blocks. Here we report the noncanonical self-assembly of multifunctional DNA nanostructures, termed as nanoflowers (NFs), and the versatile biomedical applications. These NFs were assembled from long DNA building blocks generated via rolling circle replication (RCR) of a designer template. NF assembly was driven by liquid crystallization and dense packaging of building blocks, without relying on Watson-Crick base-pairing between DNA strands, thereby avoiding the otherwise conventional complicated DNA sequence design. NF sizes were readily tunable in a wide range, by simply adjusting such parameters as assembly time and template sequences. NFs were exceptionally resistant to nuclease degradation, denaturation, or dissociation at extremely low concentration, presumably resulting from the dense DNA packaging in NFs. The exceptional biostability is critical for biomedical applications. By rational design, NFs can be readily incorporated with myriad functional moieties. All these properties make NFs promising for versatile applications. As a proof-of-principle demonstration, in this study, NFs were integrated with aptamers, bioimaging agents, and drug loading sites, and the resultant multifunctional NFs were demonstrated for selective cancer cell recognition, bioimaging, and targeted anticancer drug delivery.

Noncanonical self-assembly of multifunctional DNA nanoflowers for biomedical applications

PubMed Central

Zhu, Guizhi; Hu, Rong; Zhao, Zilong; Chen, Zhuo; Zhang, Xiaobing; Tan, Weihong

2013-01-01

DNA nanotechnology has been extensively explored to assemble various functional nanostructures for versatile applications. Mediated by Watson-Crick base-pairing, these DNA nanostructures have been conventionally assembled through hybridization of many short DNA building blocks. Here we report the noncanonical self-assembly of multifunctional DNA nanostructures, termed as nanoflowers (NFs), and the versatile biomedical applications. These NFs were assembled from long DNA building blocks generated via Rolling Circle Replication (RCR) of a designer template. NF assembly was driven by liquid crystallization and dense packaging of building blocks, without relying on Watson-Crick base-pairing between DNA strands, thereby avoiding the otherwise conventional complicated DNA sequence design. NF sizes were readily tunable in a wide range, by simply adjusting such parameters as assembly time and template sequences. NFs were exceptionally resistant to nuclease degradation, denaturation, or dissociation at extremely low concentration, presumably resulting from the dense DNA packaging in NFs. The exceptional biostability is critical for biomedical applications. By rational design, NFs can be readily incorporated with myriad functional moieties. All these properties make NFs promising for versatile applications. As a proof-of-principle demonstration, in this study, NFs were integrated with aptamers, bioimaging agents, and drug loading sites, and the resultant multifunctional NFs were demonstrated for selective cancer cell recognition, bioimaging, and targeted anticancer drug delivery. PMID:24164620

A simple nucleic acid hybridization/latex agglutination assay for the rapid detection of polymerase chain reaction amplicons.

PubMed

Vollenhofer-Schrumpf, Sabine; Buresch, Ronald; Schinkinger, Manfred

2007-03-01

We have developed a new method for the detection of nucleic acid hybridization, based on a simple latex agglutination test that can be evaluated by the unaided eye. Nucleic acid, e.g., a polymerase chain reaction (PCR) product, is denatured and incubated with polystyrene beads carrying covalently bound complementary oligonucleotide sequences. Hybridization of the nucleic acids leads to aggregation of the latex particles, thereby verifying the presence of target sequence. The test is performed at room temperature, and results are available within 10 min. As a proof of principle, the hybridization/latex agglutination assay was applied to the detection of purified PCR fragments either specific for Salmonella spp. or a synthetic sequence, and to the detection of Salmonella enterica in artificially contaminated chicken samples. A few nanograms of purified PCR fragments were detectable. In artificially contaminated chicken samples, 3 colony-forming units (cfu)/25 g were detected in one of three replicates, and 30 cfu/25 g were detected in both of two replicates when samples for PCR were taken directly from primary enrichment, demonstrating the practical applicability of this test system. Even multiplex detection might be achievable. This novel kind of assay could be useful for a range of applications where hybridization of nucleic acids, e.g., PCR fragments, is to be detected.

Experimental extraction of secure correlations from a noisy private state.

PubMed

Dobek, K; Karpiński, M; Demkowicz-Dobrzański, R; Banaszek, K; Horodecki, P

2011-01-21

We report experimental generation of a noisy entangled four-photon state that exhibits a separation between the secure key contents and distillable entanglement, a hallmark feature of the recently established quantum theory of private states. The privacy analysis, based on the full tomographic reconstruction of the prepared state, is utilized in a proof-of-principle key generation. The inferiority of distillation-based strategies to extract the key is exposed by an implementation of an entanglement distillation protocol for the produced state.

Test and Analysis of a Hyper-X Carbon-Carbon Leading Edge Chine

NASA Technical Reports Server (NTRS)

Smith, Russell W.; Sikora, Joseph G.; Lindell, Michael C.

2005-01-01

During parts production for the X43A Mach 10 hypersonic vehicle nondestructive evaluation (NDE) of a leading edge chine detected on imbedded delamination near the lower surface of the part. An ultimate proof test was conducted to verify the ultimate strength of this leading edge chine part. The ultimate proof test setup used a pressure bladder design to impose a uniform distributed pressure field over the bi-planar surface of the chine test article. A detailed description of the chine test article and experimental test setup is presented. Analysis results from a linear status model of the test article are also presented and discussed. Post-test inspection of the specimen revealed no visible failures or areas of delamination.

The Experimental Demonstration of High Efficiency Interaction-free Measurement for Quantum Counterfactual-like Communication.

PubMed

Liu, Chao; Liu, Jinhong; Zhang, Junxiang; Zhu, Shiyao

2017-09-07

We present an interaction-free measurement with quantum Zeno effect and a high efficiency η = 74.6% ± 0.15%. As a proof-of-principle demonstration, this measurement can be used to implement a quantum counterfactual-like communication protocol. Instead of a single photon state, we use a coherent light as the input source and show that the output agrees with the proposed quantum counterfactual communication protocol according to Salih et al. Although the counterfactuality is not achieved due to the presence of a few photons in the public channel, we show that the signal light is nearly absent in the public channel, which exhibits a proof-of-principle quantum counterfactual-like property of communication.

New method and installation for rapid determination of radon diffusion coefficient in various materials.

PubMed

Tsapalov, Andrey; Gulabyants, Loren; Livshits, Mihail; Kovler, Konstantin

2014-04-01

The mathematical apparatus and the experimental installation for the rapid determination of radon diffusion coefficient in various materials are developed. The single test lasts not longer than 18 h and allows testing numerous materials, such as gaseous and liquid media, as well as soil, concrete and radon-proof membranes, in which diffusion coefficient of radon may vary in an extremely wide range, from 1·10(-12) to 5·10(-5) m(2)/s. The uncertainty of radon diffusion coefficient estimation depends on the permeability of the sample and varies from about 5% (for the most permeable materials) to 40% (for less permeable materials, such as radon-proof membranes). Copyright © 2014. Published by Elsevier Ltd.

Solvent Effects on Protein Folding/Unfolding

NASA Astrophysics Data System (ADS)

García, A. E.; Hillson, N.; Onuchic, J. N.

Pressure effects on the hydrophobic potential of mean force led Hummer et al. to postulate a model for pressure denaturation of proteins in which denaturation occurs by means of water penetration into the protein interior, rather than by exposing the protein hydrophobic core to the solvent --- commonly used to describe temperature denaturation. We study the effects of pressure in protein folding/unfolding kinetics in an off-lattice minimalist model of a protein in which pressure effects have been incorporated by means of the pair-wise potential of mean force of hydrophobic groups in water. We show that pressure slows down the kinetics of folding by decreasing the reconfigurational diffusion coefficient and moves the location of the folding transition state.

The role of the AT pairs in the acid denaturation of DNA.

PubMed Central

Hermann, P; Fredericq, E

1977-01-01

It has been determined previously that the protonation of the GC pairs induces a DNA conformation change which leads to a "metastable" structure. The role of the AT pairs, however, is no well known because the protonation does not modify their spectral properties. By means of an indirect method based on the binding of proflavine, it has been determined that the AT pairs are protonated before the acid-induced denaturation and that they seem to be unable to assume a conformation change when protonated. These results would indicate that the protonated AT pairs may be responsible for the induction of the acid denaturation and not the GC pairs as it was thought previously. PMID:20604

Structure and properties of native and unfolded lysing enzyme from T. harzianum: Chemical and pH denaturation.

PubMed

Bey, Houda; Gtari, Wala; Aschi, Adel; Othman, Tahar

2016-11-01

The effect of chemical denaturants and pH on the change of the conformation of the protein Lysing Enzyme from Trichoderma Harzianum has been investigated by dynamic light scattering (DLS) and turbidimetry. Chemical denaturants are frequently used to describe the mechanisms of folding and transition states. We have analyzed the pH effect on the properties and particle size of the protein. The compaction factor CI has shown that the protein is weakly disordered. The molecular dynamics simulations confirm, at neutral pH, that the protein has a low net charge and high hydrophobicity. Copyright © 2016 Elsevier B.V. All rights reserved.

Course 12: Proteins: Structural, Thermodynamic and Kinetic Aspects

NASA Astrophysics Data System (ADS)

Finkelstein, A. V.

1 Introduction 2 Overview of protein architectures and discussion of physical background of their natural selection 2.1 Protein structures 2.2 Physical selection of protein structures 3 Thermodynamic aspects of protein folding 3.1 Reversible denaturation of protein structures 3.2 What do denatured proteins look like? 3.3 Why denaturation of a globular protein is the first-order phase transition 3.4 "Gap" in energy spectrum: The main characteristic that distinguishes protein chains from random polymers 4 Kinetic aspects of protein folding 4.1 Protein folding in vivo 4.2 Protein folding in vitro (in the test-tube) 4.3 Theory of protein folding rates and solution of the Levinthal paradox

An Inexpensive and Safe Experiment to Demonstrate Koch's Postulates Using Citrus Fruit

ERIC Educational Resources Information Center

Jakobi, Steven

2010-01-01

Citrus fruit (oranges, tangerines, grapefruit or lemons) purchased in a grocery store can be experimentally infected with readily-available sources of "Penicillium digitatum" to demonstrate the four basic steps of Koch's postulates, also known as proof of pathogenicity. The mould is isolated from naturally-infected citrus fruit into pure culture…

CDM: Teaching Discrete Mathematics to Computer Science Majors

ERIC Educational Resources Information Center

Sutner, Klaus

2005-01-01

CDM, for computational discrete mathematics, is a course that attempts to teach a number of topics in discrete mathematics to computer science majors. The course abandons the classical definition-theorem-proof model, and instead relies heavily on computation as a source of motivation and also for experimentation and illustration. The emphasis on…

Using Empirical Evidence in the Process of Proving: The Case of Dynamic Geometry

ERIC Educational Resources Information Center

Guven, Bulent; Cekmez, Erdem; Karatas, Ilhan

2010-01-01

With the emergence of Dynamic Geometry Software (DGS), a theoretical gap between the acquisition (inductive) and the justification (deductive) of a mathematical statement has started a debate. Some educators believe that deductive proof in geometry should be abandoned in favour of an experimental approach to mathematical justification. This…

Cognitive Models: The Missing Link to Learning Fraction Multiplication and Division

ERIC Educational Resources Information Center

de Castro, Belinda V.

2008-01-01

This quasi-experimental study aims to streamline cognitive models on fraction multiplication and division that contain the most worthwhile features of other existing models. Its exploratory nature and its approach to proof elicitation can be used to help establish its effectiveness in building students' understanding of fractions as compared to…

46 CFR 56.15-5 - Fluid-conditioner fittings.

Code of Federal Regulations, 2012 CFR

2012-10-01

... allowable working pressure does not exceed one-fourth of the burst pressure or produce a primary stress... of the burst pressure or produce a primary stress greater than one-fifth of the ultimate tensile... representative model to a proof test or experimental stress analysis described in paragraph A-22 of Section I of...

46 CFR 56.15-5 - Fluid-conditioner fittings.

Code of Federal Regulations, 2011 CFR

2011-10-01

... allowable working pressure does not exceed one-fourth of the burst pressure or produce a primary stress... of the burst pressure or produce a primary stress greater than one-fifth of the ultimate tensile... representative model to a proof test or experimental stress analysis described in paragraph A-22 of Section I of...

46 CFR 56.15-5 - Fluid-conditioner fittings.

Code of Federal Regulations, 2013 CFR

2013-10-01

... allowable working pressure does not exceed one-fourth of the burst pressure or produce a primary stress... of the burst pressure or produce a primary stress greater than one-fifth of the ultimate tensile... representative model to a proof test or experimental stress analysis described in paragraph A-22 of section I of...

46 CFR 56.15-5 - Fluid-conditioner fittings.

Code of Federal Regulations, 2014 CFR

2014-10-01

... allowable working pressure does not exceed one-fourth of the burst pressure or produce a primary stress... of the burst pressure or produce a primary stress greater than one-fifth of the ultimate tensile... representative model to a proof test or experimental stress analysis described in paragraph A-22 of section I of...

Epidemiology: second-rate science?

PubMed Central

Parascandola, M

1998-01-01

In recent years epidemiology has come under increasing criticism in regulatory and public arenas for being "unscientific." The tobacco industry has taken advantage of this, insisting for decades that evidence linking cigarettes and lung cancer falls short of proof. Moreover, many epidemiologists remain unduly skeptical and self-conscious about the status of their own causal claims. This situation persists in part because of a widespread belief that only the laboratory can provide evidence sufficient for scientific proof. Adherents of this view erroneously believe that there is no element of uncertainty or inductive inference in the "direct observation" of the laboratory researcher and that epidemiology provides mere "circumstantial" evidence. The historical roots of this attitude can be traced to philosopher John Stuart Mill and physiologist Claude Bernard and their influence on modern experimental thinking. The author uses the debate over cigarettes and lung cancer to examine ideas of proof in medical science and public health, concluding that inductive inference from a limited sample to a larger population is an element in all empirical science. Images p313-a p317-a p318-a PMID:9672568

27 CFR 20.161 - Withdrawals under permit.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Withdrawals under permit. 20.161 Section 20.161 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY LIQUORS DISTRIBUTION AND USE OF DENATURED ALCOHOL AND RUM Operations by Dealers and Users of Specially Denatured Spirit...

40 CFR 80.1630 - Sampling and testing requirements for refiners, gasoline importers and producers and importers of...

Code of Federal Regulations, 2014 CFR

2014-07-01

... refiners, gasoline importers and producers and importers of certified ethanol denaturant. 80.1630 Section...) REGULATION OF FUELS AND FUEL ADDITIVES Gasoline Sulfur § 80.1630 Sampling and testing requirements for refiners, gasoline importers and producers and importers of certified ethanol denaturant. (a) Sample and...

27 CFR 21.38 - Formula No. 4.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2013-04-01 2013-04-01 false Formula No. 4. 21.38... OF THE TREASURY ALCOHOL FORMULAS FOR DENATURED ALCOHOL AND RUM Specially Denatured Spirits Formulas and Authorized Uses § 21.38 Formula No. 4. (a) Formula. To every 100 gallons of alcohol, or to every...

27 CFR 21.73 - Formula No. 39-D.

Code of Federal Regulations, 2013 CFR

2013-04-01

... OF THE TREASURY ALCOHOL FORMULAS FOR DENATURED ALCOHOL AND RUM Specially Denatured Spirits Formulas and Authorized Uses § 21.73 Formula No. 39-D. (a) Formula. To every 100 gallons of alcohol add: One... 27 Alcohol, Tobacco Products and Firearms 1 2013-04-01 2013-04-01 false Formula No. 39-D. 21.73...

27 CFR 21.73 - Formula No. 39-D.

Code of Federal Regulations, 2012 CFR

2012-04-01

... OF THE TREASURY LIQUORS FORMULAS FOR DENATURED ALCOHOL AND RUM Specially Denatured Spirits Formulas and Authorized Uses § 21.73 Formula No. 39-D. (a) Formula. To every 100 gallons of alcohol add: One... 27 Alcohol, Tobacco Products and Firearms 1 2012-04-01 2012-04-01 false Formula No. 39-D. 21.73...

27 CFR 21.36 - Formula No. 3-B.

Code of Federal Regulations, 2011 CFR

2011-04-01

... OF THE TREASURY LIQUORS FORMULAS FOR DENATURED ALCOHOL AND RUM Specially Denatured Spirits Formulas and Authorized Uses § 21.36 Formula No. 3-B. (a) Formula. To every 100 gallons of alcohol add: One... 27 Alcohol, Tobacco Products and Firearms 1 2011-04-01 2011-04-01 false Formula No. 3-B. 21.36...

27 CFR 21.73 - Formula No. 39-D.

Code of Federal Regulations, 2010 CFR

2010-04-01

... OF THE TREASURY LIQUORS FORMULAS FOR DENATURED ALCOHOL AND RUM Specially Denatured Spirits Formulas and Authorized Uses § 21.73 Formula No. 39-D. (a) Formula. To every 100 gallons of alcohol add: One... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Formula No. 39-D. 21.73...

27 CFR 21.73 - Formula No. 39-D.

Code of Federal Regulations, 2014 CFR

2014-04-01

... OF THE TREASURY ALCOHOL FORMULAS FOR DENATURED ALCOHOL AND RUM Specially Denatured Spirits Formulas and Authorized Uses § 21.73 Formula No. 39-D. (a) Formula. To every 100 gallons of alcohol add: One... 27 Alcohol, Tobacco Products and Firearms 1 2014-04-01 2014-04-01 false Formula No. 39-D. 21.73...

27 CFR 21.67 - Formula No. 38-D.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2011-04-01 2011-04-01 false Formula No. 38-D. 21.67... OF THE TREASURY LIQUORS FORMULAS FOR DENATURED ALCOHOL AND RUM Specially Denatured Spirits Formulas and Authorized Uses § 21.67 Formula No. 38-D. (a) Formula. To every 100 gallons of alcohol add: Two...

27 CFR 21.38 - Formula No. 4.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2011-04-01 2011-04-01 false Formula No. 4. 21.38... OF THE TREASURY LIQUORS FORMULAS FOR DENATURED ALCOHOL AND RUM Specially Denatured Spirits Formulas and Authorized Uses § 21.38 Formula No. 4. (a) Formula. To every 100 gallons of alcohol, or to every...

27 CFR 21.58 - Formula No. 31-A.

Code of Federal Regulations, 2013 CFR

2013-04-01

... OF THE TREASURY ALCOHOL FORMULAS FOR DENATURED ALCOHOL AND RUM Specially Denatured Spirits Formulas and Authorized Uses § 21.58 Formula No. 31-A. (a) Formula. To every 100 gallons of alcohol add: One... 27 Alcohol, Tobacco Products and Firearms 1 2013-04-01 2013-04-01 false Formula No. 31-A. 21.58...

27 CFR 21.43 - Formula No. 18.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2012-04-01 2012-04-01 false Formula No. 18. 21.43... OF THE TREASURY LIQUORS FORMULAS FOR DENATURED ALCOHOL AND RUM Specially Denatured Spirits Formulas and Authorized Uses § 21.43 Formula No. 18. (a) Formula. To every 100 gallons of alcohol of not less...

27 CFR 21.67 - Formula No. 38-D.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Formula No. 38-D. 21.67... OF THE TREASURY LIQUORS FORMULAS FOR DENATURED ALCOHOL AND RUM Specially Denatured Spirits Formulas and Authorized Uses § 21.67 Formula No. 38-D. (a) Formula. To every 100 gallons of alcohol add: Two...

27 CFR 21.39 - Formula No. 6-B.

Code of Federal Regulations, 2012 CFR

2012-04-01

... OF THE TREASURY LIQUORS FORMULAS FOR DENATURED ALCOHOL AND RUM Specially Denatured Spirits Formulas and Authorized Uses § 21.39 Formula No. 6-B. (a) Formula. To every 100 gallons of alcohol add: One... 27 Alcohol, Tobacco Products and Firearms 1 2012-04-01 2012-04-01 false Formula No. 6-B. 21.39...

27 CFR 21.58 - Formula No. 31-A.

Code of Federal Regulations, 2011 CFR

2011-04-01

... OF THE TREASURY LIQUORS FORMULAS FOR DENATURED ALCOHOL AND RUM Specially Denatured Spirits Formulas and Authorized Uses § 21.58 Formula No. 31-A. (a) Formula. To every 100 gallons of alcohol add: One... 27 Alcohol, Tobacco Products and Firearms 1 2011-04-01 2011-04-01 false Formula No. 31-A. 21.58...

27 CFR 21.36 - Formula No. 3-B.

Code of Federal Regulations, 2013 CFR

2013-04-01

... OF THE TREASURY ALCOHOL FORMULAS FOR DENATURED ALCOHOL AND RUM Specially Denatured Spirits Formulas and Authorized Uses § 21.36 Formula No. 3-B. (a) Formula. To every 100 gallons of alcohol add: One... 27 Alcohol, Tobacco Products and Firearms 1 2013-04-01 2013-04-01 false Formula No. 3-B. 21.36...

27 CFR 21.73 - Formula No. 39-D.

Code of Federal Regulations, 2011 CFR

2011-04-01

... OF THE TREASURY LIQUORS FORMULAS FOR DENATURED ALCOHOL AND RUM Specially Denatured Spirits Formulas and Authorized Uses § 21.73 Formula No. 39-D. (a) Formula. To every 100 gallons of alcohol add: One... 27 Alcohol, Tobacco Products and Firearms 1 2011-04-01 2011-04-01 false Formula No. 39-D. 21.73...