INTERNATIONAL COMPARISON EXERCISE ON NEUTRON SPECTRA UNFOLDING IN BONNER SPHERES SPECTROMETRY: PROBLEM DESCRIPTION AND PRELIMINARY ANALYSIS.

PubMed

Gómez-Ros, J M; Bedogni, R; Domingo, C; Eakins, J S; Roberts, N; Tanner, R J

2018-01-29

This article describes the purpose, the proposed problems and the reference solutions of an international comparison on neutron spectra unfolding in Bonner spheres spectrometry, organised within the activities of EURADOS working group 6: computational dosimetry. The exercise considered four realistic situations: a medical accelerator, a workplace field, an irradiation room and a skyshine scenario. Although a detailed analysis of the submitted solutions is under preparation, the preliminary discussion of some physical aspects of the problem, e.g. the changes in the unfolding results due to the perturbation of the neutron field by the Bonner spheres, is presented. © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Evaluation of a new neutron energy spectrum unfolding code based on an Adaptive Neuro-Fuzzy Inference System (ANFIS).

PubMed

Hosseini, Seyed Abolfazl; Esmaili Paeen Afrakoti, Iman

2018-01-17

The purpose of the present study was to reconstruct the energy spectrum of a poly-energetic neutron source using an algorithm developed based on an Adaptive Neuro-Fuzzy Inference System (ANFIS). ANFIS is a kind of artificial neural network based on the Takagi-Sugeno fuzzy inference system. The ANFIS algorithm uses the advantages of both fuzzy inference systems and artificial neural networks to improve the effectiveness of algorithms in various applications such as modeling, control and classification. The neutron pulse height distributions used as input data in the training procedure for the ANFIS algorithm were obtained from the simulations performed by MCNPX-ESUT computational code (MCNPX-Energy engineering of Sharif University of Technology). Taking into account the normalization condition of each energy spectrum, 4300 neutron energy spectra were generated randomly. (The value in each bin was generated randomly, and finally a normalization of each generated energy spectrum was performed). The randomly generated neutron energy spectra were considered as output data of the developed ANFIS computational code in the training step. To calculate the neutron energy spectrum using conventional methods, an inverse problem with an approximately singular response matrix (with the determinant of the matrix close to zero) should be solved. The solution of the inverse problem using the conventional methods unfold neutron energy spectrum with low accuracy. Application of the iterative algorithms in the solution of such a problem, or utilizing the intelligent algorithms (in which there is no need to solve the problem), is usually preferred for unfolding of the energy spectrum. Therefore, the main reason for development of intelligent algorithms like ANFIS for unfolding of neutron energy spectra is to avoid solving the inverse problem. In the present study, the unfolded neutron energy spectra of 252Cf and 241Am-9Be neutron sources using the developed computational code were found to have excellent agreement with the reference data. Also, the unfolded energy spectra of the neutron sources as obtained using ANFIS were more accurate than the results reported from calculations performed using artificial neural networks in previously published papers. © The Author(s) 2018. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.

Modeling the thermal unfolding 2DIR spectra of a β-hairpin peptide based on the implicit solvent MD simulation.

PubMed

Wu, Tianmin; Yang, Lijiang; Zhang, Ruiting; Shao, Qiang; Zhuang, Wei

2013-07-25

We simulated the equilibrium isotope-edited FTIR and 2DIR spectra of a β-hairpin peptide trpzip2 at a series of temperatures. The simulation was based on the configuration distributions generated using the GB(OBC) implicit solvent model and the integrated tempering sampling (ITS) technique. A soaking procedure was adapted to generate the peptide in explicit solvent configurations for the spectroscopy calculations. The nonlinear exciton propagation (NEP) method was then used to calculate the spectra. Agreeing with the experiments, the intensities and ellipticities of the isotope-shifted peaks in our simulated signals have the site-specific temperature dependences, which suggest the inhomogeneous local thermal stabilities along the peptide chain. Our simulation thus proposes a cost-effective means to understand a peptide's conformational change and related IR spectra across its thermal unfolding transition.

Measurement and validation of benchmark-quality thick-target tungsten X-ray spectra below 150 kVp.

PubMed

Mercier, J R; Kopp, D T; McDavid, W D; Dove, S B; Lancaster, J L; Tucker, D M

2000-11-01

Pulse-height distributions of two constant potential X-ray tubes with fixed anode tungsten targets were measured and unfolded. The measurements employed quantitative alignment of the beam, the use of two different semiconductor detectors (high-purity germanium and cadmium-zinc-telluride), two different ion chamber systems with beam-specific calibration factors, and various filter and tube potential combinations. Monte Carlo response matrices were generated for each detector for unfolding the pulse-height distributions into spectra incident on the detectors. These response matrices were validated for the low error bars assigned to the data. A significant aspect of the validation of spectra, and a detailed characterization of the X-ray tubes, involved measuring filtered and unfiltered beams at multiple tube potentials (30-150 kVp). Full corrections to ion chamber readings were employed to convert normalized fluence spectra into absolute fluence spectra. The characterization of fixed anode pitting and its dominance over exit window plating and/or detector dead layer was determined. An Appendix of tabulated benchmark spectra with assigned error ranges was developed for future reference.

Measurement of neutron spectra in the AWE workplace using a Bonner sphere spectrometer.

PubMed

Danyluk, Peter

2010-12-01

A Bonner sphere spectrometer has been used to measure the neutron spectra in eight different workplace areas at AWE (Atomic Weapons Establishment). The spectra were analysed by the National Physical Laboratory using their principal unfolding code STAY'SL and the results were also analysed by AWE using a bespoke parametrised unfolding code. The bespoke code was designed specifically for the AWE workplace and is very simple to use. Both codes gave results, in good agreement. It was found that the measured fluence rate varied from 2 to 70 neutrons cm⁻² s⁻¹ (± 10%) and the ambient dose equivalent H*(10) varied from 0.5 to 57 µSv h⁻¹ (± 20%). A detailed description of the development and use of the bespoke code is presented.

Filter-fluorescer measurement of low-voltage simulator x-ray energy spectra

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

Baldwin, G.T.; Craven, R.E.

X-ray energy spectra of the Maxwell Laboratories MBS and Physics International Pulserad 737 were measured using an eight-channel filter-fluorescer array. The PHOSCAT computer code was used to calculate channel response functions, and the UFO code to unfold spectrum.

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

Measurement of angularly dependent spectra of betatron gamma-rays from a laser plasma accelerator with quadrant-sectored range filters

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

Jeon, Jong Ho, E-mail: jhjeon07@ibs.re.kr; Nakajima, Kazuhisa, E-mail: naka115@dia-net.ne.jp; Rhee, Yong Joo

Measurement of angularly dependent spectra of betatron gamma-rays radiated by GeV electron beams from laser wakefield accelerators (LWFAs) are presented. The angle-resolved spectrum of betatron radiation was deconvolved from the position dependent data measured for a single laser shot with a broadband gamma-ray spectrometer comprising four-quadrant sectored range filters and an unfolding algorithm, based on the Monte Carlo code GEANT4. The unfolded gamma-ray spectra in the photon energy range of 0.1–10 MeV revealed an approximately isotropic angular dependence of the peak photon energy and photon energy-integrated fluence. As expected by the analysis of betatron radiation from LWFAs, the results indicate thatmore » unpolarized gamma-rays are emitted by electrons undergoing betatron motion in isotropically distributed orbit planes.« less

Broad Energy Range Neutron Spectroscopy using a Liquid Scintillator and a Proportional Counter: Application to a Neutron Spectrum Similar to that from an Improvised Nuclear Device

PubMed Central

Randers-Pehrson, Gerhard; Marino, Stephen A.; Garty, Guy; Harken, Andrew; Brenner, David J.

2015-01-01

A novel neutron irradiation facility at the Radiological Research Accelerator Facility (RARAF) has been developed to mimic the neutron radiation from an Improvised Nuclear Device (IND) at relevant distances (e.g. 1.5 km) from the epicenter. The neutron spectrum of this IND-like neutron irradiator was designed according to estimations of the Hiroshima neutron spectrum at 1.5 km. It is significantly different from a standard reactor fission spectrum, because the spectrum changes as the neutrons are transported through air, and it is dominated by neutron energies from 100 keV up to 9 MeV. To verify such wide energy range neutron spectrum, detailed here is the development of a combined spectroscopy system. Both a liquid scintillator detector and a gas proportional counter were used for the recoil spectra measurements, with the individual response functions estimated from a series of Monte Carlo simulations. These normalized individual response functions were formed into a single response matrix for the unfolding process. Several accelerator-based quasi-monoenergetic neutron source spectra were measured and unfolded to test this spectroscopy system. These reference neutrons were produced from two reactions: T(p,n)3He and D(d,n)3He, generating neutron energies in the range between 0.2 and 8 MeV. The unfolded quasi-monoenergetic neutron spectra indicated that the detection system can provide good neutron spectroscopy results in this energy range. A broad-energy neutron spectrum from the 9Be(d,n) reaction using a 5 MeV deuteron beam, measured at 60 degrees to the incident beam was measured and unfolded with the evaluated response matrix. The unfolded broad neutron spectrum is comparable with published time-of-flight results. Finally, the pair of detectors were used to measure the neutron spectrum generated at the RARAF IND-like neutron facility and a comparison is made to the neutron spectrum of Hiroshima. PMID:26273118

Broad Energy Range Neutron Spectroscopy using a Liquid Scintillator and a Proportional Counter: Application to a Neutron Spectrum Similar to that from an Improvised Nuclear Device.

PubMed

Xu, Yanping; Randers-Pehrson, Gerhard; Marino, Stephen A; Garty, Guy; Harken, Andrew; Brenner, David J

2015-09-11

A novel neutron irradiation facility at the Radiological Research Accelerator Facility (RARAF) has been developed to mimic the neutron radiation from an Improvised Nuclear Device (IND) at relevant distances (e.g. 1.5 km) from the epicenter. The neutron spectrum of this IND-like neutron irradiator was designed according to estimations of the Hiroshima neutron spectrum at 1.5 km. It is significantly different from a standard reactor fission spectrum, because the spectrum changes as the neutrons are transported through air, and it is dominated by neutron energies from 100 keV up to 9 MeV. To verify such wide energy range neutron spectrum, detailed here is the development of a combined spectroscopy system. Both a liquid scintillator detector and a gas proportional counter were used for the recoil spectra measurements, with the individual response functions estimated from a series of Monte Carlo simulations. These normalized individual response functions were formed into a single response matrix for the unfolding process. Several accelerator-based quasi-monoenergetic neutron source spectra were measured and unfolded to test this spectroscopy system. These reference neutrons were produced from two reactions: T(p,n) 3 He and D(d,n) 3 He, generating neutron energies in the range between 0.2 and 8 MeV. The unfolded quasi-monoenergetic neutron spectra indicated that the detection system can provide good neutron spectroscopy results in this energy range. A broad-energy neutron spectrum from the 9 Be(d,n) reaction using a 5 MeV deuteron beam, measured at 60 degrees to the incident beam was measured and unfolded with the evaluated response matrix. The unfolded broad neutron spectrum is comparable with published time-of-flight results. Finally, the pair of detectors were used to measure the neutron spectrum generated at the RARAF IND-like neutron facility and a comparison is made to the neutron spectrum of Hiroshima.

FRUIT: An operational tool for multisphere neutron spectrometry in workplaces

NASA Astrophysics Data System (ADS)

Bedogni, Roberto; Domingo, Carles; Esposito, Adolfo; Fernández, Francisco

2007-10-01

FRUIT (Frascati Unfolding Interactive Tool) is an unfolding code for Bonner sphere spectrometers (BSS) developed, under the Labview environment, at the INFN-Frascati National Laboratory. It models a generic neutron spectrum as the superposition of up to four components (thermal, epithermal, fast and high energy), fully defined by up to seven positive parameters. Different physical models are available to unfold the sphere counts, covering the majority of the neutron spectra encountered in workplaces. The iterative algorithm uses Monte Carlo methods to vary the parameters and derive the final spectrum as limit of a succession of spectra fulfilling the established convergence criteria. Uncertainties on the final results are evaluated taking into consideration the different sources of uncertainty affecting the input data. Relevant features of FRUIT are (1) a high level of interactivity, allowing the user to follow the convergence process, (2) the possibility to modify the convergence tolerances during the run, allowing a rapid achievement of meaningful solutions and (3) the reduced dependence of the results from the initial hypothesis. This provides a useful instrument for spectrometric measurements in workplaces, where detailed a priori information is usually unavailable. This paper describes the characteristics of the code and presents the results of performance tests over a significant variety of reference and workplace neutron spectra ranging from thermal up to hundreds MeV neutrons.

Designing an extended energy range single-sphere multi-detector neutron spectrometer

NASA Astrophysics Data System (ADS)

Gómez-Ros, J. M.; Bedogni, R.; Moraleda, M.; Esposito, A.; Pola, A.; Introini, M. V.; Mazzitelli, G.; Quintieri, L.; Buonomo, B.

2012-06-01

This communication describes the design specifications for a neutron spectrometer consisting of 31 thermal neutron detectors, namely Dysprosium activation foils, embedded in a 25 cm diameter polyethylene sphere which includes a 1 cm thick lead shell insert that degrades the energy of neutrons through (n,xn) reactions, thus allowing to extension of the energy range of the response up to hundreds of MeV neutrons. The new spectrometer, called SP2 (SPherical SPectrometer), relies on the same detection mechanism as that of the Bonner Sphere Spectrometer, but with the advantage of determining the whole neutron spectrum in a single exposure. The Monte Carlo transport code MCNPX was used to design the spectrometer in terms of sphere diameter, number and position of the detectors, position and thickness of the lead shell, as well as to obtain the response matrix for the final configuration. This work focuses on evaluating the spectrometric capabilities of the SP2 design by simulating the exposure of SP2 in neutron fields representing different irradiation conditions (test spectra). The simulated SP2 readings were then unfolded with the FRUIT unfolding code, in the absence of detailed pre-information, and the unfolded spectra were compared with the known test spectra. The results are satisfactory and allowed approving the production of a prototypal spectrometer.

Discernment of lint trash in raw cotton using multivariate analysis of excitation-emission luminescence spectra

USDA-ARS?s Scientific Manuscript database

Excitation-Emission luminescence spectra of basic (pH 12.5) phosphate buffer solution extracts were used to distinguish among botanical components of trash within seed cotton. All components were separated from whole plants removed from a field in southern New Mexico. Unfolded Principal Component An...

Search for Functional Flexible Regions in the G-protein Family: New Reading of the FoldUnfold Program.

PubMed

Galzitskaya, Oxana; Deryusheva, Eugenia; Machulin, Andrey; Nemashkalova, Ekaterina; Glyakina, Anna

2018-06-21

High prediction accuracy of flexible loops in different protein families is a challenge because of the crucial functions associated with these regions. Results of the currently available programs for prediction of loops vary from protein to protein. For prediction of flexible regions in the G-domain for 23 representatives of G-proteins with the known 3D structure we have used eight programs. The results of predictions demonstrate that the FoldUnfold program predicts better loop positions than the PONDR, RОNN, DisEMBL, IUPred, GlobPlot 2, FoldIndex, and MobiDB programs. When classifying the predicted loops (rigid/flexible) according to the Debye-Waller fluctuation factors, our data reveal the existing weak correlation between the B-factors and the average number of closed residues according to the FoldUnfold program; the percentage of overlapping characteristics (residue fold/unfold status) of the protein residues from the two methods is about 60-70%. According to the FoldUnfold program, for G-proteins with the posttranslational modifications, the surrounding binding site residues by disordered-promoting glycine and alanine residues conduces to a more flexible position of the binding sites for fatty acid, while methionine, cysteine and isoleucine residues provide more rigid binding sites. Thus, our research demonstrates additional possibilities of the FoldUnfold program for prediction of flexible regions and characteristics of individual residues in a different protein family. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Analysis of airborne radiometric data. Volume 3. Topical reports

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

Reed, J.H.; Shreve, D.C.; Sperling, M.

1978-05-01

This volume consists of four topical reports: a general discussion of the philosophy of unfolding spectra with continuum and discrete components, a mathematical treatment of the effects of various physical parameters on the uncollided gamma-ray spectrum at aircraft elevations, a discussion of the application of the unfolding code MAZNAI to airborne data, and a discussion of the effects of the nonlinear relationship between energy deposited and pulse height in NaI(T1) detectors.

Warhead verification as inverse problem: Applications of neutron spectrum unfolding from organic-scintillator measurements

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

Lawrence, Chris C.; Flaska, Marek; Pozzi, Sara A.

2016-08-14

Verification of future warhead-dismantlement treaties will require detection of certain warhead attributes without the disclosure of sensitive design information, and this presents an unusual measurement challenge. Neutron spectroscopy—commonly eschewed as an ill-posed inverse problem—may hold special advantages for warhead verification by virtue of its insensitivity to certain neutron-source parameters like plutonium isotopics. In this article, we investigate the usefulness of unfolded neutron spectra obtained from organic-scintillator data for verifying a particular treaty-relevant warhead attribute: the presence of high-explosive and neutron-reflecting materials. Toward this end, several improvements on current unfolding capabilities are demonstrated: deuterated detectors are shown to have superior response-matrixmore » condition to that of standard hydrogen-base scintintillators; a novel data-discretization scheme is proposed which removes important detector nonlinearities; and a technique is described for re-parameterizing the unfolding problem in order to constrain the parameter space of solutions sought, sidestepping the inverse problem altogether. These improvements are demonstrated with trial measurements and verified using accelerator-based time-of-flight calculation of reference spectra. Then, a demonstration is presented in which the elemental compositions of low-Z neutron-attenuating materials are estimated to within 10%. These techniques could have direct application in verifying the presence of high-explosive materials in a neutron-emitting test item, as well as other for treaty verification challenges.« less

Warhead verification as inverse problem: Applications of neutron spectrum unfolding from organic-scintillator measurements

NASA Astrophysics Data System (ADS)

Lawrence, Chris C.; Febbraro, Michael; Flaska, Marek; Pozzi, Sara A.; Becchetti, F. D.

2016-08-01

Verification of future warhead-dismantlement treaties will require detection of certain warhead attributes without the disclosure of sensitive design information, and this presents an unusual measurement challenge. Neutron spectroscopy—commonly eschewed as an ill-posed inverse problem—may hold special advantages for warhead verification by virtue of its insensitivity to certain neutron-source parameters like plutonium isotopics. In this article, we investigate the usefulness of unfolded neutron spectra obtained from organic-scintillator data for verifying a particular treaty-relevant warhead attribute: the presence of high-explosive and neutron-reflecting materials. Toward this end, several improvements on current unfolding capabilities are demonstrated: deuterated detectors are shown to have superior response-matrix condition to that of standard hydrogen-base scintintillators; a novel data-discretization scheme is proposed which removes important detector nonlinearities; and a technique is described for re-parameterizing the unfolding problem in order to constrain the parameter space of solutions sought, sidestepping the inverse problem altogether. These improvements are demonstrated with trial measurements and verified using accelerator-based time-of-flight calculation of reference spectra. Then, a demonstration is presented in which the elemental compositions of low-Z neutron-attenuating materials are estimated to within 10%. These techniques could have direct application in verifying the presence of high-explosive materials in a neutron-emitting test item, as well as other for treaty verification challenges.

Unfolding study of a trimeric membrane protein AcrB.

PubMed

Ye, Cui; Wang, Zhaoshuai; Lu, Wei; Wei, Yinan

2014-07-01

The folding of a multi-domain trimeric α-helical membrane protein, Escherichia coli inner membrane protein AcrB, was investigated. AcrB contains both a transmembrane domain and a large periplasmic domain. Protein unfolding in sodium dodecyl sulfate (SDS) and urea was monitored using the intrinsic fluorescence and circular dichroism spectroscopy. The SDS denaturation curve displayed a sigmoidal profile, which could be fitted with a two-state unfolding model. To investigate the unfolding of separate domains, a triple mutant was created, in which all three Trp residues in the transmembrane domain were replaced with Phe. The SDS unfolding profile of the mutant was comparable to that of the wild type AcrB, suggesting that the observed signal change was largely originated from the unfolding of the soluble domain. Strengthening of trimer association through the introduction of an inter-subunit disulfide bond had little effect on the unfolding profile, suggesting that trimer dissociation was not the rate-limiting step in unfolding monitored by fluorescence emission. Under our experimental condition, AcrB unfolding was not reversible. Furthermore, we experimented with the refolding of a monomeric mutant, AcrBΔloop , from the SDS unfolded state. The CD spectrum of the refolded AcrBΔloop superimposed well onto the spectra of the original folded protein, while the fluorescence spectrum was not fully recovered. In summary, our results suggested that the unfolding of the trimeric AcrB started with a local structural rearrangement. While the refolding of secondary structure in individual monomers could be achieved, the re-association of the trimer might be the limiting factor to obtain folded wild-type AcrB. © 2014 The Protein Society.

Calculation of response matrix of CaSO 4:Dy based neutron dosimeter using Monte Carlo code FLUKA and measurement of 241Am-Be spectra

NASA Astrophysics Data System (ADS)

Chatterjee, S.; Bakshi, A. K.; Tripathy, S. P.

2010-09-01

Response matrix for CaSO 4:Dy based neutron dosimeter was generated using Monte Carlo code FLUKA in the energy range thermal to 20 MeV for a set of eight Bonner spheres of diameter 3-12″ including the bare one. Response of the neutron dosimeter was measured for the above set of spheres for 241Am-Be neutron source covered with 2 mm lead. An analytical expression for the response function was devised as a function of sphere mass. Using Frascati Unfolding Iteration Tool (FRUIT) unfolding code, the neutron spectrum of 241Am-Be was unfolded and compared with standard IAEA spectrum for the same.

Simulation of the neutron response matrix of an EJ309 liquid scintillator

NASA Astrophysics Data System (ADS)

Bai, Huaiyong; Wang, Zhimin; Zhang, Luyu; Jiang, Haoyu; Lu, Yi; Chen, Jinxiang; Zhang, Guohui

2018-04-01

The neutron response matrix is the basis for measuring the neutron energy spectrum through unfolding the pulse height spectrum detected with a liquid scintillator. Based on the light output of the EJ309 liquid scintillator and the related reaction cross sections, a Monte Carlo code is developed to obtain the neutron response matrix. The effects of the related reactions, the contributions of different number of neutron interactions and the wall effect of the recoil proton are discussed. With the obtained neutron response matrix and the GRAVEL iterative unfolding method, the neutron energy spectra of the 252Cf and the 241AmBe neutron sources are measured, and the results are respectively compared with the theoretical prediction of the 252Cf neutron energy spectrum and the previous results of the 241AmBe neutron energy spectra.

Characterization of Deuterated-xylene Scintillator as a Neutron Spectrometer

DOE PAGES

Di Fulvio, Angela; Becchetti, F. D.; Raymond, R. S.; ...

2016-11-16

We have experimentally characterized the neutron light output response functions of a deuterated-xylene scintillator for neutron energies lower than 10 MeV. We then used the response matrix to unfold the energy distribution of neutrons produced via several reactions, i.e. spontaneous fission, d(d,n)3He, 27Al(d,n)28Si, and 9Be(alpha,n)12C. Organic scintillators based on deuterated compounds show a fast response and good gamma-neutron discrimination capability, similar to proton-based scintillators. Deuterated scintillators can also effectively provide neutron spectra by unfolding measured data with the detector response matrix, without the need of time-of-flight. Deuteron recoils, produced by elastic collisions between deuterium and impinging neutrons, are preferentially forward-scattered.more » This non-isotropic reaction results in distinct peaks in the response functions to monoenergetic neutrons. In this work, we evaluated a custom-fabricated 7.62 cm x 7.62 cm deuterated-xylene (EJ301D) liquid scintillator. This liquid has a low volatility and higher flash point, compared to benzene-based deuterated detectors, e.g. EJ315 and NE230. We measured the EJ301D detector neutron response matrix (up to 6 MeV neutron energy) using an intense Cf252 source and the time-of-flight technique. The number of response functions obtained using our method is only limited by counting statistics and by the experimentally achievable energy resolution. Multi-channel unfolding was performed successfully for neutron spectra with different energy spectra.« less

The use of spin desalting columns in DMSO-quenched H/D-exchange NMR experiments

PubMed Central

Chandak, Mahesh S; Nakamura, Takashi; Takenaka, Toshio; Chaudhuri, Tapan K; Yagi-Utsumi, Maho; Chen, Jin; Kato, Koichi; Kuwajima, Kunihiro

2013-01-01

Dimethylsulfoxide (DMSO)-quenched hydrogen/deuterium (H/D)-exchange is a powerful method to characterize the H/D-exchange behaviors of proteins and protein assemblies, and it is potentially useful for investigating non-protected fast-exchanging amide protons in the unfolded state. However, the method has not been used for studies on fully unfolded proteins in a concentrated denaturant or protein solutions at high salt concentrations. In all of the current DMSO-quenched H/D-exchange studies of proteins so far reported, lyophilization was used to remove D2O from the protein solution, and the lyophilized protein was dissolved in the DMSO solution to quench the H/D exchange reactions and to measure the amide proton signals by two-dimensional nuclear magnetic resonance (2D NMR) spectra. The denaturants or salts remaining after lyophilization thus prevent the measurement of good NMR spectra. In this article, we report that the use of spin desalting columns is a very effective alternative to lyophilization for the medium exchange from the D2O buffer to the DMSO solution. We show that the medium exchange by a spin desalting column takes only about 10 min in contrast to an overnight length of time required for lyophilization, and that the use of spin desalting columns has made it possible to monitor the H/D-exchange behavior of a fully unfolded protein in a concentrated denaturant. We report the results of unfolded ubiquitin in 6.0M guanidinium chloride. PMID:23339068

UFO (UnFold Operator) computer program abstract

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

Kissel, L.; Biggs, F.

UFO (UnFold Operator) is an interactive user-oriented computer program designed to solve a wide range of problems commonly encountered in physical measurements. This document provides a summary of the capabilities of version 3A of UFO.

Advances in Neutron Spectroscopy with Deuterated Organic Scintillators

NASA Astrophysics Data System (ADS)

Febbraro, Michael; Pain, Steve; Becchetti, Frederick

2015-10-01

Deuterated organic scintillators have shown promise as neutron detectors for nuclear science as well as applications in nuclear non-proliferation and safeguards. In particular, they can extract neutron spectra without the use of neutron time-of-flight measurement (n-ToF) utilizing spectrum unfolding techniques. This permits the measure of cross sections of bound and unbound states with high efficiency and angular coverage. In the case of measurements with radioactive ion beams where low beam intensities limit long path n-ToF, short path n-ToF can be used to discriminate neutrons of interest from room return and background neutrons. This presentation will provide recent advances with these types of detectors. Digital pulse-shape discrimination using fast waveform digitizers, spectrum unfolding methods for extraction of neutron spectra, and a new safer deuterated-xylene formulation EJ-301D will be discussed. In addition, experimental results from measurements of discrete and continuous neutron spectra which illustrate the advantage of these detectors for certain applications in nuclear physics research and nuclear security will be shown. This work is supported by NSF and DOE.

Unfolding the neutron spectrum of a NE213 scintillator using artificial neural networks.

PubMed

Sharghi Ido, A; Bonyadi, M R; Etaati, G R; Shahriari, M

2009-10-01

Artificial neural networks technology has been applied to unfold the neutron spectra from the pulse height distribution measured with NE213 liquid scintillator. Here, both the single and multi-layer perceptron neural network models have been implemented to unfold the neutron spectrum from an Am-Be neutron source. The activation function and the connectivity of the neurons have been investigated and the results have been analyzed in terms of the network's performance. The simulation results show that the neural network that utilizes the Satlins transfer function has the best performance. In addition, omitting the bias connection of the neurons improve the performance of the network. Also, the SCINFUL code is used for generating the response functions in the training phase of the process. Finally, the results of the neural network simulation have been compared with those of the FORIST unfolding code for both (241)Am-Be and (252)Cf neutron sources. The results of neural network are in good agreement with FORIST code.

Stabilization of Particle Discrimination Efficiencies for Neutron Spectrum Unfolding With Organic Scintillators

NASA Astrophysics Data System (ADS)

Lawrence, Chris C.; Polack, J. K.; Febbraro, Michael; Kolata, J. J.; Flaska, Marek; Pozzi, S. A.; Becchetti, F. D.

2017-02-01

The literature discussing pulse-shape discrimination (PSD) in organic scintillators dates back several decades. However, little has been written about PSD techniques that are optimized for neutron spectrum unfolding. Variation in n-γ misclassification rates and in γ/n ratio of incident fields can distort the neutron pulse-height response of scintillators and these distortions can in turn cause large errors in unfolded spectra. New applications in arms-control verification call for detection of lower-energy neutrons, for which PSD is particularly problematic. In this article, we propose techniques for removing distortions on pulse-height response that result from the merging of PSD distributions in the low-pulse-height region. These techniques take advantage of the repeatable shapes of PSD distributions that are governed by the counting statistics of scintillation-photon populations. We validate the proposed techniques using accelerator-based time-of-flight measurements and then demonstrate them by unfolding the Watt spectrum from measurement with a 252Cf neutron source.

Estimation of neutron spectrum in the low-level gamma spectroscopy system using unfolding procedure

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

Knežević, D., E-mail: david.knezevic@df.uns.ac.rs; Jovančević, N.; Krmar, M.

2016-03-25

The radiation resulting from neutron interactions with Ge nuclei in active volume of HPGe detectors is one of the main concerns in low-level gamma spectroscopy measurements [1,2]. It is usually not possible to measure directly spectrum of neutrons which strike detector. This paper explore the possibility of estimation of neutron spectrum using measured activities of certain Ge(n,γ) and Ge(n,n’) reactions (obtained from low-level gamma measurements), available ENDF cross section data and unfolding procedures. In this work HPGe detector with passive shield made from commercial low background lead was used for the measurement. The most important objective of this study wasmore » to reconstruct muon induced neutron spectrum created in the shield of the HPGe detector. MAXED [3] and GRAVEL [4] algorithms for neutron spectra unfolding were used. The results of those two algorithms were compared and we analyzed the sensitivity of the unfolding procedure to the various input parameters.« less

A new response matrix for a 6LiI scintillator BSS system

NASA Astrophysics Data System (ADS)

Lacerda, M. A. S.; Méndez-Villafañe, R.; Lorente, A.; Ibañez, S.; Gallego, E.; Vega-Carrillo, H. R.

2017-10-01

A new response matrix was calculated for a Bonner Sphere Spectrometer (BSS) with a 6 LiI(Eu) scintillator, using the Monte Carlo N-Particle radiation transport code MCNPX. Responses were calculated for 6 spheres and the bare detector, for energies varying from 1.059E(-9) MeV to 105.9 MeV, with 20 equal-log(E)-width bins per energy decade, totalizing 221 energy groups. A comparison was done among the responses obtained in this work and other published elsewhere, for the same detector model. The calculated response functions were inserted in the response input file of the MAXED code and used to unfold the total and direct neutron spectra generated by the 241Am-Be source of the Universidad Politécnica de Madrid (UPM). These spectra were compared with those obtained using the same unfolding code with the Mares and Schraube matrix response.

Performance of Large Neutron Detectors Containing Lithium-Gadolinium-Borate Scintillator

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

Slaughter, David M.; Stuart, Cory R.; Klaass, R. Fred

2015-07-01

This paper describes the development and testing of a neutron counter, spectrometer, and dosimeter that is compact, efficient, and accurate. A self-contained neutron detection instrument has wide applications in health physics, scientific research, and programs to detect, monitor, and control strategic nuclear materials (SNM). The 1.3 liter detector head for this instrument is a composite detector with an organic scintillator containing uniformly distributed {sup 6}Li{sub 6}{sup nat}Gd{sup 10}B{sub 3}O{sub 9}:Ce (LGB:Ce) microcrystals. The plastic scintillator acts to slow impinging neutrons and emits light proportional to the energy lost by the neutrons as they moderate in the detector body. Moderating neutronsmore » that have slowed sufficiently capture in one of the Lithium-6, Boron-10, or Gadolinium-157 atoms in the LGB:Ce scintillator, which then releases the capture energy in a characteristic cerium emission pulse. The measured captured pulses indicate the presence of neutrons. When a scintillating fluor is present in the plastic, the light pulse resulting from the neutron moderating in the plastic is paired with the LGB:Ce capture pulse to identify the energy of the neutron. About 2% of the impinging neutrons lose all of their energy in a single collision with the detector. There is a linear relationship between the pulse areas of this group of neutrons and energy. The other 98% of neutrons have a wide range of collision histories within the detector body. When these neutrons are 'binned' into energy groups, each group contains a distribution of pulse areas. This data was used to assist in the unfolding of the neutron spectra. The unfolded spectra were then validated with known spectra, at both neutron emitting isotopes and fission/accelerator facilities. Having validated spectra, the dose equivalent and dose rate are determined by applying standard, regulatory damage coefficients to the measured neutron counts for each energy bin of the spectra. Testing at the Los Alamos Neutron Science Center (LANSCE), Edwards Accelerator Laboratory (EAL) at Ohio University and the Radiation Center at University of Massachusetts-Lowell has demonstrated that the instrument can measure neutrons and their spectra over the range between 0.8 MeV and 150 MeV with an uncertainty of only ± 8%. An independent test of the LGB:Ce neutron spectrometer was conducted by a US Defense Threat Reduction Agency (DTRA) team at the Idaho National Laboratory (INL). The results of this evaluation showed that the neutron spectrometer accurately identified bare radioactive isotopes by their spectra. Further, masking and shielding materials alter those spectra in predictable ways that permit an extrapolation from the observed spectra back to the identity of the isotopic spectrum. (authors)« less

Neutron spectrometry and dosimetry in 100 and 300 MeV quasi-mono-energetic neutron field at RCNP, Osaka University, Japan

NASA Astrophysics Data System (ADS)

Mares, Vladimir; Trinkl, Sebastian; Iwamoto, Yosuke; Masuda, Akihiko; Matsumoto, Tetsuro; Hagiwara, Masayuki; Satoh, Daiki; Yashima, Hiroshi; Shima, Tatsushi; Nakamura, Takashi

2017-09-01

This paper describes the results of neutron spectrometry and dosimetry measurements using an extended range Bonner Sphere Spectrometer (ERBSS) with 3He proportional counter performed in quasi-mono-energetic neutron fields at the ring cyclotron facility of the Research Center for Nuclear Physics (RCNP), Osaka University, Japan. Using 100 MeV and 296 MeV proton beams, neutron fields with nominal peak energies of 96 MeV and 293 MeV were generated via 7Li(p,n)7Be reactions. Neutrons produced at 0° and 25° emission angles were extracted into the 100 m long time-of-flight (TOF) tunnel, and the energy spectra were measured at a distance of 35 m from the target. To deduce the corresponding neutron spectra from thermal to the nominal maximum energy, the ERBSS data were unfolded using the MSANDB unfolding code. At high energies, the neutron spectra were also measured by means of the TOF method using NE213 organic liquid scintillators. The results are discussed in terms of ambient dose equivalent, H*(10), and compared with the readings of other instruments operated during the experiment.

Developing a Novel, Interdisciplinary Approach to Study Protein Unfolding

NASA Astrophysics Data System (ADS)

Bentley, Ian; Link, Justin

2013-03-01

The ability of a protein to function is a direct result of its ability to properly obtain its native, folded structure. In order to determine the structural stability of proteins and to gain knowledge of their folding mechanism, we must develop protocols that allow us to monitor the controlled unfolding of proteins. Here, we investigate the stability of cytochrome c, a well-studied, model protein, under denaturing conditions using circular dichroism (CD) and fluorescence. Using either a chemical denaturant (Guanidine HCl) or heat, we can cause a protein to gradually unfold. The changes in the fluorescence and CD spectra can provide insight into the stability of proteins by providing us with thermodynamic parameters such as the Gibbs free energy, melting temperature and enthalpy. Research in this lab has been explored with mutant proteins and change in CD signal, however further work must still be done to observe their unfolding monitored by fluorescence. This technique will allow us to determine which regions of native cytochrome c have the greatest impact on the protein folding process. The objective of this session is to present recent work in developing a protocol to observe the unfolding of wild type and mutant proteins with fluorescence. The Borcer Fund, The John A. Hauck Foundation, and Xavier University

Conformational stability of apoflavodoxin.

PubMed Central

Genzor, C. G.; Beldarraín, A.; Gómez-Moreno, C.; López-Lacomba, J. L.; Cortijo, M.; Sancho, J.

1996-01-01

Flavodoxins are alpha/beta proteins that mediate electron transfer reactions. The conformational stability of apoflavodoxin from Anaboena PCC 7119 has been studied by calorimetry and urea denaturation as a function of pH and ionic strength. At pH > 12, the protein is unfolded. Between pH 11 and pH 6, the apoprotein is folded properly as judged from near-ultraviolet (UV) circular dichroism (CD) and high-field 1H NMR spectra. In this pH interval, apoflavodoxin is a monomer and its unfolding by urea or temperature follows a simple two-state mechanism. The specific heat capacity of unfolding for this native conformation is unusually low. Near its isoelectric point (3.9), the protein is highly insoluble. At lower pH values (pH 3.5-2.0), apoflavodoxin adopts a conformation with the properties of a molten globule. Although apoflavodoxin at pH 2 unfolds cooperatively with urea in a reversible fashion and the fluorescence and far-UV CD unfolding curves coincide, the transition midpoint depends on the concentration of protein, ruling out a simple two-state process at acidic pH. Apoflavodoxin constitutes a promising system for the analysis of the stability and folding of alpha/beta proteins and for the study of the interaction between apoflavoproteins and their corresponding redox cofactors. PMID:8819170

A novel neutron energy spectrum unfolding code using particle swarm optimization

NASA Astrophysics Data System (ADS)

Shahabinejad, H.; Sohrabpour, M.

2017-07-01

A novel neutron Spectrum Deconvolution using Particle Swarm Optimization (SDPSO) code has been developed to unfold the neutron spectrum from a pulse height distribution and a response matrix. The Particle Swarm Optimization (PSO) imitates the bird flocks social behavior to solve complex optimization problems. The results of the SDPSO code have been compared with those of the standard spectra and recently published Two-steps Genetic Algorithm Spectrum Unfolding (TGASU) code. The TGASU code have been previously compared with the other codes such as MAXED, GRAVEL, FERDOR and GAMCD and shown to be more accurate than the previous codes. The results of the SDPSO code have been demonstrated to match well with those of the TGASU code for both under determined and over-determined problems. In addition the SDPSO has been shown to be nearly two times faster than the TGASU code.

A Bayesian Approach for Measurements of Stray Neutrons at Proton Therapy Facilities: Quantifying Neutron Dose Uncertainty.

PubMed

Dommert, M; Reginatto, M; Zboril, M; Fiedler, F; Helmbrecht, S; Enghardt, W; Lutz, B

2017-11-28

Bonner sphere measurements are typically analyzed using unfolding codes. It is well known that it is difficult to get reliable estimates of uncertainties for standard unfolding procedures. An alternative approach is to analyze the data using Bayesian parameter estimation. This method provides reliable estimates of the uncertainties of neutron spectra leading to rigorous estimates of uncertainties of the dose. We extend previous Bayesian approaches and apply the method to stray neutrons in proton therapy environments by introducing a new parameterized model which describes the main features of the expected neutron spectra. The parameterization is based on information that is available from measurements and detailed Monte Carlo simulations. The validity of this approach has been validated with results of an experiment using Bonner spheres carried out at the experimental hall of the OncoRay proton therapy facility in Dresden. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Investigation of structural changes of β-casein and lysozyme at the gas-liquid interface during foam fractionation.

PubMed

Barackov, Ivana; Mause, Anika; Kapoor, Shobhna; Winter, Roland; Schembecker, Gerhard; Burghoff, Bernhard

2012-10-15

Purification and separation of proteins play a major role in biotechnology. Nowadays, alternatives to multistep operations suffering from low product yields and high costs are investigated closely amidst which one of the promising options is foam fractionation. The molecular behavior at the gas-liquid interface plays an important role in the formation and stabilization of enriched foam. This study for the first time correlates the physico-chemical parameters to the molecular structure in view of protein enrichment during foam fractionation of the two relatively different proteins lysozyme and β-casein employing biophysical techniques such as circular dichroism (CD) spectroscopy and infrared reflection absorption spectroscopy (IRRAS). In case of lysozyme, high enrichment was achieved at pH

PHOTON SPECTRA IN NPL STANDARD RADIONUCLIDE NEUTRON FIELDS.

PubMed

Roberts, N J

2017-09-23

A HPGe detector has been used to measure the photon spectra from the majority of radionuclide neutron sources in use at NPL (252Cf, 241Am-Be, 241Am-Li, 241Am-B). The HPGe was characterised then modelled to produce a response matrix. The measured pulse height spectra were then unfolded to produce photon fluence spectra. Changes in the photon spectrum with time from a 252Cf source are evident. Spectra from a 2-year-old and 42-year-old 252Cf source are presented showing the change from a continuum to peaks from long-lived isotopes of Cf. Other radionuclide neutron source spectra are also presented and discussed. The new spectra were used to improve the photon to neutron dose equivalent ratios from some earlier work at NPL with GM tubes and EPDs. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Comparison of neutron spectra measured with three sizes of organic liquid scintillators using differentiation analysis

NASA Technical Reports Server (NTRS)

Shook, D. F.; Pierce, C. R.

1972-01-01

Proton recoil distributions were obtained by using organic liquid scintillators of different size. The measured distributions are converted to neutron spectra by differentiation analysis for comparison to the unfolded spectra of the largest scintillator. The approximations involved in the differentiation analysis are indicated to have small effects on the precision of neutron spectra measured with the smaller scintillators but introduce significant error for the largest scintillator. In the case of the smallest cylindrical scintillator, nominally 1.2 by 1.3 cm, the efficiency is shown to be insensitive to multiple scattering and to the angular distribution to the incident flux. These characteristics of the smaller scintillator make possible its use to measure scalar flux spectra within media high efficiency is not required.

Characterization of the Caliban and Prospero Critical Assemblies Neutron Spectra for Integral Measurements Experiments

NASA Astrophysics Data System (ADS)

Casoli, P.; Authier, N.; Jacquet, X.; Cartier, J.

2014-04-01

Caliban and Prospero are two highly enriched uranium metallic core reactors operated on the CEA Center of Valduc. These critical assemblies are suitable for integral experiments, such as fission yields measurements or perturbation measurements, which have been carried out recently on the Caliban reactor. Different unfolding methods, based on activation foils and fission chambers measurements, are used to characterize the reactor spectra and especially the Caliban spectrum, which is very close to a pure fission spectrum.

Measurement of protein unfolding/refolding kinetics and structural characterization of hidden intermediates by NMR relaxation dispersion

PubMed Central

Meinhold, Derrick W.; Wright, Peter E.

2011-01-01

Detailed understanding of protein function and malfunction hinges on the ability to characterize transiently populated states and the transitions between them. Here, we use 15N, , and 13CO NMR R2 relaxation dispersion to investigate spontaneous unfolding and refolding events of native apomyoglobin. Above pH 5.0, dispersion is dominated by processes involving fluctuations of the F-helix region, which is invisible in NMR spectra. Measurements of R2 dispersion for residues contacted by the F-helix region in the native (N) structure reveal a transient state formed by local unfolding of helix F and undocking from the protein core. A similar state was detected at pH 4.75–4.95 and determined to be an on-pathway intermediate (I1) in a linear three-state unfolding scheme (N⇆I1⇆MG) leading to a transiently populated molten globule (MG) state. The slowest steps in unfolding and refolding are N → I1 (36 s-1) and MG → I1 (26 s-1), respectively. Differences in chemical shift between N and I1 are very small, except in regions adjacent to helix F, showing that their core structures are similar. Chemical shift changes between the N and MG states, obtained from R2 dispersion, reveal that the transient MG state is structurally similar to the equilibrium MG observed previously at high temperature and low pH. Analysis of MG state chemical shifts shows the location of residual helical structure in the transient intermediate and identifies regions that unfold or rearrange into nonnative structure during the N → MG transition. The experiments also identify regions of energetic frustration that “crack” during unfolding and impede the refolding process. PMID:21562212

Upgrades of DARWIN, a dose and spectrum monitoring system applicable to various types of radiation over wide energy ranges

NASA Astrophysics Data System (ADS)

Sato, Tatsuhiko; Satoh, Daiki; Endo, Akira; Shigyo, Nobuhiro; Watanabe, Fusao; Sakurai, Hiroki; Arai, Yoichi

2011-05-01

A dose and spectrum monitoring system applicable to neutrons, photons and muons over wide ranges of energy, designated as DARWIN, has been developed for radiological protection in high-energy accelerator facilities. DARWIN consists of a phoswitch-type scintillation detector, a data-acquisition (DAQ) module for digital waveform analysis, and a personal computer equipped with a graphical-user-interface (GUI) program for controlling the system. The system was recently upgraded by introducing an original DAQ module based on a field programmable gate array, FPGA, and also by adding a function for estimating neutron and photon spectra based on an unfolding technique without requiring any specific scientific background of the user. The performance of the upgraded DARWIN was examined in various radiation fields, including an operational field in J-PARC. The experiments revealed that the dose rates and spectra measured by the upgraded DARWIN are quite reasonable, even in radiation fields with peak structures in terms of both spectrum and time variation. These results clearly demonstrate the usefulness of DARWIN for improving radiation safety in high-energy accelerator facilities.

SU-E-T-272: Direct Verification of a Treatment Planning System Megavoltage Linac Beam Photon Spectra Models, and Analysis of the Effects On Patient Plans

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

Leheta, D; Shvydka, D; Parsai, E

2015-06-15

Purpose: For the photon dose calculation Philips Pinnacle Treatment Planning System (TPS) uses collapsed cone convolution algorithm, which relies on energy spectrum of the beam in computing the scatter component. The spectrum is modeled based on Linac’s standard commissioning data and typically is not independently verified. We explored a methodology of using transmission measurements in combination with regularization data processing to unfold Linac spectra. The measured spectra were compared to those modeled by the TPS, and the effect on patient plans was evaluated. Methods: Transmission measurements were conducted in narrow-beam geometry using a standard Farmer ionization chamber. Two attenuating materialsmore » and two build -up caps, having different atomic numbers, served to enhance discrimination between absorption of low and high-energy portions of the spectra, thus improving the accuracy of the results. The data was analyzed using a regularization technique implemented through spreadsheet-based calculations. Results: The unfolded spectra were found to deviate from the TPS beam models. The effect of such deviations on treatment planning was evaluated for patient plans through dose distribution calculations with either TPS modeled or measured energy spectra. The differences were reviewed through comparison of isodose distributions, and quantified based on maximum dose values for critical structures. While in most cases no drastic differences in the calculated doses were observed, plans with deviations of 4 to 8% in the maximum dose values for critical structures were discovered. The anatomical sites with large scatter contributions are the most vulnerable to inaccuracies in the modeled spectrum. Conclusion: An independent check of the TPS model spectrum is highly desirable and should be included as part of commissioning of a new Linac. The effect is particularly important for dose calculations in high heterogeneity regions. The developed approach makes acquisition of megavoltage Linac beam spectra achievable in a typical radiation oncology clinic.« less

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.

Formation of insulin amyloid fibrils followed by FTIR simultaneously with CD and electron microscopy.

PubMed Central

Bouchard, M.; Zurdo, J.; Nettleton, E. J.; Dobson, C. M.; Robinson, C. V.

2000-01-01

Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD), and electron microscopy (EM) have been used simultaneously to follow the temperature-induced formation of amyloid fibrils by bovine insulin at acidic pH. The FTIR and CD data confirm that, before heating, insulin molecules in solution at pH 2.3 have a predominantly native-like alpha-helical structure. On heating to 70 degrees C, partial unfolding occurs and results initially in aggregates that are shown by CD and FT-IR spectra to retain a predominantly helical structure. Following this step, changes in the CD and FTIR spectra occur that are indicative of the extensive conversion of the molecular conformation from alpha-helical to beta-sheet structure. At later stages, EM shows the development of fibrils with well-defined repetitive morphologies including structures with a periodic helical twist of approximately 450 A. The results indicate that formation of fibrils by insulin requires substantial unfolding of the native protein, and that the most highly ordered structures result from a slow evolution of the morphology of the initially formed fibrillar species. PMID:11106169

Measured neutron and gamma spectra from californium-252 in a tissue-equivalent medium.

PubMed

Elson, H R; Stupar, T A; Shapiro, A; Kereiakes, J G

1979-01-01

A method of experimentally obtaining both neutron and gamma-ray spectra in a scattering medium is described. The method utilizes a liquid-organic scintillator (NE-213) coupled with a pulse-shape discrimination circuit. This allows the separation of the neutron-induced pulse-height data from the gamma-ray pulse-height data. Using mathematical unfolding techniques, the two sets of pulse-height data were transformed to obtain the neutron and gamma-ray energy spectra. A small spherical detector was designed and constructed to reduce the errors incurred by attempting spectral measurements in a scattering medium. Demonstration of the utility of the system to obtain the neutron and gamma-ray spectra in a scattering medium was performed by characterizing the neutron and gamma-ray spectra at various sites about a 3.7-microgram (1.5 cm active length) californium-252 source in a tissue-equivalent medium.

Study of protein folding under native conditions by rapidly switching the hydrostatic pressure inside an NMR sample cell

PubMed Central

Charlier, Cyril; Alderson, T. Reid; Courtney, Joseph M.; Ying, Jinfa; Anfinrud, Philip

2018-01-01

In general, small proteins rapidly fold on the timescale of milliseconds or less. For proteins with a substantial volume difference between the folded and unfolded states, their thermodynamic equilibrium can be altered by varying the hydrostatic pressure. Using a pressure-sensitized mutant of ubiquitin, we demonstrate that rapidly switching the pressure within an NMR sample cell enables study of the unfolded protein under native conditions and, vice versa, study of the native protein under denaturing conditions. This approach makes it possible to record 2D and 3D NMR spectra of the unfolded protein at atmospheric pressure, providing residue-specific information on the folding process. 15N and 13C chemical shifts measured immediately after dropping the pressure from 2.5 kbar (favoring unfolding) to 1 bar (native) are close to the random-coil chemical shifts observed for a large, disordered peptide fragment of the protein. However, 15N relaxation data show evidence for rapid exchange, on a ∼100-μs timescale, between the unfolded state and unstable, structured states that can be considered as failed folding events. The NMR data also provide direct evidence for parallel folding pathways, with approximately one-half of the protein molecules efficiently folding through an on-pathway kinetic intermediate, whereas the other half fold in a single step. At protein concentrations above ∼300 μM, oligomeric off-pathway intermediates compete with folding of the native state. PMID:29666248

Thermal and urea-induced unfolding in T7 RNA polymerase: Calorimetry, circular dichroism and fluorescence study

PubMed Central

Griko, Yuri; Sreerama, Narasimha; Osumi-Davis, Patricia; Woody, Robert W.; Woody, A-Young Moon

2001-01-01

Structural changes in T7 RNA polymerase (T7RNAP) induced by temperature and urea have been studied over a wide range of conditions to obtain information about the structural organization and the stability of the enzyme. T7RNAP is a large monomeric enzyme (99 kD). Calorimetric studies of the thermal transitions in T7RNAP show that the enzyme consists of three cooperative units that may be regarded as structural domains. Interactions between these structural domains and their stability strongly depend on solvent conditions. The unfolding of T7RNAP under different solvent conditions induces a highly stable intermediate state that lacks specific tertiary interactions, contains a significant amount of residual secondary structure, and undergoes further cooperative unfolding at high urea concentrations. Circular dichroism (CD) studies show that thermal unfolding leads to an intermediate state that has increased β-sheet and reduced α-helix content relative to the native state. Urea-induced unfolding at 25°C reveals a two-step process. The first transition centered near 3 M urea leads to a plateau from 3.5 to 5.0 M urea, followed by a second transition centered near 6.5 M urea. The CD spectrum of the enzyme in the plateau region, which is similar to that of the enzyme thermally unfolded in the absence of urea, shows little temperature dependence from 15° to 60°C. The second transition leads to a mixture of poly(Pro)II and unordered conformations. As the temperature increases, the ellipticity at 222 nm becomes more negative because of conversion of poly(Pro)II to the unordered conformation. Near-ultraviolet CD spectra at 25°C at varying concentrations of urea are consistent with this picture. Both thermal and urea denaturation are irreversible, presumably because of processes that follow unfolding. PMID:11274475

Characterization of gamma rays existing in the NMIJ standard neutron field.

PubMed

Harano, H; Matsumoto, T; Ito, Y; Uritani, A; Kudo, K

2004-01-01

Our laboratory provides national standards on fast neutron fluence. Neutron fields are always accompanied by gamma rays produced in neutron sources and surroundings. We have characterised these gamma rays in the 5.0 MeV standard neutron field. Gamma ray measurement was performed using an NE213 liquid scintillator. Pulse shape discrimination was incorporated to separate the events induced by gamma rays from those by neutrons. The measured gamma ray spectra were unfolded with the HEPRO program package to obtain the spectral fluences using the response matrix prepared with the EGS4 code. Corrections were made for the gamma rays produced by neutrons in the detector assembly using the MCNP4C code. The effective dose equivalents were estimated to be of the order of 25 microSv at the neutron fluence of 10(7) neutrons cm(-2).

Study of dynamical process of heat denaturation in optically trapped single microorganisms by near-infrared Raman spectroscopy

NASA Astrophysics Data System (ADS)

Xie, Changan; Li, Yong-qing; Tang, Wei; Newton, Ronald J.

2003-11-01

The development of laser traps has made it possible to investigate single cells and record real-time Raman spectra during a heat-denaturation process when the temperature of the surrounding medium is increased. Large changes in the phenylalanine band (1004 cm-1) of near-infrared spectra between living and heat-treated cells were observed in yeast and Escerichia coli and Enterobacter aerogenes bacteria. This change appears to reflect the change in environment of phenylalanine as proteins within the cells unfold as a result of increasing temperatures. As a comparison, we measured Raman spectra of native and heat-denatured solutions of bovine serum albumin proteins, and a similar change in the phenylalanine band of spectra was observed. In addition, we measured Raman spectra of native and heat-treated solutions of pure phenylalanine molecules; no observable difference in vibrational spectra was observed. These findings may make it possible to study conformational changes in proteins within single cells.

Neutron and gamma-ray energy reconstruction for characterization of special nuclear material

DOE PAGES

Clarke, Shaun D.; Hamel, Michael C.; Di fulvio, Angela; ...

2017-06-30

Characterization of special nuclear material may be performed using energy spectroscopy of either the neutron or gamma-ray emissions from the sample. Gamma-ray spectroscopy can be performed relatively easily using high-resolution semiconductors such as high-purity germanium. Neutron spectroscopy, by contrast, is a complex inverse problem. Here, results are presented for 252Cf and PuBe energy spectra unfolded using a single EJ309 organic scintillator; excellent agreement is observed with the reference spectra. Neutron energy spectroscopy is also possible using a two-plane detector array, whereby time-offlight kinematics can be used. With this system, energy spectra can also be obtained as a function of position.more » Finally, spatial-dependent energy spectra are presented for neutron and gamma-ray sources that are in excellent agreement with expectations.« less

Neutron and gamma-ray energy reconstruction for characterization of special nuclear material

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

Clarke, Shaun D.; Hamel, Michael C.; Di fulvio, Angela

Characterization of special nuclear material may be performed using energy spectroscopy of either the neutron or gamma-ray emissions from the sample. Gamma-ray spectroscopy can be performed relatively easily using high-resolution semiconductors such as high-purity germanium. Neutron spectroscopy, by contrast, is a complex inverse problem. Here, results are presented for 252Cf and PuBe energy spectra unfolded using a single EJ309 organic scintillator; excellent agreement is observed with the reference spectra. Neutron energy spectroscopy is also possible using a two-plane detector array, whereby time-offlight kinematics can be used. With this system, energy spectra can also be obtained as a function of position.more » Finally, spatial-dependent energy spectra are presented for neutron and gamma-ray sources that are in excellent agreement with expectations.« less

Measurement of jet spectra in Pb-Pb collisions at √{sNN} = 2.76TeV with the ALICE detector at the LHC

NASA Astrophysics Data System (ADS)

Verweij, Marta

2013-08-01

We report a measurement of transverse momentum spectra of jets detected with the ALICE detector in Pb-Pb collisions at √{sNN} = 2.76TeV. Jets are reconstructed from charged particles using the anti-kT jet algorithm. The background from soft particle production is determined for each event and subtracted. The remaining influence of underlying event fluctuations is quantified by embedding different probes into heavy-ion data. The reconstructed transverse momentum spectrum is corrected for background fluctuations by unfolding. We compare the inclusive jet spectra reconstructed with R = 0.2 and R = 0.3 for different centrality classes and compare the jet yield in Pb-Pb and pp events.

Poster - 25: Neutron Spectral Measurements around a Scanning Proton Beam

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

Kildea, John; Enger, Shirin; Maglieri, Robert

We describe the measurements of neutron spectra that we undertook around a scanning proton beam at the Skandion proton therapy clinic in Uppsala, Sweden. Measurements were undertaken using an extended energy range Nested Neutron Spectrometer (NNS, Detec Inc., Gatineau, QC) operated in pulsed and current mode. Spectra were measured as a function of location in the treatment room and for various Bragg peak depths. Our preliminary unfolded data clearly show the direct, evaporation and thermal neutron peaks and we can show the effect on the neutron spectrum of a water phantom in the primary proton beam.

Unfolding the mechanism of the AAA+ unfoldase VAT by a combined cryo-EM, solution NMR study.

PubMed

Huang, Rui; Ripstein, Zev A; Augustyniak, Rafal; Lazniewski, Michal; Ginalski, Krzysztof; Kay, Lewis E; Rubinstein, John L

2016-07-19

The AAA+ (ATPases associated with a variety of cellular activities) enzymes play critical roles in a variety of homeostatic processes in all kingdoms of life. Valosin-containing protein-like ATPase of Thermoplasma acidophilum (VAT), the archaeal homolog of the ubiquitous AAA+ protein Cdc48/p97, functions in concert with the 20S proteasome by unfolding substrates and passing them on for degradation. Here, we present electron cryomicroscopy (cryo-EM) maps showing that VAT undergoes large conformational rearrangements during its ATP hydrolysis cycle that differ dramatically from the conformational states observed for Cdc48/p97. We validate key features of the model with biochemical and solution methyl-transverse relaxation optimized spectroscopY (TROSY) NMR experiments and suggest a mechanism for coupling the energy of nucleotide hydrolysis to substrate unfolding. These findings illustrate the unique complementarity between cryo-EM and solution NMR for studies of molecular machines, showing that the structural properties of VAT, as well as the population distributions of conformers, are similar in the frozen specimens used for cryo-EM and in the solution phase where NMR spectra are recorded.

Uncertainty analysis technique for OMEGA Dante measurements

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

May, M. J.; Widmann, K.; Sorce, C.

2010-10-15

The Dante is an 18 channel x-ray filtered diode array which records the spectrally and temporally resolved radiation flux from various targets (e.g., hohlraums, etc.) at x-ray energies between 50 eV and 10 keV. It is a main diagnostic installed on the OMEGA laser facility at the Laboratory for Laser Energetics, University of Rochester. The absolute flux is determined from the photometric calibration of the x-ray diodes, filters and mirrors, and an unfold algorithm. Understanding the errors on this absolute measurement is critical for understanding hohlraum energetic physics. We present a new method for quantifying the uncertainties on the determinedmore » flux using a Monte Carlo parameter variation technique. This technique combines the uncertainties in both the unfold algorithm and the error from the absolute calibration of each channel into a one sigma Gaussian error function. One thousand test voltage sets are created using these error functions and processed by the unfold algorithm to produce individual spectra and fluxes. Statistical methods are applied to the resultant set of fluxes to estimate error bars on the measurements.« less

Uncertainty Analysis Technique for OMEGA Dante Measurements

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

May, M J; Widmann, K; Sorce, C

2010-05-07

The Dante is an 18 channel X-ray filtered diode array which records the spectrally and temporally resolved radiation flux from various targets (e.g. hohlraums, etc.) at X-ray energies between 50 eV to 10 keV. It is a main diagnostics installed on the OMEGA laser facility at the Laboratory for Laser Energetics, University of Rochester. The absolute flux is determined from the photometric calibration of the X-ray diodes, filters and mirrors and an unfold algorithm. Understanding the errors on this absolute measurement is critical for understanding hohlraum energetic physics. We present a new method for quantifying the uncertainties on the determinedmore » flux using a Monte-Carlo parameter variation technique. This technique combines the uncertainties in both the unfold algorithm and the error from the absolute calibration of each channel into a one sigma Gaussian error function. One thousand test voltage sets are created using these error functions and processed by the unfold algorithm to produce individual spectra and fluxes. Statistical methods are applied to the resultant set of fluxes to estimate error bars on the measurements.« less

Browns Ferry Unit-3 cavity neutron spectral analysis. Final report

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

Martin, G.C.

1981-08-01

This report describes neutron dosimetry measurements performed in the Browns Ferry Unit-3 reactor cavity using multiple dosimeter and spectrum unfolding techniques to assess radiation-induced degradation of nuclear plant pressure vessels. Test results and conclusions indicating the feasibility of determining neutron flux spectra and the densities in the pressure vessel cavity region via dosimetric measurements are presented.

OneG: A Computational Tool for Predicting Cryptic Intermediates in the Unfolding Kinetics of Proteins under Native Conditions

PubMed Central

Richa, Tambi; Sivaraman, Thirunavukkarasu

2012-01-01

Understanding the relationships between conformations of proteins and their stabilities is one key to address the protein folding paradigm. The free energy change (ΔG) of unfolding reactions of proteins is measured by traditional denaturation methods and native hydrogen-deuterium (H/D) exchange methods. However, the free energy of unfolding (ΔGU) and the free energy of exchange (ΔGHX) of proteins are not in good agreement, though the experimental conditions of both methods are well matching to each other. The anomaly is due to any one or combinations of the following reasons: (i) effects of cis-trans proline isomerisation under equilibrium unfolding reactions of proteins (ii) inappropriateness in accounting the baselines of melting curves (iii) presence of cryptic intermediates, which may elude the melting curve analysis and (iv) existence of higher energy metastable states in the H/D exchange reactions of proteins. Herein, we have developed a novel computational tool, OneG, which accounts the discrepancy between ΔGU and ΔGHX of proteins by systematically accounting all the four factors mentioned above. The program is fully automated and requires four inputs: three-dimensional structures of proteins, ΔGU, ΔGU * and residue-specific ΔGHX determined under EX2-exchange conditions in the absence of denaturants. The robustness of the program has been validated using experimental data available for proteins such as cytochrome c and apocytochrome b562 and the data analyses revealed that cryptic intermediates of the proteins detected by the experimental methods and the cryptic intermediates predicted by the OneG for those proteins were in good agreement. Furthermore, using OneG, we have shown possible existence of cryptic intermediates and metastable states in the unfolding pathways of cardiotoxin III and cobrotoxin, respectively, which are homologous proteins. The unique application of the program to map the unfolding pathways of proteins under native conditions have been brought into fore and the program is publicly available at http://sblab.sastra.edu/oneg.html PMID:22412877

Study on the keV neutron capture reaction in 56Fe and 57Fe

NASA Astrophysics Data System (ADS)

Wang, Taofeng; Lee, Manwoo; Kim, Guinyun; Ro, Tae-Ik; Kang, Yeong-Rok; Igashira, Masayuki; Katabuchi, Tatsuya

2014-03-01

The neutron capture cross-sections and the radiative capture gamma-ray spectra from the broad resonances of 56Fe and 57Fe in the neutron energy range from 10 to 90keV and 550keV have been measured with an anti-Compton NaI(Tl) detector. Pulsed keV neutrons were produced from the 7Li 7Be reaction by bombarding the lithium target with the 1.5ns bunched proton beam from the 3MV Pelletron accelerator. The incident neutron spectrum on a capture sample was measured by means of a time-of-flight (TOF) method with a 6Li -glass detector. The number of weighted capture counts of the iron or gold sample was obtained by applying a pulse height weighting technique to the corresponding capture gamma-ray pulse height spectrum. The neutron capture gamma-ray spectra were obtained by unfolding the observed capture gamma-ray pulse height spectra. To achieve further understanding on the mechanism of neutron radiative capture reaction and study on physics models, theoretical calculations of the -ray spectra for 56Fe and 57Fe with the POD program have been performed by applying the Hauser-Feshbach statistical model. The dominant ingredients to perform the statistical calculation were the Optical Model Potential (OMP), the level densities described by the Mengoni-Nakajima approach, and the -ray transmission coefficients described by -ray strength functions. The comparison of the theoretical calculations, performed only for the 550keV point, show a good agreement with the present experimental results.

A TLD-based ten channel system for the spectrometry of bremsstrahlung generated by laser-matter interaction

NASA Astrophysics Data System (ADS)

Horst, Felix; Fehrenbacher, Georg; Radon, Torsten; Kozlova, Ekaterina; Rosmej, Olga; Czarnecki, Damian; Schrenk, Oliver; Breckow, Joachim; Zink, Klemens

2015-05-01

This work presents a thermoluminescence dosimetry based method for the measurement of bremsstrahlung spectra in the energy range from 30 keV to 100 MeV, resolved in ten different energy intervals and for the photon ambient dosimetry in ultrashort pulsed radiation fields as e.g. generated during operation of the PHELIX laser at the GSI Helmholtzzentrum für Schwerionenforschung. The method is a routine-oriented development by application of a multi-filter technique. The data analysis takes around 1 h. The spectral information is obtained by the unfolding of the response of ten thermoluminescence dosimeters with absorbers of different materials and thicknesses arranged as a stack each with a different response function to photon radiation. These response functions were simulated by the use of the Monte Carlo code FLUKA. An algorithm was developed to unfold bremsstrahlung spectra from the readings of the ten dosimeters. The method has been validated by measurements at a clinical electron linear accelerator (6 MV and 18 MV bremsstrahlung). First measurements at the PHELIX laser system were carried out in December 2013 and January 2014. Spectra with photon energies up to 10 MeV and mean energies up to 420 keV were observed at laser-intensities around 1019 W /cm2 on a titanium foil target. The measurement results imply that the steel walls of the target chamber might be an additional bright x-ray source.

Gamma signatures of the C-BORD Tagged Neutron Inspection System

NASA Astrophysics Data System (ADS)

Sardet, A.; Pérot, B.; Carasco, C.; Sannié, G.; Moretto, S.; Nebbia, G.; Fontana, C.; Pino, F.; Iovene, A.; Tintori, C.

2018-01-01

In the frame of C-BORD project (H2020 program of the EU), a Rapidly relocatable Tagged Neutron Inspection System (RRTNIS) is being developed to non-intrusively detect explosives, chemical threats, and other illicit goods in cargo containers. Material identification is performed through gamma spectroscopy, using twenty NaI detectors and four LaBr3 detectors, to determine the different elements composing the inspected item from their specific gamma signatures induced by fast neutrons. This is performed using an unfolding algorithm to decompose the energy spectrum of a suspect item, selected by X-ray radiography and on which the RRTNIS inspection is focused, on a database of pure element gamma signatures. This paper reports on simulated signatures for the NaI and LaBr3 detectors, constructed using the MCNP6 code. First experimental spectra of a few elements of interest are also presented.

Markov Chain Monte Carlo Estimation of Item Parameters for the Generalized Graded Unfolding Model

ERIC Educational Resources Information Center

de la Torre, Jimmy; Stark, Stephen; Chernyshenko, Oleksandr S.

2006-01-01

The authors present a Markov Chain Monte Carlo (MCMC) parameter estimation procedure for the generalized graded unfolding model (GGUM) and compare it to the marginal maximum likelihood (MML) approach implemented in the GGUM2000 computer program, using simulated and real personality data. In the simulation study, test length, number of response…

Unfolding the mechanism of the AAA+ unfoldase VAT by a combined cryo-EM, solution NMR study

PubMed Central

Huang, Rui; Ripstein, Zev A.; Augustyniak, Rafal; Lazniewski, Michal; Ginalski, Krzysztof; Kay, Lewis E.; Rubinstein, John L.

2016-01-01

The AAA+ (ATPases associated with a variety of cellular activities) enzymes play critical roles in a variety of homeostatic processes in all kingdoms of life. Valosin-containing protein-like ATPase of Thermoplasma acidophilum (VAT), the archaeal homolog of the ubiquitous AAA+ protein Cdc48/p97, functions in concert with the 20S proteasome by unfolding substrates and passing them on for degradation. Here, we present electron cryomicroscopy (cryo-EM) maps showing that VAT undergoes large conformational rearrangements during its ATP hydrolysis cycle that differ dramatically from the conformational states observed for Cdc48/p97. We validate key features of the model with biochemical and solution methyl-transverse relaxation optimized spectroscopY (TROSY) NMR experiments and suggest a mechanism for coupling the energy of nucleotide hydrolysis to substrate unfolding. These findings illustrate the unique complementarity between cryo-EM and solution NMR for studies of molecular machines, showing that the structural properties of VAT, as well as the population distributions of conformers, are similar in the frozen specimens used for cryo-EM and in the solution phase where NMR spectra are recorded. PMID:27402735

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.

A comprehensive spectrometry study of a stray neutron radiation field in scanning proton therapy.

PubMed

Mares, Vladimir; Romero-Expósito, Maite; Farah, Jad; Trinkl, Sebastian; Domingo, Carles; Dommert, Martin; Stolarczyk, Liliana; Van Ryckeghem, Laurent; Wielunski, Marek; Olko, Pawel; Harrison, Roger M

2016-06-07

The purpose of this study is to characterize the stray neutron radiation field in scanning proton therapy considering a pediatric anthropomorphic phantom and a clinically-relevant beam condition. Using two extended-range Bonner sphere spectrometry systems (ERBSS), Working Group 9 of the European Radiation Dosimetry Group measured neutron spectra at ten different positions around a pediatric anthropomorphic phantom irradiated for a brain tumor with a scanning proton beam. This study compares the different systems and unfolding codes as well as neutron spectra measured in similar conditions around a water tank phantom. The ten spectra measured with two ERBSS systems show a generally similar thermal component regardless of the position around the phantom while high energy neutrons (above 20 MeV) were only registered at positions near the beam axis (at 0°, 329° and 355°). Neutron spectra, fluence and ambient dose equivalent, H (*)(10), values of both systems were in good agreement (<15%) while the unfolding code proved to have a limited effect. The highest H (*)(10) value of 2.7 μSv Gy(-1) was measured at 329° to the beam axis and 1.63 m from the isocenter where high-energy neutrons (E  ⩾  20 MeV) contribute with about 53%. The neutron mapping within the gantry room showed that H (*)(10) values significantly decreased with distance and angular position with respect to the beam axis dropping to 0.52 μSv Gy(-1) at 90° and 3.35 m. Spectra at angles of 45° and 135° with respect to the beam axis measured here with an anthropomorphic phantom showed a similar peak structure at the thermal, fast and high energy range as in the previous water-tank experiments. Meanwhile, at 90°, small differences at the high-energy range were observed. Using ERBSS systems, neutron spectra mapping was performed to characterize the exposure of scanning proton therapy patients. The ten measured spectra provide precise information about the exposure of healthy organs to thermal, epithermal, evaporation and intra-nuclear cascade neutrons. This comprehensive spectrometry analysis can also help in understanding the tremendous literature data based rem-counters while also being of great value for general neutron shielding and radiation safety studies.

Application of the BINS superheated drop detector spectrometer to the 9Be(p,xn) neutron energy spectrum determination

NASA Astrophysics Data System (ADS)

Di Fulvio, A.; Ciolini, R.; Mirzajani, N.; Romei, C.; d'Errico, F.; Bedogni, R.; Esposito, J.; Zafiropoulos, D.; Colautti, P.

2013-07-01

In the framework of TRASCO-BNCT project, a Bubble Interactive Neutron Spectrometer (BINS) device was applied to the characterization of the angle-and energy-differential neutron spectra generated by the 9Be(p,xn)reaction. The BINS spectrometer uses two superheated emulsion detectors, sequentially operated at different temperatures and thus provides a series of six sharp threshold responses, covering the 0.1-10 MeV neutron energy range. Spectrum unfolding of the data was performed by means of MAXED code. The obtained angle, energy-differential spectra were compared with those measured with a Bonner sphere spectrometer, a silicon telescope spectrometer and literature data.

Sorbitol counteracts high hydrostatic pressure-induced denaturation of inulin fructotransferase.

PubMed

Li, Yungao; Miao, Ming; Liu, Miao; Jiang, Bo; Zhang, Tao; Chen, Xiangyin

2014-09-01

Inulin fructotransferase (IFTase), a novel hydrolase for inulin, was exposed to high hydrostatic pressure (HHP) at 400 and 600 MPa for 15 min in the presence or absence of sorbitol. Sorbitol protected the enzyme against HHP-induced activity loss. The relative residual activity increased nearly 3.1- and 3.8-fold in the presence of 3 mol/L sorbitol under 400 MPa and 600 MPa for 15 min, respectively. Circular dichroism results indicated that the original chaotic unfolding conformation of the enzyme under HHP shifted toward more ordered and impact with 3 mol/L sorbitol. Fluorescence and UV spectra results suggested that sorbitol prevented partially the unfolding of the enzyme and stabilized the conformation under high pressure. These results might be attributed to the binding of sorbitol on the surface of IFTase to rearrange and strengthen intra- and intermolecular hydrogen bonds. Copyright © 2014 Elsevier B.V. All rights reserved.

Nature of autofluorescence in human serum albumin under its native, unfolding and digested forms

NASA Astrophysics Data System (ADS)

Manjunath, S.; Rao, Bola Sadashiva Satish; Satyamoorthy, Kapaettu; Mahato, Krishna Kishore

2014-02-01

Autofluorescence characteristics of human serum albumin (HSA) are highly sensitive to its local environment. Identification and characterization of the proteins in normal and disease conditions may have great clinical implications. Aim of the present study was to understand how autofluorescence properties of HSA varies with denaturation under urea (3.0M, 6.0M, 9.0M) and guanidine hydrochloride (GnHCl) (2.0M, 4.0M, 6.0M) as well as digestion with trypsin. Towards this, we have recorded the corresponding autofluorescence spectra of HSA at 281nm laser excitation and compared the outcomes. Although, HSA contains 1 tryptophan and 17 tyrosine residues, it has shown intense autofluorescence due to tryptophan as compared to the tyrosine in native form, which may be due to the fluorescence resonance energy transfer (FRET) from tyrosine to tryptophan. As the unfolding progresses in denatured and digested forms of the protein, a clear increase in tyrosine fluorescence as compared to tryptophan was observed, which may be due to the increase of tryptophan - tyrosine separation disturbing the FRET between them resulting in differences in the overall autofluorescence properties. The decrease in tryptophan fluorescence of around 17% in urea denatured, 32% in GnHCl denatured and 96% in tryptic digested HSA was observed as compared to its native form. The obtained results show a clear decrease in FRET between tyrosine and tryptophan residues with the progression of unfolding and urea seems to be less efficient than GnHCl in unfolding of HSA. These results demonstrate the potential of autofluorescence in characterizing proteins in general and HSA in particular.

Theoretical models for electrochemical impedance spectroscopy and local ζ-potential of unfolded proteins in nanopores

NASA Astrophysics Data System (ADS)

Vitarelli, Michael J.; Talaga, David S.

2013-09-01

Single solid-state nanopores find increasing use for electrical detection and/or manipulation of macromolecules. These applications exploit the changes in signals due to the geometry and electrical properties of the molecular species found within the nanopore. The sensitivity and resolution of such measurements are also influenced by the geometric and electrical properties of the nanopore. This paper continues the development of an analytical theory to predict the electrochemical impedance spectra of nanopores by including the influence of the presence of an unfolded protein using the variable topology finite Warburg impedance model previously published by the authors. The local excluded volume of, and charges present on, the segment of protein sampled by the nanopore are shown to influence the shape and peak frequency of the electrochemical impedance spectrum. An analytical theory is used to relate the capacitive response of the electrical double layer at the surface of the protein to both the charge density at the protein surface and the more commonly measured zeta potential. Illustrative examples show how the theory predicts that the varying sequential regions of surface charge density and excluded volume dictated by the protein primary structure may allow for an impedance-based approach to identifying unfolded proteins.

Thermal- and urea-induced unfolding processes of glutathione S-transferase by molecular dynamics simulation.

PubMed

Li, Jiahuang; Chen, Yuan; Yang, Jie; Hua, Zichun

2015-05-01

The Schistosoma juponicum 26 kDa glutathione S-transferase (sj26GST) consists of the N-terminal domain (N-domain), containing three alpha-helices (named H1-H3) and four anti-parallel beta-strands (S1-S4), and the C-terminal domain (C-domain), comprising five alpha-helices (named H4-H8). In present work, molecular dynamics simulations and fluorescence spectroscopic were used to gain insights into the unfolding process of sj26GST. The molecular dynamics simulations on sj26GST subunit both in water and in 8 M urea were carried out at 300 K, 400 K and 500 K, respectively. Spectroscopic measurements were employed to monitor structural changes. Molecular dynamics simulations of sj26GST subunit induced by urea and temperature showed that the initial unfolding step of sj26GST both in water and urea occurred on N-domain, involving the disruption of helices H2, H3 and strands S3 and S4, whereas H6 was the last region exposed to solution and was the last helix to unfold. Moreover, simulations analyses combining with fluorescence and circular dichroism spectra indicated that N-domain could not fold independent, suggesting that correct folding of N-domain depended on its interactions with C-domain. We further proposed that the folding of GSTs could begin with the hydrophobic collapse of C-domain whose H4, H5, H6 and H7 could move close to each other and form a hydrophobic core, especially H6 wrapped in the hydrophobic center and beginning spontaneous formation of the helix. S3, S4, H3, and H2 could form in the wake of the interaction between C-domain and N-domain. The paper can offer insights into the molecular mechanism of GSTs unfolding. © 2014 Wiley Periodicals, Inc.

The Emergence and Unfolding of Telemonitoring Practices in Different Healthcare Organizations

PubMed Central

2018-01-01

Telemonitoring, a sub-category of telemedicine, is promoted as a solution to meet the challenges in Western healthcare systems in terms of an increasing population of people with chronic conditions and fragmentation issues. Recent findings from large-scale telemonitoring programs reveal that these promises are difficult to meet in complex real-life settings which may be explained by concentrating on the practices that emerge when telemonitoring is used to treat patients with chronic conditions. This paper explores the emergence and unfolding of telemonitoring practices in relation to a large-scale, inter-organizational home telemonitoring program which involved 5 local health centers, 10 district nurse units, four hospitals, and 225 general practice clinics in Denmark. Twenty-eight interviews and 28 h of observations of health professionals and administrative staff were conducted over a 12-month period from 2014 to 2015. This study’s findings reveal how telemonitoring practices emerged and unfolded differently among various healthcare organizations. This study suggests that the emergence and unfolding of novel practices is the result of complex interplay between existing work practices, alterations of core tasks, inscriptions in the technology, and the power to either adopt or ignore such novel practices. The study enhances our understanding of how novel technology like telemonitoring impacts various types of healthcare organizations when implemented in a complex inter-organizational context. PMID:29301384

A measurement of global event shape distributions in the hadronic decays of the Z 0

NASA Astrophysics Data System (ADS)

Akrawy, M. Z.; Alexander, G.; Allison, J.; Allport, P. P.; Anderson, K. J.; Armitage, J. C.; Arnison, G. T. J.; Ashton, P.; Azuelos, G.; Baines, J. T. M.; Ball, A. H.; Banks, J.; Barker, G. J.; Barlow, R. J.; Batley, J. R.; Becker, J.; Behnke, T.; Bell, K. W.; Bella, G.; Bethke, S.; Biebel, O.; Binder, U.; Bloodworth, L. J.; Bock, P.; Breuker, H.; Brown, R. M.; Brun, R.; Buijs, A.; Burckhart, H. J.; Capiluppi, P.; Carnegie, R. K.; Carter, A. A.; Carter, J. R.; Chang, C. Y.; Charlton, D. G.; Chrin, J. T. M.; Cohen, I.; Collins, W. J.; Conboy, J. E.; Couch, M.; Coupland, M.; Cuffiani, M.; Dado, S.; Dallavalle, G. M.; Debu, P.; Deninno, M. M.; Dieckmann, A.; Dittmar, M.; Dixit, M. S.; Duchovni, E.; Duerdoth, I. P.; Dumas, D.; El Mamouni, H.; Elcombe, P. A.; Estabrooks, P. G.; Etzion, E.; Fabbri, F.; Farthouat, P.; Fischer, H. M.; Fong, D. G.; French, M. T.; Fukunaga, C.; Gaidot, A.; Ganel, O.; Gary, J. W.; Gascon, J.; Geddes, N. I.; Gee, C. N. P.; Geich-Gimbel, C.; Gensler, S. W.; Gentit, F. X.; Giacomelli, G.; Gibson, V.; Gibson, W. R.; Gillies, J. D.; Goldberg, J.; Goodrick, M. J.; Gorn, W.; Granite, D.; Gross, E.; Grosse-Wiesmann, P.; Grunhaus, J.; Hagedorn, H.; Hagemann, J.; Hansroul, M.; Hargrove, C. K.; Hart, J.; Hattersley, P. M.; Hauschild, M.; Hawkes, C. M.; Heflin, E.; Hemingway, R. J.; Heuer, R. D.; Hill, J. C.; Hillier, S. J.; Ho, C.; Hobbs, J. D.; Hobson, P. R.; Hochman, D.; Holl, B.; Homer, R. J.; Hou, S. R.; Howarth, C. P.; Hughes-Jones, R. E.; Igo-Kemenes, P.; Ihssen, H.; Imrie, D. C.; Jawahery, A.; Jeffreys, P. W.; Jeremie, H.; Jimack, M.; Jobes, M.; Jones, R. W. L.; Jovanovic, P.; Karlen, D.; Kawagoe, K.; Kawamoto, T.; Kellogg, R. G.; Kennedy, B. W.; Kleinwort, C.; Klem, D. E.; Knop, G.; Kobayashi, T.; Kokott, T. P.; Köpke, L.; Kowalewski, R.; Kreutzmann, H.; von Krogh, J.; Kroll, J.; Kuwano, M.; Kyberd, P.; Lafferty, G. D.; Lamarche, F.; Larson, W. J.; Lasota, M. M. B.; Layter, J. G.; Le Du, P.; Leblanc, P.; Lee, A. M.; Lellouch, D.; Lennert, P.; Lessard, L.; Levinson, L.; Lloyd, S. L.; Loebinger, F. K.; Lorah, J. M.; Lorazo, B.; Losty, M. J.; Ludwig, J.; Lupu, N.; Ma, J.; MacBeth, A. A.; Mannelli, M.; Marcellini, S.; Maringer, G.; Martin, A. J.; Martin, J. P.; Mashimo, T.; Mättig, P.; Maur, U.; McMahon, T. J.; McPherson, A. C.; Meijers, F.; Menszner, D.; Merritt, F. S.; Mes, H.; Michelini, A.; Middleton, R. P.; Mikenberg, G.; Miller, D. J.; Milstene, C.; Minowa, M.; Mohr, W.; Montanari, A.; Mori, T.; Moss, M. W.; Murphy, P. G.; Murray, W. J.; Nellen, B.; Nguyen, H. H.; Nozaki, M.; O'Dowd, A. J. P.; O'Neale, S. W.; O'Neill, B. P.; Oakham, F. G.; Odorici, F.; Ogg, M.; Oh, H.; Oreglia, M. J.; Orito, S.; Pansart, J. P.; Patrick, G. N.; Pawley, S. J.; Pfister, P.; Pilcher, J. E.; Pinfold, J. L.; Plane, D. E.; Poli, B.; Pouladdej, A.; Pritchard, P. W.; Quast, G.; Raab, J.; Redmond, M. W.; Rees, D. L.; Regimbald, M.; Riles, K.; Roach, C. M.; Robins, S. A.; Rollnik, A.; Roney, J. M.; Rossberg, S.; Rossi, A. M.; Routenburg, P.; Runge, K.; Runolfsson, O.; Sanghera, S.; Sansum, R. A.; Sasaki, M.; Saunders, B. J.; Schaile, A. D.; Schaile, O.; Schappert, W.; Scharff-Hansen, P.; von der Schmitt, H.; Schreiber, S.; Schwarz, J.; Shapira, A.; Shen, B. C.; Sherwood, P.; Simon, A.; Siroli, G. P.; Skuja, A.; Smith, A. M.; Smith, T. J.; Snow, G. A.; Spreadbury, E. J.; Springer, R. W.; Sproston, M.; Stephens, K.; Stier, H. E.; Ströhmer, R.; Strom, D.; Takeda, H.; Takeshita, T.; Tsukamoto, T.; Turner, M. F.; Tysarczyk-Niemeyer, G.; van den Plas, D.; Vandalen, G. J.; Vasseur, G.; Virtue, C. J.; Wagner, A.; Wahl, C.; Ward, C. P.; Ward, D. R.; Waterhouse, J.; Watkins, P. M.; Watson, A. T.; Watson, N. K.; Weber, M.; Weisz, S.; Wermes, N.; Weymann, M.; Wilson, G. W.; Wilson, J. A.; Wingerter, I.; Winterer, V.-H.; Wood, N. C.; Wotton, S.; Wuensch, B.; Wyatt, T. R.; Yaari, R.; Yang, Y.; Yekutieli, G.; Yoshida, T.; Zeuner, W.; Zorn, G. T.

1990-12-01

We present measurements of global event shape distributions in the hadronic decays of the Z 0. The data sample, corresponding to an integrated luminosity of about 1.3 pb-1, was collected with the OPAL detector at LEP. Most of the experimental distributions we present are unfolded for the finite acceptance and resolution of the OPAL detector. Through comparison with our unfolded data, we tune the parameter values of several Monte Carlo computer programs which simulate perturbative QCD and the hadronization of partons. Jetset version 7.2, Herwig version 3.4 and Ariadne version 3.1 all provide good descriptions of the experimental distributions. They in addition describe lower energy data with the parameter values adjusted at the Z 0 energy. A complete second order matrix element Monte Carlo program with a modified perturbation scale is also compared to our 91 GeV data and its parameter values are adjusted. We obtained an unfolded value for the mean charged multiplicity of 21.28±0.04±0.84, where the first error is statistical and the second is systematic.

Scaffolding Interprofessional Education: Unfolding Case Studies, Virtual World Simulations, and Patient-Centered Care.

PubMed

Zook, Sharon Strang; Hulton, Linda J; Dudding, Carol C; Stewart, Anne L; Graham, Amy C

Fragmentation of health care negatively impacts quality; one of the contributing factors may be ineffective collaboration among health care professionals. This article describes the implementation of an interprofessional education curriculum for graduate students enrolled in nursing, psychology, and speech-language pathology programs. Over 3 semesters, students engaged in interprofessional collaboration modules, unfolding case studies, virtual simulation, and shared case planning experiences. The curriculum's impact on students' attitudes and values toward interprofessional collaborative practice was measured.

Physical characterization of MxiH and PrgI, the needle component of the type III secretion apparatus from Shigella and Salmonella

PubMed Central

Darboe, Numukunda; Kenjale, Roma; Picking, Wendy L.; Picking, William D.; Middaugh, C. Russell

2006-01-01

Shigella and Salmonella use similar type III secretion systems for delivering effector proteins into host cells. This secretion system consists of a base anchored in both bacterial membranes and an extracellular “needle” that forms a rod-like structure exposed on the pathogen surface. The needle is composed of multiple subunits of a single protein and makes direct contact with host cells to facilitate protein delivery. The proteins that make up the needle of Shigella and Salmonella are MxiH and PrgI, respectively. These proteins are attractive vaccine candidates because of their essential role in virulence and surface exposure. We therefore isolated, purified, and characterized the monomeric forms of MxiH and PrgI. Their far-UV circular dichroism spectra show structural similarities with hints of subtle differences in their secondary structure. Both proteins are highly helical and thermally unstable, with PrgI having a midpoint of thermal unfolding (Tm) near 37°C and MxiH having a value near 42°C. The two proteins also have comparable intrinsic stabilities as measured by chemically induced (urea) unfolding. MxiH, however, with a free energy of unfolding (ΔG°0,un) of 1.6 kcal/mol, is slightly more stable than PrgI (1.2 kcal/mol). The relatively low m-values obtained for the urea-induced unfolding of the proteins suggest that they undergo only a small change in solvent-accessible surface area. This argues that when MxiH and PrgI are incorporated into the needle complex, they obtain a more stable structural state through the introduction of protein–protein interactions. PMID:16501225

Physical characterization of MxiH and PrgI, the needle component of the type III secretion apparatus from Shigella and Salmonella.

PubMed

Darboe, Numukunda; Kenjale, Roma; Picking, Wendy L; Picking, William D; Middaugh, C Russell

2006-03-01

Shigella and Salmonella use similar type III secretion systems for delivering effector proteins into host cells. This secretion system consists of a base anchored in both bacterial membranes and an extracellular "needle" that forms a rod-like structure exposed on the pathogen surface. The needle is composed of multiple subunits of a single protein and makes direct contact with host cells to facilitate protein delivery. The proteins that make up the needle of Shigella and Salmonella are MxiH and PrgI, respectively. These proteins are attractive vaccine candidates because of their essential role in virulence and surface exposure. We therefore isolated, purified, and characterized the monomeric forms of MxiH and PrgI. Their far-UV circular dichroism spectra show structural similarities with hints of subtle differences in their secondary structure. Both proteins are highly helical and thermally unstable, with PrgI having a midpoint of thermal unfolding (Tm) near 37 degrees C and MxiH having a value near 42 degrees C. The two proteins also have comparable intrinsic stabilities as measured by chemically induced (urea) unfolding. MxiH, however, with a free energy of unfolding (DeltaG degrees 0,un) of 1.6 kcal/mol, is slightly more stable than PrgI (1.2 kcal/mol). The relatively low m-values obtained for the urea-induced unfolding of the proteins suggest that they undergo only a small change in solvent-accessible surface area. This argues that when MxiH and PrgI are incorporated into the needle complex, they obtain a more stable structural state through the introduction of protein-protein interactions.

Kinetically trapped metastable intermediate of a disulfide-deficient mutant of the starch-binding domain of glucoamylase.

PubMed

Sugimoto, Hayuki; Nakaura, Miho; Nishimura, Shigenori; Karita, Shuichi; Miyake, Hideo; Tanaka, Akiyoshi

2009-08-01

Refolding of a thermally unfolded disulfide-deficient mutant of the starch-binding domain of glucoamylase was investigated using differential scanning calorimetry, isothermal titration calorimetry, CD, and (1)H NMR. When the protein solution was rapidly cooled from a higher temperature, a kinetic intermediate was formed during refolding. The intermediate was unexpectedly stable compared with typical folding intermediates that have short half-lives. It was shown that this intermediate contained substantial secondary structure and tertiary packing and had the same binding ability with beta-cyclodextrin as the native state, suggesting that the intermediate is highly-ordered and native-like on the whole. These characteristics differ from those of partially folded intermediates such as molten globule states. Far-UV CD spectra showed that the secondary structure was once disrupted during the transition from the intermediate to the native state. These results suggest that the intermediate could be an off-pathway type, possibly a misfolded state, that has to undergo unfolding on its way to the native state.

VARIAN CLINAC 6 MeV Photon Spectra Unfolding using a Monte Carlo Meshed Model

NASA Astrophysics Data System (ADS)

Morató, S.; Juste, B.; Miró, R.; Verdú, G.

2017-09-01

Energy spectrum is the best descriptive function to determine photon beam quality of a Medical Linear Accelerator (LinAc). The use of realistic photon spectra in Monte Carlo simulations has a great importance to obtain precise dose calculations in Radiotherapy Treatment Planning (RTP). Reconstruction of photon spectra emitted by medical accelerators from measured depth dose distributions in a water cube is an important tool for commissioning a Monte Carlo treatment planning system. Regarding this, the reconstruction problem is an inverse radiation transport function which is ill conditioned and its solution may become unstable due to small perturbations in the input data. This paper presents a more stable spectral reconstruction method which can be used to provide an independent confirmation of source models for a given machine without any prior knowledge of the spectral distribution. Monte Carlo models used in this work are built with unstructured meshes to simulate with realism the linear accelerator head geometry.

The Transformation of the Supplemental Nutrition Assistance Program

ERIC Educational Resources Information Center

Klerman, Jacob Alex; Danielson, Caroline

2011-01-01

Between 2000 and 2005, the Supplemental Nutrition Assistance Program (SNAP, until recently, the Food Stamp Program) caseload increased by half. As the Great Recession unfolded, the SNAP caseload grew even more rapidly. Further, over the past two decades the composition of the caseload has shifted sharply away from families combining food and cash…

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.

Evaluating the performance of two neutron spectrum unfolding codes based on iterative procedures and artificial neural networks

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

Ortiz-Rodriguez, J. M.; Reyes Alfaro, A.; Reyes Haro, A.

In this work the performance of two neutron spectrum unfolding codes based on iterative procedures and artificial neural networks is evaluated. The first one code based on traditional iterative procedures and called Neutron spectrometry and dosimetry from the Universidad Autonoma de Zacatecas (NSDUAZ) use the SPUNIT iterative algorithm and was designed to unfold neutron spectrum and calculate 15 dosimetric quantities and 7 IAEA survey meters. The main feature of this code is the automated selection of the initial guess spectrum trough a compendium of neutron spectrum compiled by the IAEA. The second one code known as Neutron spectrometry and dosimetrymore » with artificial neural networks (NDSann) is a code designed using neural nets technology. The artificial intelligence approach of neural net does not solve mathematical equations. By using the knowledge stored at synaptic weights on a neural net properly trained, the code is capable to unfold neutron spectrum and to simultaneously calculate 15 dosimetric quantities, needing as entrance data, only the rate counts measured with a Bonner spheres system. Similarities of both NSDUAZ and NSDann codes are: they follow the same easy and intuitive user's philosophy and were designed in a graphical interface under the LabVIEW programming environment. Both codes unfold the neutron spectrum expressed in 60 energy bins, calculate 15 dosimetric quantities and generate a full report in HTML format. Differences of these codes are: NSDUAZ code was designed using classical iterative approaches and needs an initial guess spectrum in order to initiate the iterative procedure. In NSDUAZ, a programming routine was designed to calculate 7 IAEA instrument survey meters using the fluence-dose conversion coefficients. NSDann code use artificial neural networks for solving the ill-conditioned equation system of neutron spectrometry problem through synaptic weights of a properly trained neural network. Contrary to iterative procedures, in neural net approach it is possible to reduce the rate counts used to unfold the neutron spectrum. To evaluate these codes a computer tool called Neutron Spectrometry and dosimetry computer tool was designed. The results obtained with this package are showed. The codes here mentioned are freely available upon request to the authors.« less

Evaluating the performance of two neutron spectrum unfolding codes based on iterative procedures and artificial neural networks

NASA Astrophysics Data System (ADS)

Ortiz-Rodríguez, J. M.; Reyes Alfaro, A.; Reyes Haro, A.; Solís Sánches, L. O.; Miranda, R. Castañeda; Cervantes Viramontes, J. M.; Vega-Carrillo, H. R.

2013-07-01

In this work the performance of two neutron spectrum unfolding codes based on iterative procedures and artificial neural networks is evaluated. The first one code based on traditional iterative procedures and called Neutron spectrometry and dosimetry from the Universidad Autonoma de Zacatecas (NSDUAZ) use the SPUNIT iterative algorithm and was designed to unfold neutron spectrum and calculate 15 dosimetric quantities and 7 IAEA survey meters. The main feature of this code is the automated selection of the initial guess spectrum trough a compendium of neutron spectrum compiled by the IAEA. The second one code known as Neutron spectrometry and dosimetry with artificial neural networks (NDSann) is a code designed using neural nets technology. The artificial intelligence approach of neural net does not solve mathematical equations. By using the knowledge stored at synaptic weights on a neural net properly trained, the code is capable to unfold neutron spectrum and to simultaneously calculate 15 dosimetric quantities, needing as entrance data, only the rate counts measured with a Bonner spheres system. Similarities of both NSDUAZ and NSDann codes are: they follow the same easy and intuitive user's philosophy and were designed in a graphical interface under the LabVIEW programming environment. Both codes unfold the neutron spectrum expressed in 60 energy bins, calculate 15 dosimetric quantities and generate a full report in HTML format. Differences of these codes are: NSDUAZ code was designed using classical iterative approaches and needs an initial guess spectrum in order to initiate the iterative procedure. In NSDUAZ, a programming routine was designed to calculate 7 IAEA instrument survey meters using the fluence-dose conversion coefficients. NSDann code use artificial neural networks for solving the ill-conditioned equation system of neutron spectrometry problem through synaptic weights of a properly trained neural network. Contrary to iterative procedures, in neural net approach it is possible to reduce the rate counts used to unfold the neutron spectrum. To evaluate these codes a computer tool called Neutron Spectrometry and dosimetry computer tool was designed. The results obtained with this package are showed. The codes here mentioned are freely available upon request to the authors.

Differential Cross Sections for the Electron Impact Excitation of the A(sup 3)(Sigma)(sub u)(sup +), B(sup 3)Pi(sub g), W(sup 3)(Delta)(sub u), B'(sup 3)(Sigma)(sub u)(sup -), a'(sup 1)Sigma(sub u)(sup -), a(sup 1)Pi(sub g), w(sup 1)Delta(sub u), and C(sup 3)Pi(sub u) States of N(sub 2)

NASA Technical Reports Server (NTRS)

Khakoo, M. A.; Johnson, P. V.; Ozkay, I.; Yan, P.; Trajmar, S.; Kanik, I.

2005-01-01

Measurements of differential cross sections for the electron-impact excitation of molecular nitrogen from the ground X(sup 1)(Sigma)(sub g)(sup +)(v''=0)level to the A(sup 3)(Sigma)(sub u)(sup +)(v'), B(sup 3)Pi(sub g)(v'), W(sup 3)(Delta)(sub u)(v'),B'(sup 3)(Sigma)(sub u)(sup -)(v'), a(sup 1)(Pi)(sub g)(v'), w(sup 1)(Delta)(sub u)(v'), and C(sup 3)(Pi)(sub u)(v') levels are presented. The data are obtained at the incident energies of 10, 12.5, 15, 17.5, 20, 30, 50, and 100 eV over the angular range of 5(deg)-130(deg) in 5(deg) intervals. The individual electronic state excitation differential cross sections are obtained by unfolding electron energy-loss spectra of molecular nitrogen using available semiempirical Frank-Condon factors. The data are compared to previous measurements and to available theory. We also make several important suggestions regarding future work that, like the present, relies on the unfolding of electron energy-loss spectra for obtaining differential cross sections.

High-Temperature unfolding of a trp-Cage mini-protein: a molecular dynamics simulation study

PubMed Central

Seshasayee, Aswin Sai Narain

2005-01-01

Background Trp cage is a recently-constructed fast-folding miniprotein. It consists of a short helix, a 3,10 helix and a C-terminal poly-proline that packs against a Trp in the alpha helix. It is known to fold within 4 ns. Results High-temperature unfolding molecular dynamics simulations of the Trp cage miniprotein have been carried out in explicit water using the OPLS-AA force-field incorporated in the program GROMACS. The radius of gyration (Rg) and Root Mean Square Deviation (RMSD) have been used as order parameters to follow the unfolding process. Distributions of Rg were used to identify ensembles. Conclusion Three ensembles could be identified. While the native-state ensemble shows an Rg distribution that is slightly skewed, the second ensemble, which is presumably the Transition State Ensemble (TSE), shows an excellent fit. The denatured ensemble shows large fluctuations, but a Gaussian curve could be fitted. This means that the unfolding process is two-state. Representative structures from each of these ensembles are presented here. PMID:15760474

The Secret to the "Best" Ratings from Any Evaluation Scale

ERIC Educational Resources Information Center

Berk, Ronald A.

2010-01-01

Most faculty developers have a wide variety of rating scales that fly across their desk tops as their incremental program activities unfold during the academic year. The primary issue for this column is: What is the quality of those ratings used for decisions about people and programs? When students, faculty, and administrators rate a program or…

Measurements of the neutral particle spectra on Mars by MSL/RAD from 2015-11-15 to 2016-01-15

NASA Astrophysics Data System (ADS)

Guo, Jingnan; Zeitlin, Cary; Wimmer-Schweingruber, Robert; Hassler, Donald M.; Köhler, Jan; Ehresmann, Bent; Böttcher, Stephan; Böhm, Eckart; Brinza, David E.

2017-08-01

The Radiation Assessment Detector (RAD), onboard the Mars Science Laboratory (MSL) rover Curiosity, has been measuring the energetic charged and neutral particles and the radiation dose rate on the surface of Mars since the landing of the rover in August 2012. In contrast to charged particles, neutral particles (neutrons and γ-rays) are measured indirectly: the energy deposition spectra produced by neutral particles are complex convolutions of the incident particle spectra with the detector response functions. An inversion technique has been developed and applied to jointly unfold the deposited energy spectra measured in two scintillators of different types (CsI for high γ detection efficiency, and plastic for neutrons) to obtain the neutron and γ-ray spectra. This result is important for determining the biological impact of the Martian surface radiation contributed by neutrons, which interact with materials differently from the charged particles. These first in-situ measurements on Mars provide (1) an important reference for assessing the radiation-associated health risks for future manned missions to the red planet and (2) an experimental input for validating the particle transport codes used to model the radiation environments within spacecraft or on the surface of planets. Here we present neutral particle spectra as well as the corresponding dose and dose equivalent rates derived from RAD measurement during a period (November 15, 2015 to January 15, 2016) for which the surface particle spectra have been simulated via different transport models.

Measurement of keV-neutron capture cross sections and capture gamma-ray spectra of Cs-133 and I-127

NASA Astrophysics Data System (ADS)

Umezawa, Seigo; Igashira, Masayuki; Katabuchi, Tatuya; Dominic, Moraru; Yanagida, Shotaro; Okamiya, Tomohiro

2017-09-01

The neutron capture cross sections and the capture gamma-ray spectra of 127I and 133Cs at incident neutron energies from 15 to 100 keV have been measured by the time-of-flight method. Capture gamma-rays were detected with an anti-Compton NaI(Tl) spectrometer, and the pulse-height weighting technique was applied to derive capture yields. The capture cross sections of 127I and 133Cs were determined using the standard capture cross section of 197Au. The total errors of the cross sections were 3.8-5.1%. The obtained cross sections were compared with evaluated values in JENDL-4.0 and ENDF/B-VII.1. For 127I, the energy dependence is different between the present results and the evaluations. For 133Cs, the evaluated values in JENDL-4.0 agree with the present results but the evaluated values in ENDF/B-VII.1 are smaller than the present results by 14%-18%. The capture gamma-ray spectra of 133Cs and 127I were derived by unfolding the pulse height spectra with detector response functions.

Monte Carlo analysis of a time-dependent neutron and secondary gamma-ray integral experiment on a thick concrete and steel shield

NASA Astrophysics Data System (ADS)

Cramer, S. N.; Roussin, R. W.

1981-11-01

A Monte Carlo analysis of a time-dependent neutron and secondary gamma-ray integral experiment on a thick concrete and steel shield is presented. The energy range covered in the analysis is 15-2 MeV for neutron source energies. The multigroup MORSE code was used with the VITAMIN C 171-36 neutron-gamma-ray cross-section data set. Both neutron and gamma-ray count rates and unfolded energy spectra are presented and compared, with good general agreement, with experimental results.

StarBright Learning Exchange

ERIC Educational Resources Information Center

Kalinowski, Michael

2007-01-01

This article features StarBright Learning Exchange, a program that provides a cross-cultural exchange between Australian and South African early childhood educators. The program was originated when its president, Carol Allen, and her colleague, Karen Williams, decided that they could no longer sit by and watch the unfolding social catastrophe that…

Federal Spending for Means Tested Programs, 2007 to 2027

DTIC Science & Technology

2017-02-01

child tax credits (which are refundable), the Supplemental Nutrition Assistance Program (SNAP), and Supplemental Security Income. The largest non...child tax credits, and SNAP—have been or will be significantly affected by program changes that unfold over time: B Medicaid spending rose by 35...total outlays in the Part D program). Increases in the number of beneficiaries account for about one- third of that growth; the introduction of new

Two-way and three-way approaches to ultra high performance liquid chromatography-photodiode array dataset for the quantitative resolution of a two-component mixture containing ciprofloxacin and ornidazole.

PubMed

Dinç, Erdal; Ertekin, Zehra Ceren; Büker, Eda

2016-09-01

Two-way and three-way calibration models were applied to ultra high performance liquid chromatography with photodiode array data with coeluted peaks in the same wavelength and time regions for the simultaneous quantitation of ciprofloxacin and ornidazole in tablets. The chromatographic data cube (tensor) was obtained by recording chromatographic spectra of the standard and sample solutions containing ciprofloxacin and ornidazole with sulfadiazine as an internal standard as a function of time and wavelength. Parallel factor analysis and trilinear partial least squares were used as three-way calibrations for the decomposition of the tensor, whereas three-way unfolded partial least squares was applied as a two-way calibration to the unfolded dataset obtained from the data array of ultra high performance liquid chromatography with photodiode array detection. The validity and ability of two-way and three-way analysis methods were tested by analyzing validation samples: synthetic mixture, interday and intraday samples, and standard addition samples. Results obtained from two-way and three-way calibrations were compared to those provided by traditional ultra high performance liquid chromatography. The proposed methods, parallel factor analysis, trilinear partial least squares, unfolded partial least squares, and traditional ultra high performance liquid chromatography were successfully applied to the quantitative estimation of the solid dosage form containing ciprofloxacin and ornidazole. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Differences in chewing sounds of dry-crisp snacks by multivariate data analysis

NASA Astrophysics Data System (ADS)

De Belie, N.; Sivertsvik, M.; De Baerdemaeker, J.

2003-09-01

Chewing sounds of different types of dry-crisp snacks (two types of potato chips, prawn crackers, cornflakes and low calorie snacks from extruded starch) were analysed to assess differences in sound emission patterns. The emitted sounds were recorded by a microphone placed over the ear canal. The first bite and the first subsequent chew were selected from the time signal and a fast Fourier transformation provided the power spectra. Different multivariate analysis techniques were used for classification of the snack groups. This included principal component analysis (PCA) and unfold partial least-squares (PLS) algorithms, as well as multi-way techniques such as three-way PLS, three-way PCA (Tucker3), and parallel factor analysis (PARAFAC) on the first bite and subsequent chew. The models were evaluated by calculating the classification errors and the root mean square error of prediction (RMSEP) for independent validation sets. It appeared that the logarithm of the power spectra obtained from the chewing sounds could be used successfully to distinguish the different snack groups. When different chewers were used, recalibration of the models was necessary. Multi-way models distinguished better between chewing sounds of different snack groups than PCA on bite or chew separately and than unfold PLS. From all three-way models applied, N-PLS with three components showed the best classification capabilities, resulting in classification errors of 14-18%. The major amount of incorrect classifications was due to one type of potato chips that had a very irregular shape, resulting in a wide variation of the emitted sounds.

Differential cross sections for the electron impact excitation of the A {sup 3}{sigma}{sub u}{sup +}, B {sup 3}{pi}{sub g}, W {sup 3}{delta}{sub u}, B{sup '} {sup 3}{sigma}{sub u}{sup -}, a{sup '} {sup 1}{sigma}{sub u}{sup -}, a {sup 1}{pi}{sub g}, w {sup 1}{delta}{sub u}, and C {sup 3}{pi}{sub u} states of N{sub 2}

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

Khakoo, M.A.; Ozkay, I.; Johnson, P.V.

2005-06-15

Measurements of differential cross sections for the electron-impact excitation of molecular nitrogen from the ground X {sup 1}{sigma}{sub g}{sup +}(v{sup ''}=0) level to the A {sup 3}{sigma}{sub u}{sup +}(v{sup '}), B {sup 3}{pi}{sub g}(v{sup '}), W {sup 3}{delta}{sub u}(v{sup '}), B{sup '} {sup 3}{sigma}{sub u}{sup -}(v{sup '}), a{sup '} {sup 1}{sigma}{sub u}{sup -}(v{sup '}), a {sup 1}{pi}{sub g}(v{sup '}), w {sup 1}{delta}{sub u}(v{sup '}), and C {sup 3}{pi}{sub u}(v{sup '}) levels are presented. The data are obtained at the incident energies of 10, 12.5, 15, 17.5, 20, 30, 50, and 100 eV over the angular range of 5 deg. -130more » deg. in 5 deg. intervals. The individual electronic state excitation differential cross sections are obtained by unfolding electron energy-loss spectra of molecular nitrogen using available semiempirical Frank-Condon factors. The data are compared to previous measurements and to available theory. We also make several important suggestions regarding future work that, like the present, relies on the unfolding of electron energy-loss spectra for obtaining differential cross sections.« less

Technical Assistance and Innovation in Science Education.

ERIC Educational Resources Information Center

Maybury, Robert H.

1975-01-01

This study examines programs for improving science teaching in the schools of Argentina, Brazil, Lebanon, the Philippines, and Turkey. Appearing in two parts, the first contains five case histories--descriptive and uninterpreted accounts of the events as they have unfolded over time in the programs. The second part of the study compares, analyzes,…

Neutron spectrometry with a monolithic silicon telescope.

PubMed

Agosteo, S; D'Angelo, G; Fazzi, A; Para, A Foglio; Pola, A; Zotto, P

2007-01-01

A neutron spectrometer was set-up by coupling a polyethylene converter with a monolithic silicon telescope, consisting of a DeltaE and an E stage-detector (about 2 and 500 microm thick, respectively). The detection system was irradiated with monoenergetic neutrons at INFN-Laboratori Nazionali di Legnaro (Legnaro, Italy). The maximum detectable energy, imposed by the thickness of the E stage, is about 8 MeV for the present detector. The scatter plots of the energy deposited in the two stages were acquired using two independent electronic chains. The distributions of the recoil-protons are well-discriminated from those due to secondary electrons for energies above 0.350 MeV. The experimental spectra of the recoil-protons were compared with the results of Monte Carlo simulations using the FLUKA code. An analytical model that takes into account the geometrical structure of the silicon telescope was developed, validated and implemented in an unfolding code. The capability of reproducing continuous neutron spectra was investigated by irradiating the detector with neutrons from a thick beryllium target bombarded with protons. The measured spectra were compared with data taken from the literature. Satisfactory agreement was found.

Intrinsic fluorescence spectroscopy of glutamate dehydrogenase: Integrated behavior and deconvolution analysis

NASA Astrophysics Data System (ADS)

Pompa, P. P.; Cingolani, R.; Rinaldi, R.

2003-07-01

In this paper, we present a deconvolution method aimed at spectrally resolving the broad fluorescence spectra of proteins, namely, of the enzyme bovine liver glutamate dehydrogenase (GDH). The analytical procedure is based on the deconvolution of the emission spectra into three distinct Gaussian fluorescing bands Gj. The relative changes of the Gj parameters are directly related to the conformational changes of the enzyme, and provide interesting information about the fluorescence dynamics of the individual emitting contributions. Our deconvolution method results in an excellent fitting of all the spectra obtained with GDH in a number of experimental conditions (various conformational states of the protein) and describes very well the dynamics of a variety of phenomena, such as the dependence of hexamers association on protein concentration, the dynamics of thermal denaturation, and the interaction process between the enzyme and external quenchers. The investigation was carried out by means of different optical experiments, i.e., native enzyme fluorescence, thermal-induced unfolding, and fluorescence quenching studies, utilizing both the analysis of the “average” behavior of the enzyme and the proposed deconvolution approach.

Study of multiple unfolding trajectories and unfolded states of the protein GB1 under the physical property space.

PubMed

Wang, Jihua; Zhao, Liling; Dou, Xianghua; Zhang, Zhiyong

2008-06-01

Forty nine molecular dynamics simulations of unfolding trajectories of the segment B1 of streptococcal protein G (GB1) provide a direct demonstration of the diversity of unfolding pathway and give a statistically utmost unfolding pathway under the physical property space. Twelve physical properties of the protein were chosen to construct a 12-dimensional property space. Then the 12-dimensional property space was reduced to a 3-dimensional principle component property space. Under the property space, the multiple unfolding trajectories look like "trees", which have some common characters. The "root of the tree" corresponds to the native state, the "bole" homologizes the partially unfolded conformations, and the "crown" is in correspondence to the unfolded state. These unfolding trajectories can be divided into three types. The first one has the characters of straight "bole" and "crown" corresponding to a fast two-state unfolding pathway of GB1. The second one has the character of "the standstill in the middle tree bole", which may correspond to a three-state unfolding pathway. The third one has the character of "the circuitous bole" corresponding to a slow two-state unfolding pathway. The fast two-state unfolding pathway is a statistically utmost unfolding pathway or preferred pathway of GB1, which occupies 53% of 49 unfolding trajectories. In the property space all the unfolding trajectories construct a thermal unfolding pathway ensemble of GB1. The unfolding pathway ensemble resembles a funnel that is gradually emanative from the native state ensemble to the unfolded state ensemble. In the property space, the thermal unfolded state distribution looks like electronic cloud in quantum mechanics. The unfolded states of the independent unfolding simulation trajectories have substantial overlaps, indicating that the thermal unfolded states are confined by the physical property values, and the number of protein unfolded state are much less than that was believed before.

[Near infrared spectroscopy based process trajectory technology and its application in monitoring and controlling of traditional Chinese medicine manufacturing process].

PubMed

Li, Wen-Long; Qu, Hai-Bin

2016-10-01

In this paper, the principle of NIRS (near infrared spectroscopy)-based process trajectory technology was introduced.The main steps of the technique include:① in-line collection of the processes spectra of different technics; ② unfolding of the 3-D process spectra;③ determination of the process trajectories and their normal limits;④ monitoring of the new batches with the established MSPC (multivariate statistical process control) models.Applications of the technology in the chemical and biological medicines were reviewed briefly. By a comprehensive introduction of our feasibility research on the monitoring of traditional Chinese medicine technical process using NIRS-based multivariate process trajectories, several important problems of the practical applications which need urgent solutions are proposed, and also the application prospect of the NIRS-based process trajectory technology is fully discussed and put forward in the end. Copyright© by the Chinese Pharmaceutical Association.

Measurements of the neutral particle spectra on Mars by MSL/RAD from 2015-11-15 to 2016-01-15.

PubMed

Guo, Jingnan; Zeitlin, Cary; Wimmer-Schweingruber, Robert; Hassler, Donald M; Köhler, Jan; Ehresmann, Bent; Böttcher, Stephan; Böhm, Eckart; Brinza, David E

2017-08-01

The Radiation Assessment Detector (RAD), onboard the Mars Science Laboratory (MSL) rover Curiosity, has been measuring the energetic charged and neutral particles and the radiation dose rate on the surface of Mars since the landing of the rover in August 2012. In contrast to charged particles, neutral particles (neutrons and γ-rays) are measured indirectly: the energy deposition spectra produced by neutral particles are complex convolutions of the incident particle spectra with the detector response functions. An inversion technique has been developed and applied to jointly unfold the deposited energy spectra measured in two scintillators of different types (CsI for high γ detection efficiency, and plastic for neutrons) to obtain the neutron and γ-ray spectra. This result is important for determining the biological impact of the Martian surface radiation contributed by neutrons, which interact with materials differently from the charged particles. These first in-situ measurements on Mars provide (1) an important reference for assessing the radiation-associated health risks for future manned missions to the red planet and (2) an experimental input for validating the particle transport codes used to model the radiation environments within spacecraft or on the surface of planets. Here we present neutral particle spectra as well as the corresponding dose and dose equivalent rates derived from RAD measurement during a period (November 15, 2015 to January 15, 2016) for which the surface particle spectra have been simulated via different transport models. Copyright © 2017 The Committee on Space Research (COSPAR). Published by Elsevier Ltd. All rights reserved.

Measuring spacetime: from the big bang to black holes.

PubMed

Tegmark, Max

2002-05-24

Space is not a boring static stage on which events unfold over time, but a dynamic entity with curvature, fluctuations, and a rich life of its own. Spectacular measurements of the cosmic microwave background, gravitational lensing, type Ia supernovae, large-scale structure, spectra of the Lyman alpha forest, stellar dynamics, and x-ray binaries are probing the properties of spacetime over 22 orders of magnitude in scale. Current measurements are consistent with an infinite flat everlasting universe containing about 30% cold dark matter, 65% dark energy, and at least two distinct populations of black holes.

Fieldable computer system for determining gamma-ray pulse-height distributions, flux spectra, and dose rates from Little Boy

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

Moss, C.E.; Lucas, M.C.; Tisinger, E.W.

1984-01-01

Our system consists of a LeCroy 3500 data acquisition system with a built-in CAMAC crate and eight bismuth-germanate detectors 7.62 cm in diameter and 7.62 cm long. Gamma-ray pulse-height distributions are acquired simultaneously for up to eight positions. The system was very carefully calibrated and characterized from 0.1 to 8.3 MeV using gamma-ray spectra from a variety of radioactive sources. By fitting the pulse-height distributions from the sources with a function containing 17 parameters, we determined theoretical repsonse functions. We use these response functions to unfold the distributions to obtain flux spectra. A flux-to-dose-rate conversion curve based on the workmore » of Dimbylow and Francis is then used to obtain dose rates. Direct use of measured spectra and flux-to-dose-rate curves to obtain dose rates avoids the errors that can arise from spectrum dependence in simple gamma-ray dosimeter instruments. We present some gamma-ray doses for the Little Boy assembly operated at low power. These results can be used to determine the exposures of the Hiroshima survivors and thus aid in the establishment of radation exposure limits for the nuclear industry.« less

WINDOWS: a program for the analysis of spectral data foil activation measurements

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

Stallmann, F.W.; Eastham, J.F.; Kam, F.B.K.

The computer program WINDOWS together with its subroutines is described for the analysis of neutron spectral data foil activation measurements. In particular, the unfolding of the neutron differential spectrum, estimated windows and detector contributions, upper and lower bounds for an integral response, and group fluxes obtained from neutron transport calculations. 116 references. (JFP)

77 FR 35665 - Notice of Proposed Information Collection Requests; Office of Planning, Evaluation and Policy...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-06-14

... evaluations may include both formative implementation and process evaluations that evaluate a program as it is unfolding, and summative descriptive evaluations that examine changes in final outcomes in a non-causal...

On Use of Multi-Chambered Fission Detectors for In-Core, Neutron Spectroscopy

NASA Astrophysics Data System (ADS)

Roberts, Jeremy A.

2018-01-01

Presented is a short, computational study on the potential use of multichambered fission detectors for in-core, neutron spectroscopy. Motivated by the development of very small fission chambers at CEA in France and at Kansas State University in the U.S., it was assumed in this preliminary analysis that devices can be made small enough to avoid flux perturbations and that uncertainties related to measurements can be ignored. It was hypothesized that a sufficient number of chambers with unique reactants can act as a real-time, foilactivation experiment. An unfolding scheme based on maximizing (Shannon) entropy was used to produce a flux spectrum from detector signals that requires no prior information. To test the method, integral, detector responses were generated for singleisotope detectors of various Th, U, Np, Pu, Am, and Cs isotopes using a simplified, pressurized-water reactor spectrum and fluxweighted, microscopic, fission cross sections, in the WIMS-69 multigroup format. An unfolded spectrum was found from subsets of these responses that had a maximum entropy while reproducing the responses considered and summing to one (that is, they were normalized). Several nuclide subsets were studied, and, as expected, the results indicate inclusion of more nuclides leads to better spectra but with diminishing improvements, with the best-case spectrum having an average, relative, group-wise error of approximately 51%. Furthermore, spectra found from minimum-norm and Tihkonov-regularization inversion were of lower quality than the maximum entropy solutions. Finally, the addition of thermal-neutron filters (here, Cd and Gd) provided substantial improvement over unshielded responses alone. The results, as a whole, suggest that in-core, neutron spectroscopy is at least marginally feasible.

Cooperative unfolding of apolipoprotein A-1 induced by chemical denaturation.

PubMed

Eckhardt, D; Li-Blatter, X; Schönfeld, H-J; Heerklotz, H; Seelig, J

2018-05-25

Apolipoprotein A-1 (Apo A-1) plays an important role in lipid transfer and obesity. Chemical unfolding of α-helical Apo A-1 is induced with guanidineHCl and monitored with differential scanning calorimetry (DSC) and CD spectroscopy. The unfolding enthalpy and the midpoint temperature of unfolding decrease linearly with increasing guanidineHCl concentration, caused by the weak binding of denaturant. At room temperature, binding of 50-60 molecules guanidineHCl leads to a complete Apo A-1 unfolding. The entropy of unfolding decreases to a lesser extent than the unfolding enthalpy. Apo A-1 chemical unfolding is a dynamic multi-state equilibrium that is analysed with the Zimm-Bragg theory modified for chemical unfolding. The chemical Zimm-Bragg theory predicts the denaturant binding constant K D and the protein cooperativity σ. Chemical unfolding of Apo A-1 is two orders of magnitude less cooperative than thermal unfolding. The free energy of thermal unfolding is ~0.2 kcal/mol per amino acid residue and ~1.0 kcal/mol for chemical unfolding at room temperature. The Zimm-Bragg theory calculates conformational probabilities and the chemical Zimm-Bragg theory predicts stretches of α-helical segments in dynamic equilibrium, unfolding and refolding independently and fast. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Measurement And Calculation of High-Energy Neutron Spectra Behind Shielding at the CERF 120-GeV/C Hadron Beam Facility

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

Nakao, N.; /SLAC; Taniguchi, S.

Neutron energy spectra were measured behind the lateral shield of the CERF (CERN-EU High Energy Reference Field) facility at CERN with a 120 GeV/c positive hadron beam (a mixture of mainly protons and pions) on a cylindrical copper target (7-cm diameter by 50-cm long). An NE213 organic liquid scintillator (12.7-cm diameter by 12.7-cm long) was located at various longitudinal positions behind shields of 80- and 160-cm thick concrete and 40-cm thick iron. The measurement locations cover an angular range with respect to the beam axis between 13 and 133{sup o}. Neutron energy spectra in the energy range between 32 MeVmore » and 380 MeV were obtained by unfolding the measured pulse height spectra with the detector response functions which have been verified in the neutron energy range up to 380 MeV in separate experiments. Since the source term and experimental geometry in this experiment are well characterized and simple and results are given in the form of energy spectra, these experimental results are very useful as benchmark data to check the accuracies of simulation codes and nuclear data. Monte Carlo simulations of the experimental set up were performed with the FLUKA, MARS and PHITS codes. Simulated spectra for the 80-cm thick concrete often agree within the experimental uncertainties. On the other hand, for the 160-cm thick concrete and iron shield differences are generally larger than the experimental uncertainties, yet within a factor of 2. Based on source term simulations, observed discrepancies among simulations of spectra outside the shield can be partially explained by differences in the high-energy hadron production in the copper target.« less

Efficient dynamic optimization of logic programs

NASA Technical Reports Server (NTRS)

Laird, Phil

1992-01-01

A summary is given of the dynamic optimization approach to speed up learning for logic programs. The problem is to restructure a recursive program into an equivalent program whose expected performance is optimal for an unknown but fixed population of problem instances. We define the term 'optimal' relative to the source of input instances and sketch an algorithm that can come within a logarithmic factor of optimal with high probability. Finally, we show that finding high-utility unfolding operations (such as EBG) can be reduced to clause reordering.

Spatiospectral Analysis of Accelerated Protons from Sub-Micron Liquid Crystal Films

NASA Astrophysics Data System (ADS)

Willis, Christopher; Poole, Patrick; Cochran, Ginevra; van Woerkom, Linn; Schumacher, Douglass

2017-10-01

Recent studies on ion acceleration have trended towards ultra-thin (<1 μm) targets due to improved ion energies and yields from these targets. As discussed here, ultra-thin targets may exhibit unusual spatial distributions in the accelerated ions, such that ion spectrometer data may not be representative of the overall distribution. More complete characterization of the ions requires spectral unfolding of radiochromic film (RCF) data, yielding spatially dependent spectra. Spatiospectral data will be presented from several experiments using sub-micron liquid crystal film targets at the Scarlet (OSU), Texas Petawatt (UT, Austin) and PHELIX (GSI, Darmstadt) laser facilities, including evidence of >75 MeV protons from 300 nm films at PHELIX. Analysis of RCF data is supported by Monte-Carlo modeling of RCF response to ions and electrons using FLUKA. Trends in the resulting ion distributions will be discussed including spatially varying slope temperature and observation of annular ring features at moderate ion energies on many shots. This material is based upon work supported by the AFOSR under award FA9550-14-1-0085, by the DARPA PULSE program through a Grant from AMRDEC, and by the NNSA under contract DE-NA0003107.

Investigation on the protein-binding properties of icotinib by spectroscopic and molecular modeling method

NASA Astrophysics Data System (ADS)

Zhang, Hua-xin; Xiong, Hang-xing; Li, Li-wei

2016-05-01

Icotinib is a highly-selective epidermal growth factor receptor tyrosine kinase inhibitor with preclinical and clinical activity in non-small cell lung cancer, which has been developed as a new targeted anti-tumor drug in China. In this work, the interaction of icotinib and human serum albumin (HSA) were studied by three-dimensional fluorescence spectra, ultraviolet spectra, circular dichroism (CD) spectra, molecular probe and molecular modeling methods. The results showed that icotinib binds to Sudlow's site I in subdomain IIA of HSA molecule, resulting in icotinib-HSA complexes formed at ground state. The number of binding sites, equilibrium constants, and thermodynamic parameters of the reaction were calculated at different temperatures. The negative enthalpy change (ΔHθ) and entropy change (ΔSθ) indicated that the structure of new complexes was stabilized by hydrogen bonds and van der Waals power. The distance between donor and acceptor was calculated according to Förster's non-radiation resonance energy transfer theory. The structural changes of HSA caused by icotinib binding were detected by synchronous spectra and circular dichroism (CD) spectra. Molecular modeling method was employed to unfold full details of the interaction at molecular level, most of which could be supported by experimental results. The study analyzed the probability that serum albumins act as carriers for this new anticarcinogen and provided fundamental information on the process of delivering icotinib to its target tissues, which might be helpful in understanding the mechanism of icotinib in cancer therapy.

Prefibrillar Formation Conditions of β-Lactoglobulin by Titration and Chaotropes Urea and KSCN Under Thermal Load

NASA Astrophysics Data System (ADS)

Babcock, Jeremiah; Valdez, Rolando; Brancaleon, Lorenzo

2009-10-01

The harmful growth of toxic oligomers in the formation of protein amyloid fibrils have been connected to degenerative diseases like Alzheimer's and Huntington's diseases. Understanding the fundamental mechanisms behind protein unfolding and subsequent fibrillogenesis may provide a way to stop the process from occurring. The purpose of this study was to identify favorable fibril growth conditions for a globular model protein β-lactoglobulin using the chaotropes urea and KSCN, along with titration of a pH 7.04 phosphate buffer solution at 40 ^oC over five days. Time-resolved and steady-state fluorescence was used to examine the shift in emission of the tryptophan amino acids over the applied denaturation ranges. BLG, a dimer in native form, monomerized and partially unfolded at 5 M Urea, 2 M KSCN and at pH 2 in phosphate buffer in vitro. Exposure of the solutions to continuous heat over time caused a increase in the lifetimes and red shift in the emission spectra, indicating the possible beginning of nucleation. The study has provided a base for continuation of the study of oligomerization and subsequent fibrillation of BLG, which may provide a fundamental mechanism of formation transferable to other proteins in vivo.

The effects of buffers and pH on the thermal stability, unfolding and substrate binding of RecA.

PubMed

Metrick, Michael A; Temple, Joshua E; MacDonald, Gina

2013-12-31

The Escherichia coli protein RecA is responsible for catalysis of the strand transfer reaction used in DNA repair and recombination. Previous studies in our lab have shown that high concentrations of salts stabilize RecA in a reverse-anionic Hofmeister series. Here we investigate how changes in pH and buffer alter the thermal unfolding and cofactor binding. RecA in 20mM HEPES, MES, Tris and phosphate buffers was studied in the pH range from 6.5 to 8.5 using circular dichroism (CD), infrared (IR) and fluorescence spectroscopies. The results show all of the buffers studied stabilize RecA up to 50°C above the Tris melting temperature and influence RecA's ability to nucleate on double-stranded DNA. Infrared and CD spectra of RecA in the different buffers do not show that secondary structural changes are associated with increased stability or decreased ability to nucleate on dsDNA. These results suggest the differences in stability arise from decreasing positive charge and/or buffer interactions. © 2013. Published by Elsevier B.V. All rights reserved.

High-throughput single-molecule force spectroscopy for membrane proteins

NASA Astrophysics Data System (ADS)

Bosshart, Patrick D.; Casagrande, Fabio; Frederix, Patrick L. T. M.; Ratera, Merce; Bippes, Christian A.; Müller, Daniel J.; Palacin, Manuel; Engel, Andreas; Fotiadis, Dimitrios

2008-09-01

Atomic force microscopy-based single-molecule force spectroscopy (SMFS) is a powerful tool for studying the mechanical properties, intermolecular and intramolecular interactions, unfolding pathways, and energy landscapes of membrane proteins. One limiting factor for the large-scale applicability of SMFS on membrane proteins is its low efficiency in data acquisition. We have developed a semi-automated high-throughput SMFS (HT-SMFS) procedure for efficient data acquisition. In addition, we present a coarse filter to efficiently extract protein unfolding events from large data sets. The HT-SMFS procedure and the coarse filter were validated using the proton pump bacteriorhodopsin (BR) from Halobacterium salinarum and the L-arginine/agmatine antiporter AdiC from the bacterium Escherichia coli. To screen for molecular interactions between AdiC and its substrates, we recorded data sets in the absence and in the presence of L-arginine, D-arginine, and agmatine. Altogether ~400 000 force-distance curves were recorded. Application of coarse filtering to this wealth of data yielded six data sets with ~200 (AdiC) and ~400 (BR) force-distance spectra in each. Importantly, the raw data for most of these data sets were acquired in one to two days, opening new perspectives for HT-SMFS applications.

Experimental determination of the response functions of a Bonner sphere spectrometer to monoenergetic neutrons

NASA Astrophysics Data System (ADS)

Hu, Z.; Chen, Z.; Peng, X.; Du, T.; Cui, Z.; Ge, L.; Zhu, W.; Wang, Z.; Zhu, X.; Chen, J.; Zhang, G.; Li, X.; Chen, J.; Zhang, H.; Zhong, G.; Hu, L.; Wan, B.; Gorini, G.; Fan, T.

2017-06-01

A Bonner sphere spectrometer (BSS) plays an important role in characterizing neutron spectra and determining their neutron dose in a neutron-gamma mixed field. A BSS consisting of a set of nine polyethylene spheres with a 3He proportional counter was developed at Peking University to perform neutron spectrum and dosimetry measurements. Response functions (RFs) of the BSS were calculated with the general Monte Carlo code MCNP5 for the neutron energy range from thermal up to 20 MeV, and were experimentally calibrated with monoenergetic neutron beams from 144 keV to 14 MeV on a 4.5 MV Van de Graaff accelerator. The calculated RFs were corrected with the experimental values, and the whole response matrix was completely established. The spectrum of a 241Am-Be source was obtained after unfolding the measurement data of the BSS to the source and in fair agreement with the expected one. The integral ambient dose equivalent corresponding to the spectrum was 0.95 of the expected value. Results of the unfolded spectrum and the integral dose equivalent measured by the BSS verified that the RFs of the BSS were well established.

Unfolding and unfoldability of digital pulses in the z-domain

NASA Astrophysics Data System (ADS)

Regadío, Alberto; Sánchez-Prieto, Sebastián

2018-04-01

The unfolding (or deconvolution) technique is used in the development of digital pulse processing systems applied to particle detection. This technique is applied to digital signals obtained by digitization of analog signals that represent the combined response of the particle detectors and the associated signal conditioning electronics. This work describes a technique to determine if the signal is unfoldable. For unfoldable signals the characteristics of the unfolding system (unfolder) are presented. Finally, examples of the method applied to real experimental setup are discussed.

Characterization of a 6Li enriched Cs2LiYCl6:Ce scintillator and its application as a γ-ray detector

NASA Astrophysics Data System (ADS)

Qin, Jianguo; Lai, Caifeng; Lu, Xinxin; Zheng, Pu; Zhu, Tonghua; Liu, Rong; Ye, Bangjiao; Zhang, Xinwei

2018-04-01

In this work, we characterize the γ-ray response and efficiency for a cylindrical inorganic Cs2LiYCl6:Ce detector 1‧‧ in diameter and 1‧‧ in height. The energy resolution and linearity are obtained from 21 γ-rays with energies ranging from 0.026 to 2.447 MeV. In addition, the neutron γ-ray discrimination is validated by measuring a 252Cf radioisotope. Gamma-ray response functions and matrix below 7 MeV are simulated using a Monte Carlo approach and validated through the unfolded γ-ray spectra.

Dependence of the prompt fission γ-ray spectrum on the entrance channel of compound nucleus: Spontaneous vs. neutron-induced fission

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

Chyzh, A.; Jaffke, P.; Wu, C. Y.

Prompt γ-ray spectra were measured for the spontaneous fission of 240,242Pu and the neutron-induced fission of 239,241Pu with incident neutron energies ranging from thermal to about 100 keV. Measurements were made using the Detector for Advanced Neutron Capture Experiments (DANCE) array in coincidence with the detection of fission fragments using a parallel-plate avalanche counter. The unfolded prompt fission γ-ray energy spectra can be reproduced reasonably well by Monte Carlo Hauser–Feshbach statistical model for the neutron-induced fission channel but not for the spontaneous fission channel. However, this entrance-channel dependence of the prompt fission γ-ray emission can be described qualitatively by themore » model due to the very different fission-fragment mass distributions and a lower average fragment spin for spontaneous fission. The description of measurements and the discussion of results under the framework of a Monte Carlo Hauser–Feshbach statistical approach are presented.« less

Cosmic ray spectrum and composition from three years of IceTop and IceCube

NASA Astrophysics Data System (ADS)

Rawlins, K.; IceCube Collaboration

2016-05-01

IceTop is the surface component of the IceCube Observatory, composed of frozen water tanks at the top of IceCube’s strings. Data from this detector can be analyzed in different ways with the goal of measuring cosmic ray spectrum and composition. The shower size S125 from IceTop alone can be used as a proxy for primary energy, and unfolded into an all-particle spectrum. In addition, S125 from the surface can be combined with high-energy muon energy loss information from the deep IceCube detector for those air showers which pass through both. Using these coincident events in a complementary analysis, both the spectrum and mass composition of primary cosmic rays can be extracted in parallel using a neural network. Both of these analyses have been performed on three years of IceTop and IceCube data. Both all-particle spectra as well as individual spectra for elemental groups are presented.

Latest results from the KASCADE-Grande experiment

NASA Astrophysics Data System (ADS)

Chiavassa, A.; Apel, W. D.; Arteaga-Velázquez, J. C.; Bekk, K.; Bertaina, M.; Blümer, J.; Bozdog, H.; Brancus, I. M.; Cantoni, E.; Cossavella, F.; Curcio, C.; Daumiller, K.; de Souza, V.; Di Pierro, F.; Doll, P.; Engel, R.; Engler, J.; Fuchs, B.; Fuhrmann, D.; Gils, H. J.; Glasstetter, R.; Grupen, C.; Haungs, A.; Heck, D.; Hörandel, J. R.; Huber, D.; Huege, T.; Kampert, K.-H.; Kang, D.; Klages, H. O.; Link, K.; Łuczak, P.; Ludwig, M.; Mathes, H. J.; Mayer, H. J.; Melissas, M.; Milke, J.; Mitrica, B.; Morello, C.; Oehlschläger, J.; Ostapchenko, S.; Palmieri, N.; Petcu, M.; Pierog, T.; Rebel, H.; Roth, M.; Schieler, H.; Schoo, S.; Schröder, F. G.; Sima, O.; Toma, G.; Trinchero, G. C.; Ulrich, H.; Weindl, A.; Wochele, J.; Zabierowski, J.

2014-04-01

The KASCADE-Grande experiment operated at KIT from January 2004 to November 2012, measuring EAS generated by primary cosmic rays in the 1016-1018 eV energy range. The experiment detected, for each single event, with a high resolution, the total number of charged particles (Nch) and of muons (Nμ). In this contribution we present the latest results about: The measurement of the all particle energy spectrum, discussing the influence of the hadronic interaction model used to derive the energy calibration of the experimental data. The energy spectra derived separating the events according to the Nμ /Nch ratio. This technique allowed us to unveil a steepening of the spectrum of heavy primaries at E ~10 16.92 ± 0.04 eV and a hardening of the spectrum of light primaries at E ~10 17.08 ± 0.08 eV. The elemental spectra (for five mass groups) obtained applying a detailed unfolding analysis technique. A search for large scale anisotropies.

Dependence of the prompt fission γ-ray spectrum on the entrance channel of compound nucleus: Spontaneous vs. neutron-induced fission

DOE PAGES

Chyzh, A.; Jaffke, P.; Wu, C. Y.; ...

2018-06-07

Prompt γ-ray spectra were measured for the spontaneous fission of 240,242Pu and the neutron-induced fission of 239,241Pu with incident neutron energies ranging from thermal to about 100 keV. Measurements were made using the Detector for Advanced Neutron Capture Experiments (DANCE) array in coincidence with the detection of fission fragments using a parallel-plate avalanche counter. The unfolded prompt fission γ-ray energy spectra can be reproduced reasonably well by Monte Carlo Hauser–Feshbach statistical model for the neutron-induced fission channel but not for the spontaneous fission channel. However, this entrance-channel dependence of the prompt fission γ-ray emission can be described qualitatively by themore » model due to the very different fission-fragment mass distributions and a lower average fragment spin for spontaneous fission. The description of measurements and the discussion of results under the framework of a Monte Carlo Hauser–Feshbach statistical approach are presented.« less

Discriminating trpzip2 and trpzip4 peptides’ folding landscape using the two-dimensional infrared spectroscopy: A simulation study

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

Wu, Tianmin; Zhang, Ruiting; Li, Huanhuan

2014-02-07

We analyzed, based on the theoretical spectroscopic modeling, how the differences in the folding landscapes of two β-hairpin peptides trpzip2 and trpzip4 are reflected in their thermal unfolding infrared measurements. The isotope-edited equilibrium FTIR and two dimensional infrared spectra of the two peptides were calculated, using the nonlinear exciton propagation method, at a series of temperatures. The spectra calculations were based on the configuration distributions generated using the GB{sup OBC} implicit solvent MD simulation and the integrated tempering sampling technique. Conformational analysis revealed the different local thermal stabilities for these two peptides, which suggested the different folding landscapes. Our studymore » further suggested that the ellipticities of the isotope peaks in the coherent IR signals are more sensitive to these local stability differences compared with other spectral features such as the peak intensities. Our technique can thus be combined with the relevant experimental measurements to achieve a better understanding of the peptide folding behaviors.« less

Direct Observation of Markovian Behavior of the Mechanical Unfolding of Individual Proteins

PubMed Central

Cao, Yi; Kuske, Rachel; Li, Hongbin

2008-01-01

Single-molecule force-clamp spectroscopy is a valuable tool to analyze unfolding kinetics of proteins. Previous force-clamp spectroscopy experiments have demonstrated that the mechanical unfolding of ubiquitin deviates from the generally assumed Markovian behavior and involves the features of glassy dynamics. Here we use single molecule force-clamp spectroscopy to study the unfolding kinetics of a computationally designed fast-folding mutant of the small protein GB1, which shares a similar β-grasp fold as ubiquitin. By treating the mechanical unfolding of polyproteins as the superposition of multiple identical Poisson processes, we developed a simple stochastic analysis approach to analyze the dwell time distribution of individual unfolding events in polyprotein unfolding trajectories. Our results unambiguously demonstrate that the mechanical unfolding of NuG2 fulfills all criteria of a memoryless Markovian process. This result, in contrast with the complex mechanical unfolding behaviors observed for ubiquitin, serves as a direct experimental demonstration of the Markovian behavior for the mechanical unfolding of a protein and reveals the complexity of the unfolding dynamics among structurally similar proteins. Furthermore, we extended our method into a robust and efficient pseudo-dwell-time analysis method, which allows one to make full use of all the unfolding events obtained in force-clamp experiments without categorizing the unfolding events. This method enabled us to measure the key parameters characterizing the mechanical unfolding energy landscape of NuG2 with improved precision. We anticipate that the methods demonstrated here will find broad applications in single-molecule force-clamp spectroscopy studies for a wide range of proteins. PMID:18375518

Protein unfolding in detergents: effect of micelle structure, ionic strength, pH, and temperature.

PubMed Central

Otzen, Daniel E

2002-01-01

The 101-residue monomeric protein S6 unfolds in the anionic detergent sodium dodecyl sulfate (SDS) above the critical micelle concentration, with unfolding rates varying according to two different modes. Our group has proposed that spherical micelles lead to saturation kinetics in unfolding (mode 1), while cylindrical micelles prevalent at higher SDS concentrations induce a power-law dependent increase in the unfolding rate (mode 2). Here I investigate in more detail how micellar properties affect protein unfolding. High NaCl concentrations, which induce cylindrical micelles, favor mode 2. This is consistent with our model, though other effects such as electrostatic screening cannot be discounted. Furthermore, unfolding does not occur in mode 2 in the cationic detergent LTAB, which is unable to form cylindrical micelles. A strong retardation of unfolding occurs at higher LTAB concentrations, possibly due to the formation of dead-end protein-detergent complexes. A similar, albeit much weaker, effect is seen in SDS in the absence of salt. Chymotrypsin inhibitor 2 exhibits the same modes of unfolding in SDS as S6, indicating that this type of protein unfolding is not specific for S6. The unfolding process in mode 1 has an activation barrier similar in magnitude to that in water, while the activation barrier in mode 2 is strongly concentration-dependent. The strong pH-dependence of unfolding in SDS and LTAB suggests that the rate of unfolding in anionic detergent is modulated by repulsion between detergent headgroups and anionic side chains, while cationic side chains modulate unfolding rates in cationic detergents. PMID:12324439

Electrostatic effects in unfolded staphylococcal nuclease

PubMed Central

Fitzkee, Nicholas C.; García-Moreno E, Bertrand

2008-01-01

Structure-based calculations of pK a values and electrostatic free energies of proteins assume that electrostatic effects in the unfolded state are negligible. In light of experimental evidence showing that this assumption is invalid for many proteins, and with increasing awareness that the unfolded state is more structured and compact than previously thought, a detailed examination of electrostatic effects in unfolded proteins is warranted. Here we address this issue with structure-based calculations of electrostatic interactions in unfolded staphylococcal nuclease. The approach involves the generation of ensembles of structures representing the unfolded state, and calculation of Coulomb energies to Boltzmann weight the unfolded state ensembles. Four different structural models of the unfolded state were tested. Experimental proton binding data measured with a variant of nuclease that is unfolded under native conditions were used to establish the validity of the calculations. These calculations suggest that weak Coulomb interactions are an unavoidable property of unfolded proteins. At neutral pH, the interactions are too weak to organize the unfolded state; however, at extreme pH values, where the protein has a significant net charge, the combined action of a large number of weak repulsive interactions can lead to the expansion of the unfolded state. The calculated pK a values of ionizable groups in the unfolded state are similar but not identical to the values in small peptides in water. These studies suggest that the accuracy of structure-based calculations of electrostatic contributions to stability cannot be improved unless electrostatic effects in the unfolded state are calculated explicitly. PMID:18227429

Studying the unfolding process of protein G and protein L under physical property space

PubMed Central

Zhao, Liling; Wang, Jihua; Dou, Xianghua; Cao, Zanxia

2009-01-01

Background The studies on protein folding/unfolding indicate that the native state topology is an important determinant of protein folding mechanism. The folding/unfolding behaviors of proteins which have similar topologies have been studied under Cartesian space and the results indicate that some proteins share the similar folding/unfolding characters. Results We construct physical property space with twelve different physical properties. By studying the unfolding process of the protein G and protein L under the property space, we find that the two proteins have the similar unfolding pathways that can be divided into three types and the one which with the umbrella-shape represents the preferred pathway. Moreover, the unfolding simulation time of the two proteins is different and protein L unfolding faster than protein G. Additionally, the distributing area of unfolded state ensemble of protein L is larger than that of protein G. Conclusion Under the physical property space, the protein G and protein L have the similar folding/unfolding behaviors, which agree with the previous results obtained from the studies under Cartesian coordinate space. At the same time, some different unfolding properties can be detected easily, which can not be analyzed under Cartesian coordinate space. PMID:19208146

Exploring Early Stages of the Chemical Unfolding of Proteins at the Proteome Scale

PubMed Central

Candotti, Michela; Pérez, Alberto; Ferrer-Costa, Carles; Rueda, Manuel; Meyer, Tim; Gelpí, Josep Lluís; Orozco, Modesto

2013-01-01

After decades of using urea as denaturant, the kinetic role of this molecule in the unfolding process is still undefined: does urea actively induce protein unfolding or passively stabilize the unfolded state? By analyzing a set of 30 proteins (representative of all native folds) through extensive molecular dynamics simulations in denaturant (using a range of force-fields), we derived robust rules for urea unfolding that are valid at the proteome level. Irrespective of the protein fold, presence or absence of disulphide bridges, and secondary structure composition, urea concentrates in the first solvation shell of quasi-native proteins, but with a density lower than that of the fully unfolded state. The presence of urea does not alter the spontaneous vibration pattern of proteins. In fact, it reduces the magnitude of such vibrations, leading to a counterintuitive slow down of the atomic-motions that opposes unfolding. Urea stickiness and slow diffusion is, however, crucial for unfolding. Long residence urea molecules placed around the hydrophobic core are crucial to stabilize partially open structures generated by thermal fluctuations. Our simulations indicate that although urea does not favor the formation of partially open microstates, it is not a mere spectator of unfolding that simply displaces to the right of the folded←→unfolded equilibrium. On the contrary, urea actively favors unfolding: it selects and stabilizes partially unfolded microstates, slowly driving the protein conformational ensemble far from the native one and also from the conformations sampled during thermal unfolding. PMID:24348236

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.

Investigation on the protein-binding properties of icotinib by spectroscopic and molecular modeling method.

PubMed

Zhang, Hua-xin; Xiong, Hang-xing; Li, Li-wei

2016-05-15

Icotinib is a highly-selective epidermal growth factor receptor tyrosine kinase inhibitor with preclinical and clinical activity in non-small cell lung cancer, which has been developed as a new targeted anti-tumor drug in China. In this work, the interaction of icotinib and human serum albumin (HSA) were studied by three-dimensional fluorescence spectra, ultraviolet spectra, circular dichroism (CD) spectra, molecular probe and molecular modeling methods. The results showed that icotinib binds to Sudlow's site I in subdomain IIA of HSA molecule, resulting in icotinib-HSA complexes formed at ground state. The number of binding sites, equilibrium constants, and thermodynamic parameters of the reaction were calculated at different temperatures. The negative enthalpy change (ΔH(θ)) and entropy change (ΔS(θ)) indicated that the structure of new complexes was stabilized by hydrogen bonds and van der Waals power. The distance between donor and acceptor was calculated according to Förster's non-radiation resonance energy transfer theory. The structural changes of HSA caused by icotinib binding were detected by synchronous spectra and circular dichroism (CD) spectra. Molecular modeling method was employed to unfold full details of the interaction at molecular level, most of which could be supported by experimental results. The study analyzed the probability that serum albumins act as carriers for this new anticarcinogen and provided fundamental information on the process of delivering icotinib to its target tissues, which might be helpful in understanding the mechanism of icotinib in cancer therapy. Copyright © 2016 Elsevier B.V. All rights reserved.

Amyloidogenesis of Natively Unfolded Proteins

PubMed Central

Uversky, Vladimir N.

2009-01-01

Aggregation and subsequent development of protein deposition diseases originate from conformational changes in corresponding amyloidogenic proteins. The accumulated data support the model where protein fibrillogenesis proceeds via the formation of a relatively unfolded amyloidogenic conformation, which shares many structural properties with the pre-molten globule state, a partially folded intermediate first found during the equilibrium and kinetic (un)folding studies of several globular proteins and later described as one of the structural forms of natively unfolded proteins. The flexibility of this structural form is essential for the conformational rearrangements driving the formation of the core cross-beta structure of the amyloid fibril. Obviously, molecular mechanisms describing amyloidogenesis of ordered and natively unfolded proteins are different. For ordered protein to fibrillate, its unique and rigid structure has to be destabilized and partially unfolded. On the other hand, fibrillogenesis of a natively unfolded protein involves the formation of partially folded conformation; i.e., partial folding rather than unfolding. In this review recent findings are surveyed to illustrate some unique features of the natively unfolded proteins amyloidogenesis. PMID:18537543

The unfolding effects on the protein hydration shell and partial molar volume: a computational study.

PubMed

Del Galdo, Sara; Amadei, Andrea

2016-10-12

In this paper we apply the computational analysis recently proposed by our group to characterize the solvation properties of a native protein in aqueous solution, and to four model aqueous solutions of globular proteins in their unfolded states thus characterizing the protein unfolded state hydration shell and quantitatively evaluating the protein unfolded state partial molar volumes. Moreover, by using both the native and unfolded protein partial molar volumes, we obtain the corresponding variations (unfolding partial molar volumes) to be compared with the available experimental estimates. We also reconstruct the temperature and pressure dependence of the unfolding partial molar volume of Myoglobin dissecting the structural and hydration effects involved in the process.

Lethal factor unfolding is the most force-dependent step of anthrax toxin translocation

PubMed Central

Thoren, Katie L.; Worden, Evan J.; Yassif, Jaime M.; Krantz, Bryan A.

2009-01-01

Cellular compartmentalization requires machinery capable of translocating polypeptides across membranes. In many cases, transported proteins must first be unfolded by means of the proton motive force and/or ATP hydrolysis. Anthrax toxin, which is composed of a channel-forming protein and two substrate proteins, is an attractive model system to study translocation-coupled unfolding, because the applied driving force can be externally controlled and translocation can be monitored directly by using electrophysiology. By controlling the driving force and introducing destabilizing point mutations in the substrate, we identified the barriers in the transport pathway, determined which barrier corresponds to protein unfolding, and mapped how the substrate protein unfolds during translocation. In contrast to previous studies, we find that the protein's structure next to the signal tag is not rate-limiting to unfolding. Instead, a more extensive part of the structure, the amino-terminal β-sheet subdomain, must disassemble to cross the unfolding barrier. We also find that unfolding is catalyzed by the channel's phenylalanine-clamp active site. We propose a broad molecular mechanism for translocation-coupled unfolding, which is applicable to both soluble and membrane-embedded unfolding machines. PMID:19926859

Solvent sensitivity of protein unfolding: dynamical study of chicken villin headpiece subdomain in water-ethanol binary mixture.

PubMed

Ghosh, Rikhia; Roy, Susmita; Bagchi, Biman

2013-12-12

We carry out a series of long atomistic molecular dynamics simulations to study the unfolding of a small protein, chicken villin headpiece (HP-36), in water-ethanol (EtOH) binary mixture. The prime objective of this work is to explore the sensitivity of protein unfolding dynamics toward increasing concentration of the cosolvent and unravel essential features of intermediates formed in search of a dynamical pathway toward unfolding. In water-ethanol binary mixtures, HP-36 is found to unfold partially, under ambient conditions, that otherwise requires temperature as high as ∼600 K to denature in pure aqueous solvent. However, an interesting course of pathway is observed to be followed in the process, guided by the formation of unique intermediates. The first step of unfolding is essentially the separation of the cluster formed by three hydrophobic (phenylalanine) residues, namely, Phe-7, Phe-11, and Phe-18, which constitute the hydrophobic core, thereby initiating melting of helix-2 of the protein. The initial steps are similar to temperature-induced unfolding as well as chemical unfolding using DMSO as cosolvent. Subsequent unfolding steps follow a unique path. As water-ethanol shows composition-dependent anomalies, so do the details of unfolding dynamics. With an increase in cosolvent concentration, different partially unfolded intermediates are found to be formed. This is reflected in a remarkable nonmonotonic composition dependence of several order parameters, including fraction of native contacts and protein-solvent interaction energy. The emergence of such partially unfolded states can be attributed to the preferential solvation of the hydrophobic residues by the ethyl groups of ethanol. We further quantify the local dynamics of unfolding by using a Marcus-type theory.

Data Unfolding with Wiener-SVD Method

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

Tang, W.; Li, X.; Qian, X.

Here, data unfolding is a common analysis technique used in HEP data analysis. Inspired by the deconvolution technique in the digital signal processing, a new unfolding technique based on the SVD technique and the well-known Wiener filter is introduced. The Wiener-SVD unfolding approach achieves the unfolding by maximizing the signal to noise ratios in the effective frequency domain given expectations of signal and noise and is free from regularization parameter. Through a couple examples, the pros and cons of the Wiener-SVD approach as well as the nature of the unfolded results are discussed.

Data Unfolding with Wiener-SVD Method

DOE PAGES

Tang, W.; Li, X.; Qian, X.; ...

2017-10-04

Here, data unfolding is a common analysis technique used in HEP data analysis. Inspired by the deconvolution technique in the digital signal processing, a new unfolding technique based on the SVD technique and the well-known Wiener filter is introduced. The Wiener-SVD unfolding approach achieves the unfolding by maximizing the signal to noise ratios in the effective frequency domain given expectations of signal and noise and is free from regularization parameter. Through a couple examples, the pros and cons of the Wiener-SVD approach as well as the nature of the unfolded results are discussed.

Pathway Programs to Life after College

ERIC Educational Resources Information Center

McAtee, Jim F.

2012-01-01

The transition from college to whatever lies ahead can be exciting and scary for students. How the transition experience unfolds for students will be the result of a combination of factors including individual circumstances, how the student responds to change and unknown expectations, and how well they are prepared and informed for their…

Nanomechanics of Protein Unfolding outside Protease Nanopores

NASA Astrophysics Data System (ADS)

Luan, Binquan; Zhou, Ruhong

Protein folding and unfolding have been the subject of active research for decades. Most of previous studies in protein unfolding were focused on temperature, chemical and/or force (such as in AFM) induced denaturations. Recent studies on the functional roles of proteasomes (such as ClpXP) revealed a novel unfolding process in cell, during which a target protein is mechanically unfolded and pulled into a confined, pore-like geometry for degradation. While the proteasome nanomachine has been extensively studied, the mechanism for unfolding proteins with the proteasome pore is still poorly understood. Here, we investigate the mechanical unfolding process of ubiquitin with (or really outside) an idealized proteasome pore, and compare such process with that in the AFM pulling experiment. Unexpectedly, the required force by a proteosome can be much smaller than that by the AFM. Simulation results also unveiled different nanomechanics, tearing fracture vs. shearing friction, in these two distinct types of mechanical unfoldings.

Application of a Bonner sphere spectrometer for the determination of the angular neutron energy spectrum of an accelerator-based BNCT facility.

PubMed

Mirzajani, N; Ciolini, R; Di Fulvio, A; Esposito, J; d'Errico, F

2014-06-01

Experimental activities are underway at INFN Legnaro National Laboratories (LNL) (Padua, Italy) and Pisa University aimed at angular-dependent neutron energy spectra measurements produced by the (9)Be(p,xn) reaction, under a 5MeV proton beam. This work has been performed in the framework of INFN TRASCO-BNCT project. Bonner Sphere Spectrometer (BSS), based on (6)LiI (Eu) scintillator, was used with the shadow-cone technique. Proper unfolding codes, coupled to BSS response function calculated by Monte Carlo code, were finally used. The main results are reported here. Crown Copyright © 2014. Published by Elsevier Ltd. All rights reserved.

Prompt fission gamma-ray emission spectral data for 239Pu(n,f) using fast directional neutrons from the LICORNE neutron source

NASA Astrophysics Data System (ADS)

Qi, L.; Wilson, J. N.; Lebois, M.; Al-Adili, A.; Chatillon, A.; Choudhury, D.; Gatera, A.; Georgiev, G.; Göök, A.; Laurent, B.; Maj, A.; Matea, I.; Oberstedt, A.; Oberstedt, S.; Rose, S. J.; Schmitt, C.; Wasilewska, B.; Zeiser, F.

2018-03-01

Prompt fission gamma-ray spectra (PFGS) have been measured for the 239Pu(n,f) reaction using fast neutrons at Ēn=1.81 MeV produced by the LICORNE directional neutron source. The setup makes use of LaBr3 scintillation detectors and PARIS phoswich detectors to measure the emitted prompt fission gamma rays (PFG). The mean multiplicity, average total energy release per fission and average energy of photons are extracted from the unfolded PFGS. These new measurements provide complementary information to other recent work on thermal neutron induced fission of 239Pu and spontaneous fission of 252Cf.

Communication: nanosecond folding dynamics of an alpha helix: time-dependent 2D-IR cross peaks observed using polarization-sensitive dispersed pump-probe spectroscopy.

PubMed

Panman, Matthijs R; van Dijk, Chris N; Meuzelaar, Heleen; Woutersen, S

2015-01-28

We present a simple method to measure the dynamics of cross peaks in time-resolved two-dimensional vibrational spectroscopy. By combining suitably weighted dispersed pump-probe spectra, we eliminate the diagonal contribution to the 2D-IR response, so that the dispersed pump-probe signal contains the projection of only the cross peaks onto one of the axes of the 2D-IR spectrum. We apply the method to investigate the folding dynamics of an alpha-helical peptide in a temperature-jump experiment and find characteristic folding and unfolding time constants of 260 ± 30 and 580 ± 70 ns at 298 K.

The Evaluation of Teaching the Nursing Process Using Traditional Lecture, Campus Laboratory, Clinical, and the Addition of High Fidelity Human Simulation (HFHS) Unfolding Scenarios

ERIC Educational Resources Information Center

Irwin, Ruth E.

2013-01-01

It is not sufficient to just make changes in a nursing curriculum without a plan to evaluate the impact on program outcomes. This study sought to determine the outcomes of teaching the nursing process to Foundation of Nursing students in an Associate Degree Nursing program using a factorial design study. Four groups of students were taught the…

Gamma-Ray Signatures Improvement of the EURITRACK Tagged Neutron Inspection System Database

NASA Astrophysics Data System (ADS)

Kanawati, Wassila El; Carasco, Cedric; Perot, Bertrand; Mariani, Alain; Raoux, Anne-Cecile; Valkovic, Vladivoj; Sudac, Davorin; Obhodas, Jasmina; Baricevic, Martina

2010-10-01

The EURopean Illicit TRAfficking Countermeasures Kit (EURITRACK) inspection system uses 14 MeV neutrons produced by the D(T,n α) reaction to detect explosives in cargo containers. Reactions induced by fast neutrons inside the container produce gamma rays, which are detected in coincidence with the associated alpha particle, the detection of which allows the neutron direction to be determined. The neutron path length is obtained from a neutron time-of-flight measurement, thus allowing the origin of the gamma rays inside the container to be determined, while the chemical composition of the target material is correlated with their energy spectrum. Gamma-ray spectra have been collected with the inspection portal equipped with large volume NaI (Tl) detectors, in order to build a database of signatures for various elements (C, O, N, Fe, Pb, Al, Na, Si, Cl, Cu, Zn) with a low energy threshold of 0.6 MeV. The spectra are compared with previous ones, which were acquired with a 1.35 MeV threshold. The new library is currently being tested to unfold the energy spectra of transported goods into elemental contributions. Results are compared with data processed with the old 1.35 MeV threshold database, thus illustrating the improvement for material identification.

Neutron spectroscopy for pulsed beams with frame overlap using a double time-of-flight technique

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

Harrig, K. P.; Goldblum, B. L.; Brown, J. A.

A new double time-of- ight (dTOF) neutron spectroscopy technique has been developed for pulsed broad spectrum sources with a duty cycle that results in frame overlap, where fast neutrons from a given pulse overtake slower neutrons from previous pulses. Using a tunable beam at the 88-Inch Cyclotron at Lawrence Berkeley National Laboratory, neutrons were produced via thick-target breakup of 16 MeV deuterons on a beryllium target in the cyclotron vault. The breakup spectral shape was deduced from a dTOF measurement using an array of EJ-309 organic liquid scintillators. Simulation of the neutron detection efficiency of the scintillator array was performedmore » using both GEANT4 and MCNP6. The efficiency- corrected spectral shape was normalized using a foil activation technique to obtain the energy-dependent flux of the neutron beam at zero degrees with respect to the incoming deuteron beam. The dTOF neutron spectrum was compared to spectra obtained using HEPROW and GRAVEL pulse height spectrum unfolding techniques. While the unfolding and dTOF results exhibit some discrepancies in shape, the integrated flux values agree within two standard deviations. As a result, this method obviates neutron time-of-flight spectroscopy challenges posed by pulsed beams« less

Structural basis of sodium–potassium exchange of a human telomeric DNA quadruplex without topological conversion

PubMed Central

Wang, Zi-Fu; Li, Ming-Hao; Hsu, Shang-Te Danny; Chang, Ta-Chau

2014-01-01

Understanding the mechanism of Na+/K+-dependent spectral conversion of human telomeric G-quadruplex (G4) sequences has been limited not only because of the structural polymorphism but also the lack of sufficient structural information at different stages along the conversion process for one given oligonucleotide. In this work, we have determined the topology of the Na+ form of Tel23 G4, which is the same hybrid form as the K+ form of Tel23 G4 despite the distinct spectral patterns in their respective nuclear magnetic resonance (NMR) and circular dichroism spectra. The spectral difference, particularly the well-resolved imino proton NMR signals, allows us to monitor the structural conversion from Na+ form to K+ form during Na+/K+ exchange. Time-resolved NMR experiments of hydrogen–deuterium exchange and hybridization clearly exclude involvement of the global unfolding for the fast Na+/K+ spectral conversion. In addition, the K+ titration monitored by NMR reveals that the Na+/K+ exchange in Tel23 G4 is a two-step process. The addition of K+ significantly stabilizes the unfolding kinetics of Tel23 G4. These results offer a possible explanation of rapid spectral conversion of Na+/K+ exchange and insight into the mechanism of Na+/K+ structural conversion in human telomeric G4s. PMID:24476914

Neutron Spectroscopy for pulsed beams with frame overlap using a double time-of-flight technique

NASA Astrophysics Data System (ADS)

Harrig, K. P.; Goldblum, B. L.; Brown, J. A.; Bleuel, D. L.; Bernstein, L. A.; Bevins, J.; Harasty, M.; Laplace, T. A.; Matthews, E. F.

2018-01-01

A new double time-of-flight (dTOF) neutron spectroscopy technique has been developed for pulsed broad spectrum sources with a duty cycle that results in frame overlap, where fast neutrons from a given pulse overtake slower neutrons from previous pulses. Using a tunable beam at the 88-Inch Cyclotron at Lawrence Berkeley National Laboratory, neutrons were produced via thick-target breakup of 16 MeV deuterons on a beryllium target in the cyclotron vault. The breakup spectral shape was deduced from a dTOF measurement using an array of EJ-309 organic liquid scintillators. Simulation of the neutron detection efficiency of the scintillator array was performed using both GEANT4 and MCNP6. The efficiency-corrected spectral shape was normalized using a foil activation technique to obtain the energy-dependent flux of the neutron beam at zero degrees with respect to the incoming deuteron beam. The dTOF neutron spectrum was compared to spectra obtained using HEPROW and GRAVEL pulse height spectrum unfolding techniques. While the unfolding and dTOF results exhibit some discrepancies in shape, the integrated flux values agree within two standard deviations. This method obviates neutron time-of-flight spectroscopy challenges posed by pulsed beams with frame overlap and opens new opportunities for pulsed white neutron source facilities.

Neutron spectroscopy for pulsed beams with frame overlap using a double time-of-flight technique

DOE PAGES

Harrig, K. P.; Goldblum, B. L.; Brown, J. A.; ...

2017-10-16

A new double time-of- ight (dTOF) neutron spectroscopy technique has been developed for pulsed broad spectrum sources with a duty cycle that results in frame overlap, where fast neutrons from a given pulse overtake slower neutrons from previous pulses. Using a tunable beam at the 88-Inch Cyclotron at Lawrence Berkeley National Laboratory, neutrons were produced via thick-target breakup of 16 MeV deuterons on a beryllium target in the cyclotron vault. The breakup spectral shape was deduced from a dTOF measurement using an array of EJ-309 organic liquid scintillators. Simulation of the neutron detection efficiency of the scintillator array was performedmore » using both GEANT4 and MCNP6. The efficiency- corrected spectral shape was normalized using a foil activation technique to obtain the energy-dependent flux of the neutron beam at zero degrees with respect to the incoming deuteron beam. The dTOF neutron spectrum was compared to spectra obtained using HEPROW and GRAVEL pulse height spectrum unfolding techniques. While the unfolding and dTOF results exhibit some discrepancies in shape, the integrated flux values agree within two standard deviations. As a result, this method obviates neutron time-of-flight spectroscopy challenges posed by pulsed beams« less

Endoplasmic Reticulum Stress and Unfolded Protein Response in Cartilage Pathophysiology; Contributing Factors to Apoptosis and Osteoarthritis.

PubMed

Hughes, Alexandria; Oxford, Alexandra E; Tawara, Ken; Jorcyk, Cheryl L; Oxford, Julia Thom

2017-03-20

Chondrocytes of the growth plate undergo apoptosis during the process of endochondral ossification, as well as during the progression of osteoarthritis. Although the regulation of this process is not completely understood, alterations in the precisely orchestrated programmed cell death during development can have catastrophic results, as exemplified by several chondrodystrophies which are frequently accompanied by early onset osteoarthritis. Understanding the mechanisms that underlie chondrocyte apoptosis during endochondral ossification in the growth plate has the potential to impact the development of therapeutic applications for chondrodystrophies and associated early onset osteoarthritis. In recent years, several chondrodysplasias and collagenopathies have been recognized as protein-folding diseases that lead to endoplasmic reticulum stress, endoplasmic reticulum associated degradation, and the unfolded protein response. Under conditions of prolonged endoplasmic reticulum stress in which the protein folding load outweighs the folding capacity of the endoplasmic reticulum, cellular dysfunction and death often occur. However, unfolded protein response (UPR) signaling is also required for the normal maturation of chondrocytes and osteoblasts. Understanding how UPR signaling may contribute to cartilage pathophysiology is an essential step toward therapeutic modulation of skeletal disorders that lead to osteoarthritis.

A new role for HERPUD1 and ERAD activation in osteoblast differentiation and mineralization.

PubMed

Américo-Da-Silva, Luan; Diaz, Jheimmy; Bustamante, Mario; Mancilla, Georthan; Oyarzún, Ingrid; Verdejo, Hugo E; Quiroga, Clara

2018-03-23

Bone integrity depends on a finely tuned balance between bone synthesis by osteoblasts and resorption by osteoclasts. The secretion capacity of mature osteoblasts requires strict control of proteostasis. Endoplasmic reticulum-associated degradation (ERAD) prevents the accumulation of unfolded ER proteins via dislocation to the cytosol and degradation by the proteasome. The ER membrane protein, homocysteine-inducible endoplasmic reticulum protein with ubiquitin-like domain 1 (HERPUD1), is a key component of the ERAD multiprotein complex which helps to stabilize the complex and facilitate the efficient degradation of unfolded proteins. HERPUD1 expression is strongly up-regulated by the unfolded protein response and cellular stress. The aim of the current study was to establish whether HERPUD1 and ERAD play roles in osteoblast differentiation and maturation. We evaluated preosteoblastic MC3T3-E1 cell and primary rat osteoblast differentiation by measuring calcium deposit levels, alkaline phosphatase activity, and runt-related transcription factor 2 and osterix expression. We found that ERAD and proteasomal degradation were activated and that HERPUD1 expression was increased as osteoblast differentiation progressed. The absence of HERPUD1 blocked osteoblast mineralization in vitro and significantly reduced alkaline phosphatase activity. In contrast, HERPUD1 overexpression activated the osteoblast differentiation program. Our results demonstrate that HERPUD1 and ERAD are important for the activation of the osteoblast maturation program and may be useful new targets for elucidating bone physiology.-Américo-Da-Silva, L., Diaz, J., Bustamante, M., Mancilla, G., Oyarzún, I., Verdejo, H. E., Quiroga, C. A new role for HERPUD1 and ERAD activation in osteoblast differentiation and mineralization.

Folding in and out: passive morphing in flapping wings.

PubMed

Stowers, Amanda K; Lentink, David

2015-03-25

We present a new mechanism for passive wing morphing of flapping wings inspired by bat and bird wing morphology. The mechanism consists of an unactuated hand wing connected to the arm wing with a wrist joint. Flapping motion generates centrifugal accelerations in the hand wing, forcing it to unfold passively. Using a robotic model in hover, we made kinematic measurements of unfolding kinematics as functions of the non-dimensional wingspan fold ratio (2-2.5) and flapping frequency (5-17 Hz) using stereo high-speed cameras. We find that the wings unfold passively within one to two flaps and remain unfolded with only small amplitude oscillations. To better understand the passive dynamics, we constructed a computer model of the unfolding process based on rigid body dynamics, contact models, and aerodynamic correlations. This model predicts the measured passive unfolding within about one flap and shows that unfolding is driven by centrifugal acceleration induced by flapping. The simulations also predict that relative unfolding time only weakly depends on flapping frequency and can be reduced to less than half a wingbeat by increasing flapping amplitude. Subsequent dimensional analysis shows that the time required to unfold passively is of the same order of magnitude as the flapping period. This suggests that centrifugal acceleration can drive passive unfolding within approximately one wingbeat in small and large wings. Finally, we show experimentally that passive unfolding wings can withstand impact with a branch, by first folding and then unfolding passively. This mechanism enables flapping robots to squeeze through clutter without sophisticated control. Passive unfolding also provides a new avenue in morphing wing design that makes future flapping morphing wings possibly more energy efficient and light-weight. Simultaneously these results point to possible inertia driven, and therefore metabolically efficient, control strategies in bats and birds to morph or recover within a beat.

Protein unfolding under isometric tension-what force can integrins generate, and can it unfold FNIII domains?

PubMed

Erickson, Harold P

2017-02-01

Extracellular matrix fibrils of fibronectin (FN) are highly elastic, and are typically stretched three to four times their relaxed length. The mechanism of stretching has been controversial, in particular whether it involves tension-induced unfolding of FNIII domains. Recent studies have found that ∼5pN is the threshold isometric force for unfolding various protein domains. FNIII domains should therefore not be unfolded until the tension approaches 5pN. Integrins have been reported to generate forces ranging from 1 to >50pN, but I argue that studies reporting 1-2pN are the most convincing. This is not enough to unfold FNIII domains. Even if domains were unfolded, 2pN would only extend the worm-like-chain to about twice the length of the folded domain. Overall I conclude that stretching FN matrix fibrils involves primarily the compact to extended conformational change of FN dimers, with minimal contribution from unfolding FNIII domains. Copyright © 2016 Elsevier Ltd. All rights reserved.

Point Mutations in Membrane Proteins Reshape Energy Landscape and Populate Different Unfolding Pathways

PubMed Central

Sapra, K. Tanuj; Balasubramanian, G. Prakash; Labudde, Dirk; Bowie, James U.; Muller, Daniel J.

2009-01-01

Using single-molecule force spectroscopy, we investigated the effect of single point mutations on the energy landscape and unfolding pathways of the transmembrane protein bacteriorhodopsin. We show that the unfolding energy barriers in the energy landscape of the membrane protein followed a simple two-state behavior and represent a manifestation of many converging unfolding pathways. Although the unfolding pathways of wild-type and mutant bacteriorhodopsin did not change, indicating the presence of same ensemble of structural unfolding intermediates, the free energies of the rate-limiting transition states of the bacteriorhodopsin mutants decreased as the distance of those transition states to the folded intermediate states decreased. Thus, all mutants exhibited Hammond behavior and a change in the free energies of the intermediates along the unfolding reaction coordinate and, consequently, their relative occupancies. This is the first experimental proof showing that point mutations can reshape the free energy landscape of a membrane protein and force single proteins to populate certain unfolding pathways over others. PMID:18191146

Prediction of change in protein unfolding rates upon point mutations in two state proteins.

PubMed

Chaudhary, Priyashree; Naganathan, Athi N; Gromiha, M Michael

2016-09-01

Studies on protein unfolding rates are limited and challenging due to the complexity of unfolding mechanism and the larger dynamic range of the experimental data. Though attempts have been made to predict unfolding rates using protein sequence-structure information there is no available method for predicting the unfolding rates of proteins upon specific point mutations. In this work, we have systematically analyzed a set of 790 single mutants and developed a robust method for predicting protein unfolding rates upon mutations (Δlnku) in two-state proteins by combining amino acid properties and knowledge-based classification of mutants with multiple linear regression technique. We obtain a mean absolute error (MAE) of 0.79/s and a Pearson correlation coefficient (PCC) of 0.71 between predicted unfolding rates and experimental observations using jack-knife test. We have developed a web server for predicting protein unfolding rates upon mutation and it is freely available at https://www.iitm.ac.in/bioinfo/proteinunfolding/unfoldingrace.html. Prominent features that determine unfolding kinetics as well as plausible reasons for the observed outliers are also discussed. Copyright © 2016 Elsevier B.V. All rights reserved.

The Dialectics Informing Identity in an Urban Youth Digital Storytelling Workshop

ERIC Educational Resources Information Center

DeGennaro, Donna

2008-01-01

This article examines an after-school program entitled Silk City Media Workshop. Briefly, the workshop engages youth in digital storytelling as a means of enhancing both their technology and literacy skills. Transcending these goals, this workshop also provides opportunities for youth to reveal multiple aspects of their unfolding identities as…

A Multiple Case Study of Applied Scholastic's Study Technology Program

ERIC Educational Resources Information Center

Guidry, Chejuana

2017-01-01

In 1966, the Coleman Report alerted the American people to the unfolding tragedy of a dysfunctional educational system. Seventeen years later, the National Commission on Excellence in Education issued its report titled "A Nation At Risk: The Imperative for Educational Reform," on the declining quality of American schools in general. This…

Rerouting Success: Several Academic Pathways Programs Are Fueling Reform at Community Colleges

ERIC Educational Resources Information Center

Woods, Bob

2015-01-01

"Completion" has become the rallying cry at community colleges. As the 21st century unfolds, the earlier emphasis on guaranteeing greater access to a postsecondary education has evolved into a sharp focus on student success. Whether that means earning a workforcerelated certificate or an associate degree transferable to a four-year…

Citizenship Education in Croatia: At the Margins of the System

ERIC Educational Resources Information Center

Kekez, Anka; Horvat, Martina; Šalaj, Berto

2017-01-01

Purpose: This paper unfolds the ways in which Croatia, as a young post-communist democracy, has aligned its transition and consolidation with the development of education programs that would support the protection of human rights and the creation of a democratic political culture. Design/methodology/approach: By combining the existing studies with…

Unfolding pathway of CotA-laccase and the role of copper on the prevention of refolding through aggregation of the unfolded state

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

Fernandes, Andre T.; Lopes, Carlos; Martins, Ligia O.

2012-06-08

Highlights: Black-Right-Pointing-Pointer CotA-laccase unfolds with an intermediate state. Black-Right-Pointing-Pointer Copper stabilizes the native and the intermediate state. Black-Right-Pointing-Pointer Copper binding to the unfolded state prevents refolding through protein aggregation. Black-Right-Pointing-Pointer Copper incorporation in CotA-laccase occurs as a later step during folding. -- Abstract: Copper is a redox-active metal and the main player in electron transfer reactions occurring in multicopper oxidases. The role of copper in the unfolding pathway and refolding of the multicopper oxidase CotA laccase in vitro was solved using double-jump stopped-flow experiments. Unfolding of apo- and holo-CotA was described as a three-state process with accumulation of an intermediatemore » in between the native and unfolded state. Copper stabilizes the native holo-CotA but also the intermediate state showing that copper is still bound to this state. Also, copper binds to unfolded holo-CotA in a non-native coordination promoting CotA aggregation and preventing refolding to the native structure. These results gather information on unfolding/folding pathways of multicopper oxidases and show that copper incorporation in vivo should be a tight controlled process as copper binding to the unfolded state under native conditions promotes protein aggregation.« less

Probing the contribution of internal cavities to the volume change of protein unfolding under pressure.

PubMed Central

Frye, K. J.; Royer, C. A.

1998-01-01

The structural origin of the decrease in system volume upon protein denaturation by pressure has remained a puzzle for decades. This negative volume change upon unfolding is assumed to arise globally from more intimate interactions between the polypeptide chain and water, including electrostriction of buried charges that become exposed upon unfolding, hydration of the polypeptide backbone and amino acid side chains and elimination of packing defects and internal void volumes upon unfolding of the chain. However, the relative signs and magnitudes of each of these contributing factors have not been experimentally determined. Our laboratory has probed the fundamental basis for the volume change upon unfolding of staphylococcal nuclease (Snase) using variable solution conditions and point mutants of Snase (Royer CA et al., 1993, Biochemistry 32:5222-5232; Frye KJ et al., 1996, Biochemistry 35:10234-10239). Our prior results indicate that for Snase, neither electrostriction nor polar or nonpolar hydration contributes significantly to the value of the volume change of unfolding. In the present work, we investigate the pressure induced unfolding of three point mutants of Snase in which internal cavity size is altered. The experimentally determined volume changes of unfolding for the mutants suggest that loss of internal void volume upon unfolding represents the major contributing factor to the value of the volume change of Snase unfolding. PMID:9792110

Beetle wings are inflatable origami

NASA Astrophysics Data System (ADS)

Chen, Rui; Ren, Jing; Ge, Siqin; Hu, David

2015-11-01

Beetles keep their wings folded and protected under a hard shell. In times of danger, they must unfold them rapidly in order for them to fly to escape. Moreover, they must do so across a range of body mass, from 1 mg to 10 grams. How can they unfold their wings so quickly? We use high-speed videography to record wing unfolding times, which we relate to the geometry of the network of blood vessels in the wing. Larger beetles have longer unfolding times. Modeling of the flow of blood through the veins successfully accounts for the wing unfolding speed of large beetles. However, smaller beetles have anomalously short unfolding times, suggesting they have lower blood viscosity or higher driving pressure. The use of hydraulics to unfold complex objects may have implications in the design of micro-flying air vehicles.

History, rare, and multiple events of mechanical unfolding of repeat proteins

NASA Astrophysics Data System (ADS)

Sumbul, Fidan; Marchesi, Arin; Rico, Felix

2018-03-01

Mechanical unfolding of proteins consisting of repeat domains is an excellent tool to obtain large statistics. Force spectroscopy experiments using atomic force microscopy on proteins presenting multiple domains have revealed that unfolding forces depend on the number of folded domains (history) and have reported intermediate states and rare events. However, the common use of unspecific attachment approaches to pull the protein of interest holds important limitations to study unfolding history and may lead to discarding rare and multiple probing events due to the presence of unspecific adhesion and uncertainty on the pulling site. Site-specific methods that have recently emerged minimize this uncertainty and would be excellent tools to probe unfolding history and rare events. However, detailed characterization of these approaches is required to identify their advantages and limitations. Here, we characterize a site-specific binding approach based on the ultrastable complex dockerin/cohesin III revealing its advantages and limitations to assess the unfolding history and to investigate rare and multiple events during the unfolding of repeated domains. We show that this approach is more robust, reproducible, and provides larger statistics than conventional unspecific methods. We show that the method is optimal to reveal the history of unfolding from the very first domain and to detect rare events, while being more limited to assess intermediate states. Finally, we quantify the forces required to unfold two molecules pulled in parallel, difficult when using unspecific approaches. The proposed method represents a step forward toward more reproducible measurements to probe protein unfolding history and opens the door to systematic probing of rare and multiple molecule unfolding mechanisms.

Folding and unfolding pathway of chaperonin GroEL monomer and elucidation of thermodynamic parameters.

PubMed

Puri, Sarita; Chaudhuri, Tapan K

2017-03-01

The conformation and thermodynamic stability of monomeric GroEL were studied by CD and fluorescence spectroscopy. GroEL denaturation with urea and dilution in buffer leads to formation of a folded GroEL monomer. The monomeric nature of this protein was verified by size-exclusion chromatography and native PAGE. It has a well-defined secondary and tertiary structure, folding activity (prevention of aggregation) for substrate protein and is resistant to proteolysis. Being a properly folded and reversibly refoldable, monomeric GroEL is amenable for the study of thermodynamic stability by unfolding transition methods. We present the equilibrium unfolding of monomeric GroEL as studied by urea and heat mediated unfolding processes. The urea mediated unfolding shows two transitions and a single transition in the heat mediated unfolding process. In the case of thermal unfolding, some residual structure unfolds at a higher temperature (70-75°C). The process of folding/unfolding is reversible in both cases. Analysis of folding/unfolding data provides a measure of ΔG NU H 2 O , T m , ΔH van and ΔS van of monomeric GroEL. The thermodynamic stability parameter ΔG NU H 2 O is similar with both CD and intrinsic fluorescence i.e. 7.10±1.0kcal/mol. The calculated T m , ΔH van and ΔS van from the thermal unfolding transition is 46±0.5°C, 43.3±0.1kcal/mol and 143.9±0.1cal/mol/k respectively. Copyright © 2016 Elsevier B.V. All rights reserved.

Measurements of the atmospheric neutrino flux by Super-Kamiokande: Energy spectra, geomagnetic effects, and solar modulation

NASA Astrophysics Data System (ADS)

Richard, E.; Okumura, K.; Abe, K.; Haga, Y.; Hayato, Y.; Ikeda, M.; Iyogi, K.; Kameda, J.; Kishimoto, Y.; Miura, M.; Moriyama, S.; Nakahata, M.; Nakajima, T.; Nakano, Y.; Nakayama, S.; Orii, A.; Sekiya, H.; Shiozawa, M.; Takeda, A.; Tanaka, H.; Tomura, T.; Wendell, R. A.; Akutsu, R.; Irvine, T.; Kajita, T.; Kaneyuki, K.; Nishimura, Y.; Labarga, L.; Fernandez, P.; Gustafson, J.; Kachulis, C.; Kearns, E.; Raaf, J. L.; Stone, J. L.; Sulak, L. R.; Berkman, S.; Nantais, C. M.; Tanaka, H. A.; Tobayama, S.; Goldhaber, M.; Kropp, W. R.; Mine, S.; Weatherly, P.; Smy, M. B.; Sobel, H. W.; Takhistov, V.; Ganezer, K. S.; Hartfiel, B. L.; Hill, J.; Hong, N.; Kim, J. Y.; Lim, I. T.; Park, R. G.; Himmel, A.; Li, Z.; O'Sullivan, E.; Scholberg, K.; Walter, C. W.; Wongjirad, T.; Ishizuka, T.; Tasaka, S.; Jang, J. S.; Learned, J. G.; Matsuno, S.; Smith, S. N.; Friend, M.; Hasegawa, T.; Ishida, T.; Ishii, T.; Kobayashi, T.; Nakadaira, T.; Nakamura, K.; Oyama, Y.; Sakashita, K.; Sekiguchi, T.; Tsukamoto, T.; Suzuki, A. T.; Takeuchi, Y.; Yano, T.; Cao, S. V.; Hiraki, T.; Hirota, S.; Huang, K.; Kikawa, T.; Minamino, A.; Nakaya, T.; Suzuki, K.; Fukuda, Y.; Choi, K.; Itow, Y.; Suzuki, T.; Mijakowski, P.; Frankiewicz, K.; Hignight, J.; Imber, J.; Jung, C. K.; Li, X.; Palomino, J. L.; Wilking, M. J.; Yanagisawa, C.; Fukuda, D.; Ishino, H.; Kayano, T.; Kibayashi, A.; Koshio, Y.; Mori, T.; Sakuda, M.; Xu, C.; Kuno, Y.; Tacik, R.; Kim, S. B.; Okazawa, H.; Choi, Y.; Nishijima, K.; Koshiba, M.; Totsuka, Y.; Suda, Y.; Yokoyama, M.; Bronner, C.; Hartz, M.; Martens, K.; Marti, Ll.; Suzuki, Y.; Vagins, M. R.; Martin, J. F.; Konaka, A.; Chen, S.; Zhang, Y.; Wilkes, R. J.; Super-Kamiokande Collaboration

2016-09-01

A comprehensive study of the atmospheric neutrino flux in the energy region from sub-GeV up to several TeV using the Super-Kamiokande (SK) water Cherenkov detector is presented in this paper. The energy and azimuthal spectra, and variation over time, of the atmospheric νe+ν¯ e and νμ+ν¯μ fluxes are measured. The energy spectra are obtained using an iterative unfolding method by combining various event topologies with differing energy responses. The azimuthal spectra depending on energy and zenith angle, and their modulation by geomagnetic effects, are also studied. A predicted east-west asymmetry is observed in both the νe and νμ samples at 8.0 σ and 6.0 σ significance, respectively, and an indication that the asymmetry dipole angle changes depending on the zenith angle was seen at the 2.2 σ level. The measured energy and azimuthal spectra are consistent with the current flux models within the estimated systematic uncertainties. A study of the long-term correlation between the atmospheric neutrino flux and the solar magnetic activity cycle is performed, and a weak preference for a correlation was seen at the 1.1 σ level, using SK-I-SK-IV data spanning a 20-year period. For several particularly strong solar activity periods, corresponding to Forbush decrease events, no theoretical prediction is available but a deviation below the typical neutrino event rate is seen at the 2.4 σ level. The seasonal modulation of the neutrino flux is also examined, but the change in flux at the SK site is predicted to be negligible, and, as expected, no evidence for a seasonal correlation is seen.

Interaction between two discontiguous chain segments from the beta-sheet of Escherichia coli thioredoxin suggests an initiation site for folding.

PubMed

Tasayco, M L; Fuchs, J; Yang, X M; Dyalram, D; Georgescu, R E

2000-09-05

The approach of comparing folding and folding/binding processes is exquisitely poised to narrow down the regions of the sequence that drive protein folding. We have dissected the small single alpha/beta domain of oxidized Escherichia coli thioredoxin (Trx) into three complementary fragments (N, residues 1-37; M, residues 38-73; and C, residues 74-108) to study them in isolation and upon recombination by far-UV CD and NMR spectroscopy. The isolated fragments show a minimum of ellipticity of ca. 197 nm in their far-UV CD spectra without concentration dependence, chemical shifts of H(alpha) that are close to the random coil values, and no medium- and long-range NOE connectivities in their three-dimensional NMR spectra. These fragments behave as disordered monomers. Only the far-UV CD spectra of binary or ternary mixtures that contain N- and C-fragments are different from the sum of their individual spectra, which is indicative of folding and/or binding of these fragments. Indeed, the cross-peaks corresponding to the rather hydrophobic beta(2) and beta(4) regions of the beta-sheet of Trx disappear from the (1)H-(15)N HSQC spectra of isolated labeled N- and C-fragments, respectively, upon addition of the unlabeled complementary fragments. The disappearing cross-peaks indicate interactions between the beta(2) and beta(4) regions, and their reappearance at lower temperatures indicates unfolding and/or dissociation of heteromers that are predominantly held by hydrophobic forces. Our results argue that the folding of Trx begins by zippering two discontiguous and rather hydrophobic chain segments (beta(2) and beta(4)) corresponding to neighboring strands of the native beta-sheet.

Deciding Full Branching Time Logic by Program Transformation

NASA Astrophysics Data System (ADS)

Pettorossi, Alberto; Proietti, Maurizio; Senni, Valerio

We present a method based on logic program transformation, for verifying Computation Tree Logic (CTL*) properties of finite state reactive systems. The finite state systems and the CTL* properties we want to verify, are encoded as logic programs on infinite lists. Our verification method consists of two steps. In the first step we transform the logic program that encodes the given system and the given property, into a monadic ω -program, that is, a stratified program defining nullary or unary predicates on infinite lists. This transformation is performed by applying unfold/fold rules that preserve the perfect model of the initial program. In the second step we verify the property of interest by using a proof method for monadic ω-programs.

Neutron Productions from thin Be target irradiated by 50 MeV/u 238U beam

NASA Astrophysics Data System (ADS)

Lee, Hee-Seock; Oh, Joo-Hee; Jung, Nam-Suk; Oranj, Leila Mokhtari; Nakao, Noriaki; Uwamino, Yoshitomo

2017-09-01

Neutrons generated from thin beryllium target by 50 MeV/u 238U beam were measured using activation analysis at 15, 30, 45, and 90 degrees from the beam direction. A 0.085 mm-thick Be stripper of RIBF was used as the neutron generating target. Activation detectors of bismuth, cobalt, and aluminum were placed out of the stripper chamber. The threshold reactions of 209Bi(n, xn)210-xBi(x=4 8), 59Co(n, xn)60-xCO(x=2 5), 59Co(n, 2nα)54Mn, 27Al(n, α)24Na, and 27Al(n,2nα)22Na were applied to measure the production rates of radionuclides. The neutron spectra were obtained using an unfolding method with the SAND-II code. All of production rates and neutron spectra were compared with the calculated results using Monte Carlo codes, the PHITS and the FLUKA. The FLUKA results showed better agreement with the measurements than the PHITS. The discrepancy between the measurements and the calculations were discussed.

Modeling the temperature-dependent peptide vibrational spectra based on implicit-solvent model and enhance sampling technique

NASA Astrophysics Data System (ADS)

Tianmin, Wu; Tianjun, Wang; Xian, Chen; Bin, Fang; Ruiting, Zhang; Wei, Zhuang

2016-01-01

We herein review our studies on simulating the thermal unfolding Fourier transform infrared and two-dimensional infrared spectra of peptides. The peptide-water configuration ensembles, required forspectrum modeling, aregenerated at a series of temperatures using the GBOBC implicit solvent model and the integrated tempering sampling technique. The fluctuating vibrational Hamiltonians of the amide I vibrational band are constructed using the Frenkel exciton model. The signals are calculated using nonlinear exciton propagation. The simulated spectral features such as the intensity and ellipticity are consistent with the experimental observations. Comparing the signals for two beta-hairpin polypeptides with similar structures suggests that this technique is sensitive to peptide folding landscapes. Project supported by the National Natural Science Foundation of China (Grant No. 21203178), the National Natural Science Foundation of China (Grant No. 21373201), the National Natural Science Foundation of China (Grant No. 21433014), the Science and Technological Ministry of China (Grant No. 2011YQ09000505), and “Strategic Priority Research Program” of the Chinese Academy of Sciences (Grant Nos. XDB10040304 and XDB100202002).

Monitoring human leukocyte antigen class I molecules by micro-Raman spectroscopy at single-cell level

NASA Astrophysics Data System (ADS)

Das, Gobind; La Rocca, Rosanna; Lakshmikanth, Tadepally; Gentile, Francesco; Tallerico, Rossana; Zambetti, Lia P.; Devitt, J.; Candeloro, Patrizio; de Angelis, Francesco; Carbone, Ennio; di Fabrizio, Enzo

2010-03-01

Human leukocyte antigen (HLA) class I molecules are formed by three immunoglobulin-like domains (α1, α2, and α3) once folded by peptide and β2-microglobulin show the presence of two α-helix streams and one β-sheet limiting the pocket for the antigenic peptide. The loss of HLA class I expression in tumors and virus-infected cells, on one hand, prevents T cell recognition, while on the other hand, it leads to natural killer (NK) cell mediated cytotoxicity. We propose the possibility of using Raman spectroscopy to measure the relative expression of HLA class I molecules at the single-cell level. Raman spectra are recorded for three cell lines (K562, T2, and T3) and monomers (HLA class I folded, unfolded and peptide+β2-microlobulin refolded) using 830 nm laser line. Our data are consistent with the hypothesis that in the Raman spectra, ranging from 1600 to 1800 cm-1, the intensity variation of cells associated with HLA class I molecules could be measured.

A computer program to predict rotor rotational noise of a stationary rotor from blade loading coefficient

NASA Technical Reports Server (NTRS)

Ramakrishnan, R.; Randall, D.; Hosier, R. N.

1976-01-01

The programing language used is FORTRAN IV. A description of all main and subprograms is provided so that any user possessing a FORTRAN compiler and random access capability can adapt the program to his facility. Rotor blade surface-pressure spectra can be used by the program to calculate: (1) blade station loading spectra, (2) chordwise and/or spanwise integrated blade-loading spectra, and (3) far-field rotational noise spectra. Any of five standard inline functions describing the chordwise distribution of the blade loading can be chosen in order to study parametrically the acoustic predictions. The program output consists of both printed and graphic descriptions of the blade-loading coefficient spectra and far-field acoustic spectrum. The results may also be written on binary file for future processing. Examples of the application of the program along with a description of the rotational noise prediction theory on which the program is based are also provided.

The mechanochemistry of copper reports on the directionality of unfolding in model cupredoxin proteins

NASA Astrophysics Data System (ADS)

Beedle, Amy E. M.; Lezamiz, Ainhoa; Stirnemann, Guillaume; Garcia-Manyes, Sergi

2015-08-01

Understanding the directionality and sequence of protein unfolding is crucial to elucidate the underlying folding free energy landscape. An extra layer of complexity is added in metalloproteins, where a metal cofactor participates in the correct, functional fold of the protein. However, the precise mechanisms by which organometallic interactions are dynamically broken and reformed on (un)folding are largely unknown. Here we use single molecule force spectroscopy AFM combined with protein engineering and MD simulations to study the individual unfolding pathways of the blue-copper proteins azurin and plastocyanin. Using the nanomechanical properties of the native copper centre as a structurally embedded molecular reporter, we demonstrate that both proteins unfold via two independent, competing pathways. Our results provide experimental evidence of a novel kinetic partitioning scenario whereby the protein can stochastically unfold through two distinct main transition states placed at the N and C termini that dictate the direction in which unfolding occurs.

Complex Stability of Single Proteins Explored by Forced Unfolding Experiments

PubMed Central

Janovjak, Harald; Sapra, K. Tanuj; Müller, Daniel J.

2005-01-01

In the last decade atomic force microscopy has been used to measure the mechanical stability of single proteins. These force spectroscopy experiments have shown that many water-soluble and membrane proteins unfold via one or more intermediates. Recently, Li and co-workers found a linear correlation between the unfolding force of the native state and the intermediate in fibronectin, which they suggested indicated the presence of a molecular memory or multiple unfolding pathways (1). Here, we apply two independent methods in combination with Monte Carlo simulations to analyze the unfolding of α-helices E and D of bacteriorhodopsin (BR). We show that correlation analysis of unfolding forces is very sensitive to errors in force calibration of the instrument. In contrast, a comparison of relative forces provides a robust measure for the stability of unfolding intermediates. The proposed approach detects three energetically different states of α-helices E and D in trimeric BR. These states are not observed for monomeric BR and indicate that substantial information is hidden in forced unfolding experiments of single proteins. PMID:15792967

Complex stability of single proteins explored by forced unfolding experiments.

PubMed

Janovjak, Harald; Sapra, K Tanuj; Müller, Daniel J

2005-05-01

In the last decade atomic force microscopy has been used to measure the mechanical stability of single proteins. These force spectroscopy experiments have shown that many water-soluble and membrane proteins unfold via one or more intermediates. Recently, Li and co-workers found a linear correlation between the unfolding force of the native state and the intermediate in fibronectin, which they suggested indicated the presence of a molecular memory or multiple unfolding pathways (1). Here, we apply two independent methods in combination with Monte Carlo simulations to analyze the unfolding of alpha-helices E and D of bacteriorhodopsin (BR). We show that correlation analysis of unfolding forces is very sensitive to errors in force calibration of the instrument. In contrast, a comparison of relative forces provides a robust measure for the stability of unfolding intermediates. The proposed approach detects three energetically different states of alpha-helices E and D in trimeric BR. These states are not observed for monomeric BR and indicate that substantial information is hidden in forced unfolding experiments of single proteins.

Thermodynamic and hydration effects for the incorporation of a cationic 3-aminopropyl chain into DNA

PubMed Central

Soto, Ana Maria; Kankia, Besik I.; Dande, Prasad; Gold, Barry; Marky, Luis A.

2002-01-01

The introduction of cationic 5-(ω-aminoalkyl)-2′-deoxypyrimidines into duplex DNA has been shown to induce DNA bending. In order to understand the energetic and hydration contributions for the incorporation of a cationic side chain in DNA a combination of spectroscopy, calorimetry and density techniques were used. Specifically, the temperature unfolding and isothermal formation was studied for a pair of duplexes with sequence d(CGTAGUCG TGC)/d(GCACGACTACG), where U represents 2′-deoxyuridine (‘control’) or 5-(3-aminopropyl)-2′-deoxyuridine (‘modified’). Continuous variation experiments confirmed 1:1 stoichiometries for each duplex and the circular dichroism spectra show that both duplexes adopted the B conformation. UV and differential scanning calorimetry melting experiments reveal that each duplex unfolds in two-state transitions. In low salt buffer, the ‘modified’ duplex is more stable and unfolds with a lower endothermic heat and lower release of counterion and water. This electrostatic stabilization is entropy driven and disappears at higher salt concentrations. Complete thermodynamic profiles at 15°C show that the favorable formation of each duplex results from the compensation of a favorable exothermic heat with an unfavorable entropy contribution. However, the isothermal profiles yielded a differential enthalpy of 8.8 kcal/mol, which is 4.3 kcal/mol higher than the differential enthalpy observed in the unfolding profiles. This indicates that the presence of the aminopropyl chain induces an increase in base stacking interactions in the modified single strand and a decrease in base stacking interactions in the modified duplex. Furthermore, the formation of the ‘control’ duplex releases water while the ‘modified’ duplex takes up water. Relative to the control duplex, formation of the modified duplex at 15°C yielded a marginal differential ΔG° term, positive ΔΔHITC–Δ(TΔS) compensation, negative ΔΔV and a net release of counterions. The opposite signs of the differential enthalpy–entropy compensation and differential volume change terms show a net uptake of structural water around polar and non-polar groups. This indicates that incorporation of the aminopropyl chain induces a higher exposure of aromatic bases to the solvent, which may be consistent with a small and local bend in the ‘modified’ duplex. PMID:12136099

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

The unfolding mechanism of monomeric mutant SOD1 by simulated force spectroscopy.

PubMed

Habibi, Mona; Rottler, Jörg; Plotkin, Steven S

2017-11-01

Mechanical unfolding of mutated apo, disulfide-reduced, monomeric superoxide dismutase 1 protein (SOD1) has been simulated via force spectroscopy techniques, using both an all-atom (AA), explicit solvent model and a coarse-grained heavy-atom Gō (HA-Gō) model. The HA-Gō model was implemented at two different pulling speeds for comparison. The most-common sequence of unfolding in the AA model agrees well with the most-common unfolding sequence of the HA-Gō model, when the same normalized pulling rate was used. Clustering of partially-native structures as the protein unfolds shows that the AA and HA-Gō models both exhibit a dominant pathway for early unfolding, which eventually bifurcates repeatedly to multiple branches after the protein is about half-unfolded. The force-extension curve exhibits multiple force drops, which are concomitant with jumps in the local interaction potential energy between specific β-strands in the protein. These sudden jumps in the potential energy coincide with the dissociation of specific pairs of β-strands, and thus intermediate unfolding events. The most common sequence of β-strand dissociation in the unfolding pathway of the AA model is β-strands 5, 4, 8, 7, 1, 2, then finally β-strands 3 and 6. The observation that β-strand 5 is among the first to unfold here, but the last to unfold in simulations of loop-truncated SOD1, could imply the existence of an evolutionary compensation mechanism, which would stabilize β-strands flanking long loops against their entropic penalty by strengthening intramolecular interactions. This article is part of a Special Issue entitled: Biophysics in Canada, edited by Lewis Kay, John Baenziger, Albert Berghuis and Peter Tieleman. Copyright © 2017 Elsevier B.V. All rights reserved.

Chemical unfolding of chicken villin headpiece in aqueous dimethyl sulfoxide solution: cosolvent concentration dependence, pathway, and microscopic mechanism.

PubMed

Roy, Susmita; Bagchi, Biman

2013-04-25

Unfolding of a protein often proceeds through partial unfolded intermediate states (PUIS). PUIS have been detected in several experimental and simulation studies. However, complete analyses of transitions between different PUIS and the unfolding trajectory are sparse. To understand such dynamical processes, we study chemical unfolding of a small protein, chicken villin head piece (HP-36), in aqueous dimethyl sulfoxide (DMSO) solution. We carry out molecular dynamics simulations at various solution compositions under ambient conditions. In each concentration, the initial step of unfolding involves separation of two adjacent native contacts, between phenyl alanine residues (11-18 and 7-18). This first step induces, under appropriate conditions, subsequent separation among other hydrophobic contacts, signifying a high degree of cooperativity in the unfolding process. The observed sequence of structural changes in HP-36 on increasing DMSO concentration and the observed sequence of PUIS, are in approximate agreement with earlier simulation results (in pure water) and experimental observations on unfolding of HP-36. Peculiar to water-DMSO mixture, an intervening structural transformation (around 15% of DMSO) in the binary mixture solvent retards the progression of unfolding as composition is increased. This is reflected in a remarkable nonmonotonic composition dependence of RMSD, radius of gyration and the fraction of native contacts. At 30% mole fraction of DMSO, we find the extended randomly coiled structure of the unfolded protein. The molecular mechanism of DMSO induced unfolding process is attributed to the initial preferential solvation of the hydrophobic side chain atoms through the methyl groups of DMSO, followed by the hydrogen bonding of the oxygen atom of DMSO to the exposed backbone NH groups of HP-36.

Studying pressure denaturation of a protein by molecular dynamics simulations.

PubMed

Sarupria, Sapna; Ghosh, Tuhin; García, Angel E; Garde, Shekhar

2010-05-15

Many globular proteins unfold when subjected to several kilobars of hydrostatic pressure. This "unfolding-up-on-squeezing" is counter-intuitive in that one expects mechanical compression of proteins with increasing pressure. Molecular simulations have the potential to provide fundamental understanding of pressure effects on proteins. However, the slow kinetics of unfolding, especially at high pressures, eliminates the possibility of its direct observation by molecular dynamics (MD) simulations. Motivated by experimental results-that pressure denatured states are water-swollen, and theoretical results-that water transfer into hydrophobic contacts becomes favorable with increasing pressure, we employ a water insertion method to generate unfolded states of the protein Staphylococcal Nuclease (Snase). Structural characteristics of these unfolded states-their water-swollen nature, retention of secondary structure, and overall compactness-mimic those observed in experiments. Using conformations of folded and unfolded states, we calculate their partial molar volumes in MD simulations and estimate the pressure-dependent free energy of unfolding. The volume of unfolding of Snase is negative (approximately -60 mL/mol at 1 bar) and is relatively insensitive to pressure, leading to its unfolding in the pressure range of 1500-2000 bars. Interestingly, once the protein is sufficiently water swollen, the partial molar volume of the protein appears to be insensitive to further conformational expansion or unfolding. Specifically, water-swollen structures with relatively low radii of gyration have partial molar volume that are similar to that of significantly more unfolded states. We find that the compressibility change on unfolding is negligible, consistent with experiments. We also analyze hydration shell fluctuations to comment on the hydration contributions to protein compressibility. Our study demonstrates the utility of molecular simulations in estimating volumetric properties and pressure stability of proteins, and can be potentially extended for applications to protein complexes and assemblies. Proteins 2010. (c) 2009 Wiley-Liss, Inc.

Dirac cone pairs in silicene induced by interface Si-Ag hybridization: A first-principles effective band study

NASA Astrophysics Data System (ADS)

Lian, Chao; Meng, Sheng

2017-06-01

Using density functional theory combined with orbital-selective band unfolding techniques, we study the effective band structure of silicene (3 ×3 )/Ag(111) (4 ×4 ) structure. Consistent with the ARPES spectra recently obtained by [Feng et al. Proc. Natl. Acad. Sci. USA 113, 14656 (2016), 10.1073/pnas.1613434114], we observe six pairs of Dirac cones near the boundary of the Brillouin zone (BZ) of Ag (1 ×1 ) , while no Dirac cone is observed inside the BZ. Furthermore, we find that these Dirac cones are induced by the interfacial Si-Ag hybridization, mainly composed of Si pz orbitals and Ag s p bands, which is intrinsically different from the Dirac cones in free-standing silicene.

The State of the World's Children, 2001: Early Childhood.

ERIC Educational Resources Information Center

United Nations Children's Fund, New York, NY.

Although the experiences from birth to age three influence how the rest of childhood unfolds, this critical time is usually neglected in the policies, programs, and budgets of countries around the world. This report details the daily lives of parents and other caregivers who are striving to protect the rights and meet the needs of young children.…

"Stand and Unfold Your Self": New Moves for Exploring "Hamlet"

ERIC Educational Resources Information Center

Rocklin, Edward L.

2009-01-01

One way of understanding the impact of the (re)emergence of a performance approach to teaching Shakespeare's plays that was, in part, initiated by the "Shakespeare Set Free" program and the books its creators composed is to say that for many teachers their work initiated the process of making performance activities central in English classrooms.…

What Role Can School Health Providers Play in Health Care Reform?

ERIC Educational Resources Information Center

Fleming, Robin

2009-01-01

President Barack Obama is wasting no time in unfolding his plan to provide health coverage for all Americans. He started in February by signing legislation to reinstate the State Children's Health Insurance Program, which expands eligibility criteria to provide 4 million more children access to health care. This first step is one of many needed to…

Effect of temperature on the conformation of natively unfolded protein 4E-BP1 in aqueous and mixed solutions containing trifluoroethanol and hexafluoroisopropanol.

PubMed

Hackl, Ellen V

2015-02-01

Natively unfolded (intrinsically disordered) proteins have attracted growing attention due to their high abundance in nature, involvement in various signalling and regulatory pathways and direct association with many diseases. In the present work the combined effect of temperature and alcohols, trifluoroethanol (TFE) and hexafluoroisopropanol (HFIP), on the natively unfolded 4E-BP1 protein was studied to elucidate the balance between temperature-induced folding and unfolding in intrinsically disordered proteins. It was shown that elevated temperatures induce reversible partial folding of 4E-BP1 both in buffer and in the mixed solutions containing denaturants. In the mixed solutions containing TFE (HFIP) 4E-BP1 adopts a partially folded helical conformation. As the temperature increases, the initial temperature-induced protein folding is replaced by irreversible unfolding/melting only after a certain level of the protein helicity has been reached. Onset unfolding temperature decreases with TFE (HFIP) concentration in solution. It was shown that an increase in the temperature induces two divergent processes in a natively unfolded protein--hydrophobicity-driven folding and unfolding. Balance between these two processes determines thermal behaviour of a protein. The correlation between heat-induced protein unfolding and the amount of helical content in a protein is revealed. Heat-induced secondary structure formation can be a valuable test to characterise minor changes in the conformations of natively unfolded proteins as a result of site-directed mutagenesis. Mutants with an increased propensity to fold into a structured form reveal different temperature behaviour.

Unfolding of the cold shock protein studied with biased molecular dynamics.

PubMed

Morra, Giulia; Hodoscek, Milan; Knapp, Ernst-Walter

2003-11-15

The cold shock protein from Bacillus caldolyticus is a small beta-barrel protein that folds in a two-state mechanism. For the native protein and for several mutants, a wealth of experimental data are available on stability and folding, so that it is an optimal system to study this process. We compare data from unfolding simulations (trajectories of 5 and up to 12 ns) obtained with a bias potential at room temperature and from unbiased thermal unfolding simulations with experimental data. The unfolding patterns derived from the trajectories starting from different native-like conformations and subject to different unfolding conditions agree. The transition state found in the simulations of unfolding is close to the native structure in agreement with experiment. Moreover, a lower value of the free energy barrier of unfolding was found for the mutant R3E than for the mutant E46A and the native protein, as indicated by experimental data. The first unfolding event involves the three-stranded beta-sheet whose decomposition corresponds to the transition state. In contrast to conclusions drawn from experiments, we found that the two-stranded beta-strand forms the most stable substructure, which decomposes very late in the unfolding process. However, assuming that this structure forms very early in the folding process, our findings would not contradict the experiments but require a different interpretation of them. Copyright 2003 Wiley-Liss, Inc.

Application of long-range order to predict unfolding rates of two-state proteins.

PubMed

Harihar, B; Selvaraj, S

2011-03-01

Predicting the experimental unfolding rates of two-state proteins and models describing the unfolding rates of these proteins is quite limited because of the complexity present in the unfolding mechanism and the lack of experimental unfolding data compared with folding data. In this work, 25 two-state proteins characterized by Maxwell et al. (Protein Sci 2005;14:602–616) using a consensus set of experimental conditions were taken, and the parameter long-range order (LRO) derived from their three-dimensional structures were related with their experimental unfolding rates ln(k(u)). From the total data set of 30 proteins used by Maxwell et al. (Protein Sci 2005;14:602–616), five slow-unfolding proteins with very low unfolding rates were considered to be outliers and were not included in our data set. Except all beta structural class, LRO of both the all-alpha and mixed-class proteins showed a strong inverse correlation of r = -0.99 and -0.88, respectively, with experimental ln(k(u)). LRO shows a correlation of -0.62 with experimental ln(k(u)) for all-beta proteins. For predicting the unfolding rates, a simple statistical method has been used and linear regression equations were developed for individual structural classes of proteins using LRO, and the results obtained showed a better agreement with experimental results. Copyright © 2010 Wiley-Liss, Inc.

Deconvoluting Protein (Un)folding Structural Ensembles Using X-Ray Scattering, Nuclear Magnetic Resonance Spectroscopy and Molecular Dynamics Simulation

PubMed Central

Nasedkin, Alexandr; Marcellini, Moreno; Religa, Tomasz L.; Freund, Stefan M.; Menzel, Andreas; Fersht, Alan R.; Jemth, Per; van der Spoel, David; Davidsson, Jan

2015-01-01

The folding and unfolding of protein domains is an apparently cooperative process, but transient intermediates have been detected in some cases. Such (un)folding intermediates are challenging to investigate structurally as they are typically not long-lived and their role in the (un)folding reaction has often been questioned. One of the most well studied (un)folding pathways is that of Drosophila melanogaster Engrailed homeodomain (EnHD): this 61-residue protein forms a three helix bundle in the native state and folds via a helical intermediate. Here we used molecular dynamics simulations to derive sample conformations of EnHD in the native, intermediate, and unfolded states and selected the relevant structural clusters by comparing to small/wide angle X-ray scattering data at four different temperatures. The results are corroborated using residual dipolar couplings determined by NMR spectroscopy. Our results agree well with the previously proposed (un)folding pathway. However, they also suggest that the fully unfolded state is present at a low fraction throughout the investigated temperature interval, and that the (un)folding intermediate is highly populated at the thermal midpoint in line with the view that this intermediate can be regarded to be the denatured state under physiological conditions. Further, the combination of ensemble structural techniques with MD allows for determination of structures and populations of multiple interconverting structures in solution. PMID:25946337

Deconvoluting Protein (Un)folding Structural Ensembles Using X-Ray Scattering, Nuclear Magnetic Resonance Spectroscopy and Molecular Dynamics Simulation.

PubMed

Nasedkin, Alexandr; Marcellini, Moreno; Religa, Tomasz L; Freund, Stefan M; Menzel, Andreas; Fersht, Alan R; Jemth, Per; van der Spoel, David; Davidsson, Jan

2015-01-01

The folding and unfolding of protein domains is an apparently cooperative process, but transient intermediates have been detected in some cases. Such (un)folding intermediates are challenging to investigate structurally as they are typically not long-lived and their role in the (un)folding reaction has often been questioned. One of the most well studied (un)folding pathways is that of Drosophila melanogaster Engrailed homeodomain (EnHD): this 61-residue protein forms a three helix bundle in the native state and folds via a helical intermediate. Here we used molecular dynamics simulations to derive sample conformations of EnHD in the native, intermediate, and unfolded states and selected the relevant structural clusters by comparing to small/wide angle X-ray scattering data at four different temperatures. The results are corroborated using residual dipolar couplings determined by NMR spectroscopy. Our results agree well with the previously proposed (un)folding pathway. However, they also suggest that the fully unfolded state is present at a low fraction throughout the investigated temperature interval, and that the (un)folding intermediate is highly populated at the thermal midpoint in line with the view that this intermediate can be regarded to be the denatured state under physiological conditions. Further, the combination of ensemble structural techniques with MD allows for determination of structures and populations of multiple interconverting structures in solution.

Alcohol-induced versus anion-induced states of alpha-chymotrypsinogen A at low pH.

PubMed

Khan, F; Khan, R H; Muzammil, S

2000-09-29

Characterization of conformational transition and folding intermediates is central to the study of protein folding. We studied the effect of various alcohols (trifluoroethanol (TFE), butanol, propanol, ethanol and methanol) and salts (K(3)FeCN(6), Na(2)SO(4), KClO(4) and KCl) on the acid-induced state of alpha-chymotrypsinogen A, a predominantly beta-sheet protein, at pH 2.0 by near-UV circular dichroism (CD), far-UV CD and 1-anilinonaphthalene-8-sulfonic acid (ANS) fluorescence measurements. Addition of alcohols led to an increase in ellipticity value at 222 nm indicating the formation of alpha-helical structure. The order of effectiveness of alcohols was shown to be TFE>butanol>propanol>ethanol>methanol. ANS fluorescence data showed a decrease in fluorescence intensity on alcohol addition, suggesting burial of hydrophobic patches. The near-UV CD spectra showed disruption of tertiary structure on alcohol addition. No change in ellipticity was observed on addition of salts at pH 2.0, whereas in the presence of 2 M urea, salts were found to induce a molten globule-like state as evident from the increases in ellipticity at 222 nm and ANS fluorescence indicating exposure of hydrophobic regions of the protein. The effectiveness in inducing the molten globule-like state, i.e. both increase in ellipticity at 222 nm and increase in ANS fluorescence, followed the order K(3)FeCN(6)>Na(2)SO(4)>KClO(4)>KCl. The loss of signal in the near-UV CD spectrum on addition of alcohols indicating disordering of tertiary structure results suggested that the decrease in ANS fluorescence intensity may be attributed to the unfolding of the ANS binding sites. The results imply that the alcohol-induced state had characteristics of an unfolded structure and lies between the molten globule and the unfolded state. Characterization of such partially folded states has important implications for protein folding.

Unfolding thermodynamics of intramolecular G-quadruplexes: base sequence contributions of the loops.

PubMed

Olsen, Chris M; Lee, Hui-Ting; Marky, Luis A

2009-03-05

G-quadruplexes are a highly studied DNA motif with a potential role in a variety of cellular processes and more recently are considered novel targets for drug therapy in aging and anticancer research. In this work, we have investigated the thermodynamic contributions of the loops on the stable formation of G-quadruplexes. Specifically, we use a combination of UV, circular dichroism (CD) and fluorescence spectroscopies, and differential scanning calorimetry (DSC) to determine thermodynamic profiles, including the differential binding of ions and water, for the unfolding of the thrombin aptamer: d(GGT2GGTGTGGT2GG) that is referred to as G2. The sequences in italics, TGT and T2, are known to form loops. Other sequences examined contained base substitutions in the TGT loop (TAT, TCT, TTT, TAPT, and UUU), in the T2 loops (T4, U2), or in both loops (UGU and U2, UUU and U2). The CD spectra of all molecules show a positive band centered at 292 nm, which corresponds to the "chair" conformation. The UV and DSC melting curves of each G-quadruplex show monophasic transitions with transition temperatures (T(M)s) that remained constant with increasing strand concentration, confirming their intramolecular formation. These G-quadruplexes unfold with T(M)s in the range from 43.2 to 56.5 degrees C and endothermic enthalpies from 22.9 to 37.2 kcal/mol. Subtracting the contribution of a G-quartet stack from each experimental profile indicated that the presence of the loops stabilize each G-quadruplex by favorable enthalpy contributions, larger differential binding of K+ ions (0.1-0.6 mol K+/ mol), and a variable uptake/release of water molecules (-6 to 8 mol H2O/mol). The thermodynamic contributions for these specific base substitutions are discussed in terms of loop stacking (base-base stacking within the loops) and their hydration effects.

Measurements of the neutron spectrum on the Martian surface with MSL/RAD

NASA Astrophysics Data System (ADS)

Köhler, J.; Zeitlin, C.; Ehresmann, B.; Wimmer-Schweingruber, R. F.; Hassler, D. M.; Reitz, G.; Brinza, D. E.; Weigle, G.; Appel, J.; Böttcher, S.; Böhm, E.; Burmeister, S.; Guo, J.; Martin, C.; Posner, A.; Rafkin, S.; Kortmann, O.

2014-03-01

The Radiation Assessment Detector (RAD), onboard the Mars Science Laboratory (MSL) rover Curiosity, measures the energetic charged and neutral particles and the radiation dose rate on the surface of Mars. An important factor for determining the biological impact of the Martian surface radiation is the specific contribution of neutrons, with their deeper penetration depth and ensuing high biological effectiveness. This is very difficult to measure quantitatively, resulting in considerable uncertainties in the total radiation dose. In contrast to charged particles, neutral particles (neutrons and gamma rays) are generally only measured indirectly. Measured spectra are a complex convolution of the incident particle spectrum with the detector response function and must be unfolded. We apply an inversion method (based on a maximum likelihood estimation) to calculate the neutron and gamma spectra from the RAD neutral particle measurements. Here we show the first spectra on the surface of Mars and compare them to theoretical predictions. The measured neutron spectrum (ranging from 8 to 740 MeV) translates into a radiation dose rate of 14±4μGy/d and a dose equivalent rate of 61±15μSv/d. This corresponds to 7% of the measured total surface dose rate and 10% of the biologically relevant surface dose equivalent rate on Mars. Measuring the Martian neutron and gamma spectra is an essential step for determining the mutagenic influences to past or present life at or beneath the Martian surface as well as the radiation hazard for future human exploration, including the shielding design of a potential habitat.

Folding thermodynamics of pseudoknotted chain conformations

PubMed Central

Kopeikin, Zoia; Chen, Shi-Jie

2008-01-01

We develop a statistical mechanical framework for the folding thermodynamics of pseudoknotted structures. As applications of the theory, we investigate the folding stability and the free energy landscapes for both the thermal and the mechanical unfolding of pseudoknotted chains. For the mechanical unfolding process, we predict the force-extension curves, from which we can obtain the information about structural transitions in the unfolding process. In general, a pseudoknotted structure unfolds through multiple structural transitions. The interplay between the helix stems and the loops plays an important role in the folding stability of pseudoknots. For instance, variations in loop sizes can lead to the destabilization of some intermediate states and change the (equilibrium) folding pathways (e.g., two helix stems unfold either cooperatively or sequentially). In both thermal and mechanical unfolding, depending on the nucleotide sequence, misfolded intermediate states can emerge in the folding process. In addition, thermal and mechanical unfoldings often have different (equilibrium) pathways. For example, for certain sequences, the misfolded intermediates, which generally have longer tails, can fold, unfold, and refold again in the pulling process, which means that these intermediates can switch between two different average end-end extensions. PMID:16674261

Chemical probes and engineered constructs reveal a detailed unfolding mechanism for a solvent-free multi-domain protein

PubMed Central

Eschweiler, Joseph D.; Martini, Rachel M.; Ruotolo, Brandon T.

2017-01-01

Despite the growing application of gas-phase measurements in structural biology and drug discovery, the factors that govern protein stabilities and structures in a solvent-free environment are still poorly understood. Here, we examine the solvent-free unfolding pathway for a group of homologous serum albumins. Utilizing a combination of chemical probes and non-covalent reconstructions, we draw new specific conclusions regarding the unfolding of albumins in the gas-phase, as well as more-general inferences regarding the sensitivity of collision induced unfolding to changes in protein primary and tertiary structure. Our findings suggest that the general unfolding pathway of low charge state albumin ions is largely unaffected by changes in primary structure; however, the stabilities of intermediates along these pathways vary widely as sequences diverge. Additionally, we find that human albumin follows a domain associated unfolding pathway, and are able to assign each unfolded form observed in our gas-phase dataset to the disruption of specific domains within the protein. The totality of our data informs the first detailed mechanism for multi-domain protein unfolding in the gas phase, and highlights key similarities and differences from the known the solution-phase pathway. PMID:27959526

Comparative study of protein unfolding in aqueous urea and dimethyl sulfoxide solutions: surface polarity, solvent specificity, and sequence of secondary structure melting.

PubMed

Roy, Susmita; Bagchi, Biman

2014-05-29

Elucidation of possible pathways between folded (native) and unfolded states of a protein is a challenging task, as the intermediates are often hard to detect. Here, we alter the solvent environment in a controlled manner by choosing two different cosolvents of water, urea, and dimethyl sulfoxide (DMSO) and study unfolding of four different proteins to understand the respective sequence of melting by computer simulation methods. We indeed find interesting differences in the sequence of melting of α helices and β sheets in these two solvents. For example, in 8 M urea solution, β-sheet parts of a protein are found to unfold preferentially, followed by the unfolding of α helices. In contrast, 8 M DMSO solution unfolds α helices first, followed by the separation of β sheets for the majority of proteins. Sequence of unfolding events in four different α/β proteins and also in chicken villin head piece (HP-36) both in urea and DMSO solutions demonstrate that the unfolding pathways are determined jointly by relative exposure of polar and nonpolar residues of a protein and the mode of molecular action of a solvent on that protein.

Impact of ion binding on poly-L-lysine (un)folding energy landscape and kinetics.

PubMed

Xiong, Kan; Asher, Sanford A

2012-06-21

We utilize T-jump UV resonance Raman spectroscopy (UVRR) to study the impact of ion binding on the equilibrium energy landscape and on (un)folding kinetics of poly-L-lysine (PLL). We observe that the relaxation rates of the folded conformations (including π-helix (bulge), pure α-helix, and turns) of PLL are slower than those of short alanine-based peptides. The PLL pure α-helix folding time is similar to that of short alanine-based peptides. We for the first time have directly observed that turn conformations are α-helix and π-helix (bulge) unfolding intermediates. ClO(4)(-) binding to the Lys side chain -NH(3)(+) groups and the peptide backbone slows the α-helix unfolding rate compared to that in pure water, but little impacts the folding rate, resulting in an increased α-helix stability. ClO(4)(-) binding significantly increases the PLL unfolding activation barrier but little impacts the folding barrier. Thus, the PLL folding coordinate(s) differs from the unfolding coordinate(s). The-π helix (bulge) unfolding and folding coordinates do not directly go through the α-helix energy well. Our results clearly demonstrate that PLL (un)folding is not a two-state process.

KASCADE-Grande measurements of energy spectra for elemental groups of cosmic rays

NASA Astrophysics Data System (ADS)

Apel, W. D.; Arteaga-Velázquez, J. C.; Bekk, K.; Bertaina, M.; Blümer, J.; Bozdog, H.; Brancus, I. M.; Cantoni, E.; Chiavassa, A.; Cossavella, F.; Daumiller, K.; de Souza, V.; Di Pierro, F.; Doll, P.; Engel, R.; Engler, J.; Finger, M.; Fuchs, B.; Fuhrmann, D.; Gils, H. J.; Glasstetter, R.; Grupen, C.; Haungs, A.; Heck, D.; Hörandel, J. R.; Huber, D.; Huege, T.; Kampert, K.-H.; Kang, D.; Klages, H. O.; Link, K.; Łuczak, P.; Ludwig, M.; Mathes, H. J.; Mayer, H. J.; Melissas, M.; Milke, J.; Mitrica, B.; Morello, C.; Oehlschläger, J.; Ostapchenko, S.; Palmieri, N.; Petcu, M.; Pierog, T.; Rebel, H.; Roth, M.; Schieler, H.; Schoo, S.; Schröder, F. G.; Sima, O.; Toma, G.; Trinchero, G. C.; Ulrich, H.; Weindl, A.; Wochele, J.; Wommer, M.; Zabierowski, J.

2013-07-01

The KASCADE-Grande air shower experiment [1] consists of, among others, a large scintillator array for measurements of charged particles, N, and of an array of shielded scintillation counters used for muon counting, Nμ. KASCADE-Grande is optimized for cosmic ray measurements in the energy range 10 PeV to about 2000 PeV, where exploring the composition is of fundamental importance for understanding the transition from galactic to extragalactic origin of cosmic rays. Following earlier studies of the all-particle and the elemental spectra reconstructed in the knee energy range from KASCADE data [2], we have now extended these measurements to beyond 200 PeV. By analysing the two-dimensional shower size spectrum N vs. Nμ for nearly vertical events, we reconstruct the energy spectra of different mass groups by means of unfolding methods over an energy range where the detector is fully efficient. The procedure and its results, which are derived based on the hadronic interaction model QGSJET-II-02 and which yield a strong indication for a dominance of heavy mass groups in the covered energy range and for a knee-like structure in the iron spectrum at around 80 PeV, are presented. This confirms and further refines the results obtained by other analyses of KASCADE-Grande data, which already gave evidence for a knee-like structure in the heavy component of cosmic rays at about 80 PeV [3].

Distinct transcriptional responses elicited by unfolded nuclear or cytoplasmic protein in mammalian cells

PubMed Central

Miyazaki, Yusuke; Chen, Ling-chun; Chu, Bernard W; Swigut, Tomek; Wandless, Thomas J

2015-01-01

Eukaryotic cells possess a variety of signaling pathways that prevent accumulation of unfolded and misfolded proteins. Chief among these is the heat shock response (HSR), which is assumed to respond to unfolded proteins in the cytosol and nucleus alike. In this study, we probe this axiom further using engineered proteins called ‘destabilizing domains’, whose folding state we control with a small molecule. The sudden appearance of unfolded protein in mammalian cells elicits a robust transcriptional response, which is distinct from the HSR and other known pathways that respond to unfolded proteins. The cellular response to unfolded protein is strikingly different in the nucleus and the cytosol, although unfolded protein in either compartment engages the p53 network. This response provides cross-protection during subsequent proteotoxic stress, suggesting that it is a central component of protein quality control networks, and like the HSR, is likely to influence the initiation and progression of human pathologies. DOI: http://dx.doi.org/10.7554/eLife.07687.001 PMID:26314864

A method to determine residue-specific unfolded-state pKa values from analysis of stability changes in single mutant cycles.

PubMed

Shen, Jana K

2010-06-02

It is now widely recognized that the unfolded state of a protein in equilibrium with the native state under folding conditions may contain significant residual structures. However, due to technical difficulties residue-specific interactions in the unfolded state remain elusive. Here we introduce a method derived from the Wyman-Tanford theory to determine residue-specific pK(a)'s in the unfolded state. This method requires equilibrium stability measurements of the wild type and single-point mutants in which titrable residues are replaced with charge-neutral ones under two pH conditions. Application of the proposed approach reveals a highly depressed pK(a) for Asp8 in the unfolded state of the NTL9 protein. Knowledge of unfolded-state pK(a)'s enables quantitative estimation of the unfolded-state electrostatic effects on protein stability. It also provides valuable benchmarks for the improvement of force fields and validation of microscopic information from molecular dynamics simulations.

High temperature unfolding of a truncated hemoglobin by molecular dynamics simulation.

PubMed

Sharma, Ravi Datta; Kanwal, Rajnee; Lynn, Andrew M; Singh, Prerna; Pasha, Syed Tazeen; Fatma, Tasneem; Jawaid, Safdar

2013-09-01

Heme containing proteins are associated with peroxidase activity. The proteins like hemoglobin, myoglobins, cytochrome c and micro-peroxidase other than peroxidases have been shown to exhibit weak peroxidase-like activity. This weak peroxidase-like activity in hemoglobin-like molecules is due to heme moiety. We conducted molecular dynamics (MD) studies to decipher the unfolding path of Ba-Glb (a truncated hemoglobin from Bacillus anthracis) and the role of heme moiety to its unfolding path. The similar unfolding path is also observed in vitro by UV/VIS spectroscopy. The data confirmed that the unfolding of Ba-Glb follows a three state process with a meta-stable (intermediate) state between the native and unfolded conformations. The present study is supported by several unfolding parameters like root-mean-square-deviation (RMSD), dictionary of protein secondary structure (DSSP), and free energy landscape. Understanding the structure of hemoglobin like proteins in unicellular dreaded pathogens like B. anthracis will pave way for newer drug discovery targets and in the disease management of anthrax.

Mass Spectral Library with Search Program, Data Version: NIST v17

National Institute of Standards and Technology Data Gateway

SRD 1A NIST/EPA/NIH Mass Spectral Library with Search Program, Data Version: NIST v17 (PC database for purchase)   Available with full-featured NIST MS Search Program for Windows integrated tools, the NIST '98 is a fully evaluated collection of electron-ionization mass spectra. (147,198 Compounds with Spectra; 147,194 Chemical Structures; 174,948 Spectra )

A (1)H-NMR study on the effect of high pressures on beta-lactoglobulin.

PubMed

Belloque, J; López-Fandiño, R; Smith, G M

2000-09-01

1H NMR was used to study the effect of high pressure on changes in the structure of beta-lactoglobulin (beta-Lg), particularly the strongly bonded regions, the "core". beta-Lg was exposed to pressures ranging from 100 to 400 MPa at neutral pH. After depressurization and acidification to pH 2.0, (1)H NMR spectra were taken. Pressure-induced unfolding was studied by deuterium exchange. Refolding was also evaluated. Our results showed that the core was unaltered at 100 MPa but increased its conformational flexibility at >/=200 MPa. Even though the core was highly flexible at 400 MPa, its structure was found to be identical to the native structure after equilibration back to atmospheric pressure. It is suggested that pressure-induced aggregates are formed by beta-Lg molecules maintaining most of their structure, and the intermolecular -SS- bonds, formed by -SH/-SS- exchange reaction, are likely to involve C(66)-C(160) rather than C(106)-C(119). In addition, the beta-Lg variants A and B could be distinguished in a (1)H NMR spectrum from a solution made with the AB mixed variant, by the differences in chemical shifts of M(107) and C(106); structural implications are discussed. Under pressure, the core of beta-Lg A seemed to unfold faster than that of beta-LgB. The structural recovery of the core was full for both variants.

How Kinetics within the Unfolded State Affects Protein Folding: an Analysis Based on Markov State Models and an Ultra-Long MD Trajectory

PubMed Central

Deng, Nan-jie; Dai, Wei

2013-01-01

Understanding how kinetics in the unfolded state affects protein folding is a fundamentally important yet less well-understood issue. Here we employ three different models to analyze the unfolded landscape and folding kinetics of the miniprotein Trp-cage. The first is a 208 μs explicit solvent molecular dynamics (MD) simulation from D. E. Shaw Research containing tens of folding events. The second is a Markov state model (MSM-MD) constructed from the same ultra-long MD simulation; MSM-MD can be used to generate thousands of folding events. The third is a Markov state model built from temperature replica exchange MD simulations in implicit solvent (MSM-REMD). All the models exhibit multiple folding pathways, and there is a good correspondence between the folding pathways from direct MD and those computed from the MSMs. The unfolded populations interconvert rapidly between extended and collapsed conformations on time scales ≤ 40 ns, compared with the folding time of ≈ 5 μs. The folding rates are independent of where the folding is initiated from within the unfolded ensemble. About 90 % of the unfolded states are sampled within the first 40 μs of the ultra-long MD trajectory, which on average explores ~27 % of the unfolded state ensemble between consecutive folding events. We clustered the folding pathways according to structural similarity into “tubes”, and kinetically partitioned the unfolded state into populations that fold along different tubes. From our analysis of the simulations and a simple kinetic model, we find that when the mixing within the unfolded state is comparable to or faster than folding, the folding waiting times for all the folding tubes are similar and the folding kinetics is essentially single exponential despite the presence of heterogeneous folding paths with non-uniform barriers. When the mixing is much slower than folding, different unfolded populations fold independently leading to non-exponential kinetics. A kinetic partition of the Trp-cage unfolded state is constructed which reveals that different unfolded populations have almost the same probability to fold along any of the multiple folding paths. We are investigating whether the results for the kinetics in the unfolded state of the twenty-residue Trp-cage is representative of larger single domain proteins. PMID:23705683

An Application of Unfolding and Cumulative Item Response Theory Models for Noncognitive Scaling: Examining the Assumptions and Applicability of the Generalized Graded Unfolding Model

ERIC Educational Resources Information Center

Sgammato, Adrienne N.

2009-01-01

This study examined the applicability of a relatively new unidimensional, unfolding item response theory (IRT) model called the generalized graded unfolding model (GGUM; Roberts, Donoghue, & Laughlin, 2000). A total of four scaling methods were applied. Two commonly used cumulative IRT models for polytomous data, the Partial Credit Model and…

Verification of unfold error estimates in the unfold operator code

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

Fehl, D.L.; Biggs, F.

Spectral unfolding is an inverse mathematical operation that attempts to obtain spectral source information from a set of response functions and data measurements. Several unfold algorithms have appeared over the past 30 years; among them is the unfold operator (UFO) code written at Sandia National Laboratories. In addition to an unfolded spectrum, the UFO code also estimates the unfold uncertainty (error) induced by estimated random uncertainties in the data. In UFO the unfold uncertainty is obtained from the error matrix. This built-in estimate has now been compared to error estimates obtained by running the code in a Monte Carlo fashionmore » with prescribed data distributions (Gaussian deviates). In the test problem studied, data were simulated from an arbitrarily chosen blackbody spectrum (10 keV) and a set of overlapping response functions. The data were assumed to have an imprecision of 5{percent} (standard deviation). One hundred random data sets were generated. The built-in estimate of unfold uncertainty agreed with the Monte Carlo estimate to within the statistical resolution of this relatively small sample size (95{percent} confidence level). A possible 10{percent} bias between the two methods was unresolved. The Monte Carlo technique is also useful in underdetermined problems, for which the error matrix method does not apply. UFO has been applied to the diagnosis of low energy x rays emitted by Z-pinch and ion-beam driven hohlraums. {copyright} {ital 1997 American Institute of Physics.}« less

Titin domains progressively unfolded by force are homogenously distributed along the molecule.

PubMed

Bianco, Pasquale; Mártonfalvi, Zsolt; Naftz, Katalin; Kőszegi, Dorina; Kellermayer, Miklós

2015-07-21

Titin is a giant filamentous protein of the muscle sarcomere in which stretch induces the unfolding of its globular domains. However, the mechanisms of how domains are progressively selected for unfolding and which domains eventually unfold have for long been elusive. Based on force-clamp optical tweezers experiments we report here that, in a paradoxical violation of mechanically driven activation kinetics, neither the global domain unfolding rate, nor the folded-state lifetime distributions of full-length titin are sensitive to force. This paradox is reconciled by a gradient of mechanical stability so that domains are gradually selected for unfolding as the magnitude of the force field increases. Atomic force microscopic screening of extended titin molecules revealed that the unfolded domains are distributed homogenously along the entire length of titin, and this homogeneity is maintained with increasing overstretch. Although the unfolding of domains with progressively increasing mechanical stability makes titin a variable viscosity damper, the spatially randomized variation of domain stability ensures that the induced structural changes are not localized but are distributed along the molecule's length. Titin may thereby provide complex safety mechanims for protecting the sarcomere against structural disintegration under excessive mechanical conditions. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Protein unfolding with a steric trap.

PubMed

Blois, Tracy M; Hong, Heedeok; Kim, Tae H; Bowie, James U

2009-10-07

The study of protein folding requires a method to drive unfolding, which is typically accomplished by altering solution conditions to favor the denatured state. This has the undesirable consequence that the molecular forces responsible for configuring the polypeptide chain are also changed. It would therefore be useful to develop methods that can drive unfolding without the need for destabilizing solvent conditions. Here we introduce a new method to accomplish this goal, which we call steric trapping. In the steric trap method, the target protein is labeled with two biotin tags placed close in space so that both biotin tags can only be bound by streptavidin when the protein unfolds. Thus, binding of the second streptavidin is energetically coupled to unfolding of the target protein. Testing the method on a model protein, dihydrofolate reductase (DHFR), we find that streptavidin binding can drive unfolding and that the apparent binding affinity reports on changes in DHFR stability. Finally, by employing the slow off-rate of wild-type streptavidin, we find that DHFR can be locked in the unfolded state. The steric trap method provides a simple method for studying aspects of protein folding and stability in native solvent conditions, could be used to specifically unfold selected domains, and could be applicable to membrane proteins.

The Proteasomal ATPases Use a Slow but Highly Processive Strategy to Unfold Proteins

PubMed Central

Snoberger, Aaron; Anderson, Raymond T.; Smith, David M.

2017-01-01

All domains of life have ATP-dependent compartmentalized proteases that sequester their peptidase sites on their interior. ATPase complexes will often associate with these compartmentalized proteases in order to unfold and inject substrates into the protease for degradation. Significant effort has been put into understanding how ATP hydrolysis is used to apply force to proteins and cause them to unfold. The unfolding kinetics of the bacterial ATPase, ClpX, have been shown to resemble a fast motor that traps unfolded intermediates as a strategy to unfold proteins. In the present study, we sought to determine if the proteasomal ATPases from eukaryotes and archaea exhibit similar unfolding kinetics. We found that the proteasomal ATPases appear to use a different kinetic strategy for protein unfolding, behaving as a slower but more processive and efficient translocation motor, particularly when encountering a folded domain. We expect that these dissimilarities are due to differences in the ATP binding/exchange cycle, the presence of a trans-arginine finger, or the presence of a threading ring (i.e., the OB domain), which may be used as a rigid platform to pull folded domains against. We speculate that these differences may have evolved due to the differing client pools these machines are expected to encounter. PMID:28421184

The Unfolding of a Pre-Kindergarten Program for Four Year Olds.

ERIC Educational Resources Information Center

Bouchard, Ruth Ann; Mackler, Bernard

This report provides an in-depth description of an early (1965) Head Start classroom for 4-year-olds in Harlem, New York City. It is based on direct, in-class observation, but standardized tests and interviews were also utilized. The teacher is described in terms of her role in the preschool, and her behavior in interaction with children and…

Coding in Senior School Mathematics with Live Editing

ERIC Educational Resources Information Center

Thompson, Ian

2017-01-01

In this paper, an example is offered of a problem-solving task for senior secondary school students which was given in the context of a story. As the story unfolds, the task requires progressively more complex forms of linear programming to be applied. Coding in MATLAB is used throughout the task in such a way that it supports the increasing…

Delivering biodefense continuing education to military medical providers by allowing a biodefense educational curriculum to unfold in practice.

PubMed

D'Alessandro, Donna M; D'Alessandro, Michael P

2007-12-01

A challenge today is how to deliver initial and continuing education on biodefense to military medical providers in a manner that can be integrated into their workflow and lifestyle. A summative evaluation of a prototypical biodefense digital library (BDL) and learning collaboratory was performed. The BDL posted daily links to biodefense news stories from January 2004 to December 2005. Four evaluations were completed, that is, content evaluation, curriculum comparison with a biodefense graduate program, usage evaluation, and impact factor analysis. News stories (N = 678) came from a broad range of authoritative national and international news sources (N = 178). News stories covered all of the categories in the required and elective formal biodefense graduate program courses. The BDL was consistently displayed on the first page of the top three Internet search engines, meaning that it was among the top 10 authoritative Internet sites on biodefense. Presenting biodefense news stories to busy military medical providers in an organized chronological fashion produces an unstructured biodefense educational curriculum that unfolds in practice and becomes an educational resource that is ultimately well regarded and may be efficient to use.

NSDann2BS, a neutron spectrum unfolding code based on neural networks technology and two bonner spheres

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

Ortiz-Rodriguez, J. M.; Reyes Alfaro, A.; Reyes Haro, A.

In this work a neutron spectrum unfolding code, based on artificial intelligence technology is presented. The code called ''Neutron Spectrometry and Dosimetry with Artificial Neural Networks and two Bonner spheres'', (NSDann2BS), was designed in a graphical user interface under the LabVIEW programming environment. The main features of this code are to use an embedded artificial neural network architecture optimized with the ''Robust design of artificial neural networks methodology'' and to use two Bonner spheres as the only piece of information. In order to build the code here presented, once the net topology was optimized and properly trained, knowledge stored atmore » synaptic weights was extracted and using a graphical framework build on the LabVIEW programming environment, the NSDann2BS code was designed. This code is friendly, intuitive and easy to use for the end user. The code is freely available upon request to authors. To demonstrate the use of the neural net embedded in the NSDann2BS code, the rate counts of {sup 252}Cf, {sup 241}AmBe and {sup 239}PuBe neutron sources measured with a Bonner spheres system.« less

The unfolded protein response and programmed cell death are induced by expression of Garlic virus X p11 in Nicotiana benthamiana.

PubMed

Lu, Yuwen; Yin, Mingyuan; Wang, Xiaodan; Chen, Binghua; Yang, Xue; Peng, Jiejun; Zheng, Hongying; Zhao, Jinping; Lin, Lin; Yu, Chulang; MacFarlane, Stuart; He, Jianqing; Liu, Yong; Chen, Jianping; Dai, Liangying; Yan, Fei

2016-06-01

Garlic virus X (GarVX) ORF3 encodes a p11 protein, which contributes to virus cell-to-cell movement and forms granules on the endoplasmic reticulum (ER) in Nicotiana benthamiana. Expression of p11 either from a binary vector, PVX or TMV induced ER stress and the unfolded protein response (UPR), as demonstrated by an increase in transcription of the ER luminal binding protein (BiP) and bZIP60 genes. UPR-related programmed cell death (PCD) was elicited by PVX : p11 or TMV : p11 in systemic infected leaves. Examination of p11 mutants with deletions of two transmembrane domains (TM) revealed that both were required for generating granules and for inducing necrosis. TRV-based VIGS was used to investigate the correlation between bZIP60 expression and p11-induced UPR-related PCD. Less necrosis was observed on local and systemic leaves of bZIP60 knockdown plants when infected with PVXp11, suggesting that bZIP60 plays an important role in the UPR-related PCD response to p11 in N. benthamiana.

NSDann2BS, a neutron spectrum unfolding code based on neural networks technology and two bonner spheres

NASA Astrophysics Data System (ADS)

Ortiz-Rodríguez, J. M.; Reyes Alfaro, A.; Reyes Haro, A.; Solís Sánches, L. O.; Miranda, R. Castañeda; Cervantes Viramontes, J. M.; Vega-Carrillo, H. R.

2013-07-01

In this work a neutron spectrum unfolding code, based on artificial intelligence technology is presented. The code called "Neutron Spectrometry and Dosimetry with Artificial Neural Networks and two Bonner spheres", (NSDann2BS), was designed in a graphical user interface under the LabVIEW programming environment. The main features of this code are to use an embedded artificial neural network architecture optimized with the "Robust design of artificial neural networks methodology" and to use two Bonner spheres as the only piece of information. In order to build the code here presented, once the net topology was optimized and properly trained, knowledge stored at synaptic weights was extracted and using a graphical framework build on the LabVIEW programming environment, the NSDann2BS code was designed. This code is friendly, intuitive and easy to use for the end user. The code is freely available upon request to authors. To demonstrate the use of the neural net embedded in the NSDann2BS code, the rate counts of 252Cf, 241AmBe and 239PuBe neutron sources measured with a Bonner spheres system.

Probing the Structure of the Escherichia coli Periplasmic Proteins HdeA and YmgD by Molecular Dynamics Simulations.

PubMed

Socher, Eileen; Sticht, Heinrich

2016-11-23

HdeA and YmgD are structurally homologous proteins in the periplasm of Escherichia coli. HdeA has been shown to represent an acid-activated chaperone, whereas the function of YmgD has not yet been characterized. We performed pH-titrating molecular dynamics simulations (pHtMD) to investigate the structural changes of both proteins and to assess whether YmgD may also exhibit an unfolding behavior similar to that of HdeA. The unfolding pathway of HdeA includes partially unfolded dimer structures, which represent a prerequisite for subsequent dissociation. In contrast to the coupled unfolding and dissociation of HdeA, YmgD displays dissociation of the folded subunits, and the subunits do not undergo significant unfolding even at low pH values. The differences in subunit stability between HdeA and YmgD may be explained by the structural features of helix D, which represents the starting point of unfolding in HdeA. In summary, the present study suggests that YmgD either is not an acid-activated chaperone or, at least, does not require unfolding for activation.

Unfolding of Proteins: Thermal and Mechanical Unfolding

NASA Technical Reports Server (NTRS)

Hur, Joe S.; Darve, Eric

2004-01-01

We have employed a Hamiltonian model based on a self-consistent Gaussian appoximation to examine the unfolding process of proteins in external - both mechanical and thermal - force elds. The motivation was to investigate the unfolding pathways of proteins by including only the essence of the important interactions of the native-state topology. Furthermore, if such a model can indeed correctly predict the physics of protein unfolding, it can complement more computationally expensive simulations and theoretical work. The self-consistent Gaussian approximation by Micheletti et al. has been incorporated in our model to make the model mathematically tractable by signi cantly reducing the computational cost. All thermodynamic properties and pair contact probabilities are calculated by simply evaluating the values of a series of Incomplete Gamma functions in an iterative manner. We have compared our results to previous molecular dynamics simulation and experimental data for the mechanical unfolding of the giant muscle protein Titin (1TIT). Our model, especially in light of its simplicity and excellent agreement with experiment and simulation, demonstrates the basic physical elements necessary to capture the mechanism of protein unfolding in an external force field.

Unfolding of the bacterial nucleoid both in vivo and in vitro as a result of exposure to camphor.

PubMed Central

Harrington, E W; Trun, N J

1997-01-01

Both prokaryotic and eukaryotic cells are sensitive to killing by camphor; however, the mechanism by which camphor kills has not been elucidated. We report here that camphor unfolds the nucleoid of Escherichia coli and that unfolding does not require DNA replication, translation, or cell division. We show that exposure of isolated nucleoids to camphor results in unfolding of the chromosome. PMID:9079934

Machinery of protein folding and unfolding.

PubMed

Zhang, Xiaodong; Beuron, Fabienne; Freemont, Paul S

2002-04-01

During the past two years, a large amount of biochemical, biophysical and low- to high-resolution structural data have provided mechanistic insights into the machinery of protein folding and unfolding. It has emerged that dual functionality in terms of folding and unfolding might exist for some systems. The majority of folding/unfolding machines adopt oligomeric ring structures in a cooperative fashion and utilise the conformational changes induced by ATP binding/hydrolysis for their specific functions.

Single molecule unfolding and stretching of protein domains inside a solid-state nanopore by electric field.

PubMed

Freedman, Kevin J; Haq, S Raza; Edel, Joshua B; Jemth, Per; Kim, Min Jun

2013-01-01

Single molecule methods have provided a significantly new look at the behavior of biomolecules in both equilibrium and non-equilibrium conditions. Most notable are the stretching experiments performed by atomic force microscopes and laser tweezers. Here we present an alternative single molecule method that can unfold a protein domain, observed at electric fields greater than 10(6) V/m, and is fully controllable by the application of increasing voltages across the membrane of the pore. Furthermore this unfolding mechanism is characterized by measuring both the residence time of the protein within the nanopore and the current blockade. The unfolding data supports a gradual unfolding mechanism rather than the cooperative transition observed by classical urea denaturation experiments. Lastly it is shown that the voltage-mediated unfolding is a function of the stability of the protein by comparing two mutationally destabilized variants of the protein.

XAP, a program for deconvolution and analysis of complex X-ray spectra

USGS Publications Warehouse

Quick, James E.; Haleby, Abdul Malik

1989-01-01

The X-ray analysis program (XAP) is a spectral-deconvolution program written in BASIC and specifically designed to analyze complex spectra produced by energy-dispersive X-ray analytical systems (EDS). XAP compensates for spectrometer drift, utilizes digital filtering to remove background from spectra, and solves for element abundances by least-squares, multiple-regression analysis. Rather than base analyses on only a few channels, broad spectral regions of a sample are reconstructed from standard reference spectra. The effects of this approach are (1) elimination of tedious spectrometer adjustments, (2) removal of background independent of sample composition, and (3) automatic correction for peak overlaps. Although the program was written specifically to operate a KEVEX 7000 X-ray fluorescence analytical system, it could be adapted (with minor modifications) to analyze spectra produced by scanning electron microscopes, electron microprobes, and probes, and X-ray defractometer patterns obtained from whole-rock powders.

UNFOLD-SENSE: a parallel MRI method with self-calibration and artifact suppression.

PubMed

Madore, Bruno

2004-08-01

This work aims at improving the performance of parallel imaging by using it with our "unaliasing by Fourier-encoding the overlaps in the temporal dimension" (UNFOLD) temporal strategy. A self-calibration method called "self, hybrid referencing with UNFOLD and GRAPPA" (SHRUG) is presented. SHRUG combines the UNFOLD-based sensitivity mapping strategy introduced in the TSENSE method by Kellman et al. (5), with the strategy introduced in the GRAPPA method by Griswold et al. (10). SHRUG merges the two approaches to alleviate their respective limitations, and provides fast self-calibration at any given acceleration factor. UNFOLD-SENSE further includes an UNFOLD artifact suppression scheme to significantly suppress artifacts and amplified noise produced by parallel imaging. This suppression scheme, which was published previously (4), is related to another method that was presented independently as part of TSENSE. While the two are equivalent at accelerations < or = 2.0, the present approach is shown here to be significantly superior at accelerations > 2.0, with up to double the artifact suppression at high accelerations. Furthermore, a slight modification of Cartesian SENSE is introduced, which allows departures from purely Cartesian sampling grids. This technique, termed variable-density SENSE (vdSENSE), allows the variable-density data required by SHRUG to be reconstructed with the simplicity and fast processing of Cartesian SENSE. UNFOLD-SENSE is given by the combination of SHRUG for sensitivity mapping, vdSENSE for reconstruction, and UNFOLD for artifact/amplified noise suppression. The method was implemented, with online reconstruction, on both an SSFP and a myocardium-perfusion sequence. The results from six patients scanned with UNFOLD-SENSE are presented.

Salt bridge as a gatekeeper against partial unfolding.

PubMed

Hinzman, Mark W; Essex, Morgan E; Park, Chiwook

2016-05-01

Salt bridges are frequently observed in protein structures. Because the energetic contribution of salt bridges is strongly dependent on the environmental context, salt bridges are believed to contribute to the structural specificity rather than the stability. To test the role of salt bridges in enhancing structural specificity, we investigated the contribution of a salt bridge to the energetics of native-state partial unfolding in a cysteine-free version of Escherichia coli ribonuclease H (RNase H*). Thermolysin cleaves a protruding loop of RNase H(*) through transient partial unfolding under native conditions. Lys86 and Asp108 in RNase H(*) form a partially buried salt bridge that tethers the protruding loop. Investigation of the global stability of K86Q/D108N RNase H(*) showed that the salt bridge does not significantly contribute to the global stability. However, K86Q/D108N RNase H(*) is greatly more susceptible to proteolysis by thermolysin than wild-type RNase H(*) is. The free energy for partial unfolding determined by native-state proteolysis indicates that the salt bridge significantly increases the energy for partial unfolding by destabilizing the partially unfolded form. Double mutant cycles with single and double mutations of the salt bridge suggest that the partially unfolded form is destabilized due to a significant decrease in the interaction energy between Lys86 and Asp108 upon partial unfolding. This study demonstrates that, even in the case that a salt bridge does not contribute to the global stability, the salt bridge may function as a gatekeeper against partial unfolding that disturbs the optimal geometry of the salt bridge. © 2016 The Protein Society.

Neutron response function characterization of 4He scintillation detectors

DOE PAGES

Kelley, Ryan P.; Rolison, Lucas M.; Lewis, Jason M.; ...

2015-04-15

Time-of-flight measurements were conducted to characterize the neutron energy response of pressurized 4He fast neutron scintillation detectors for the first time, using the Van de Graaff generator at Ohio University. The time-of-flight spectra and pulse height distributions were measured. This data was used to determine the light output response function, which was found to be linear at energies below 3.5 MeV. The intrinsic efficiency of the detector as a function of incident energy was also calculated: the average efficiency up to 10 MeV was 3.1%, with a maximum efficiency of 6.6% at 1.05 MeV. Furthermore, these results will enable developmentmore » of neutron spectrum unfolding algorithms for neutron spectroscopy applications with these detectors.« less

An integrated radiation physics computer code system.

NASA Technical Reports Server (NTRS)

Steyn, J. J.; Harris, D. W.

1972-01-01

An integrated computer code system for the semi-automatic and rapid analysis of experimental and analytic problems in gamma photon and fast neutron radiation physics is presented. Such problems as the design of optimum radiation shields and radioisotope power source configurations may be studied. The system codes allow for the unfolding of complex neutron and gamma photon experimental spectra. Monte Carlo and analytic techniques are used for the theoretical prediction of radiation transport. The system includes a multichannel pulse-height analyzer scintillation and semiconductor spectrometer coupled to an on-line digital computer with appropriate peripheral equipment. The system is geometry generalized as well as self-contained with respect to material nuclear cross sections and the determination of the spectrometer response functions. Input data may be either analytic or experimental.

Electronic Excitation of Furan by Low Energy Electrons

NASA Astrophysics Data System (ADS)

Hargreaves, Leigh R.; Khakoo, Murtadha A.; Lopes, Maria Cristina A.; da Costa, Romarly; Bettega, Marcio H. F.; Lima, Marco A. P.

2011-10-01

We present absolute differential cross section (DCS) measurements and calculations of electron impact excitation of the lowest lying triplet 3B2 and 3A1 electronic states of furan. The incident electron energy range of the present study was 5-15eV. The experimental data were normalized to the elastic DCS data of. The cross sections were determined by unfolding electron energy loss spectra, using an open source data analysis package and the spectroscopic assignments of. The calculations employ a Multichannel Schwinger method with a 9-state closed coupling CI configuration including polarized pseudo-potentials. The preliminary theoretical results show reasonable agreement with experiment below 10eV, but differ at higher energies. Funded by the US NSF and the Brazilian funding agencies CNPq, CAPES and FAPESP.

Measurement of Differential Cross-Sections in the t$$\\bar{t}$$ → ℓ+jets Channel at √s= 1.96 TeV with the D0 Experiment at Fermilab

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

Kvita, J

2009-04-01

The analysis presented in this thesis focuses on kinematic distributions in the tmore » $$\\bar{t}$$ system and studies in detail selected differential cross sections of top quarks as well as the reconstructed t$$\\bar{t}$$ pair, namely the top quark transverse momentum and the t$$\\bar{t}$$ system mass. The structure of the thesis is organized as follows: first the Standard Model of the particle physics is briefly introduced in Chapter 1, with relevant aspects of electroweak and strong interactions discussed. The physics of the top quark and its properties are then outlined in Chapter 2, together with the motivation for measuring the transverse top quark momentum and other kinematic-related variables of the t$$\\bar{t}$$ system. The concepts of present-day high energy physics collider experiments and the explicit example of Fermilab Tevatron collider and the D0 detector in Chapters 3 and 4 are followed by the description of basic detector-level objects, i.e. tracks, leptons and jets, in Chapter 5; their identification and calibration following in next chapter with the emphasis on the jet energy scale in Chapter 6 and jet identification at the D0. The analysis itself is outlined in Chapter 7 and is structured so that first the data and simulation samples and the basic preselection are described in Chapter 8 and 9, followed by the kinematic reconstruction part in Chapter 10. Chapter 11 on background normalization and Chapter 12 with raw reconstructed spectra results (at the detector-smeared level) are followed by the purity-based background subtraction method and examples of signal-level corrected spectra in Chapter 13. Next, the procedure of correcting measured spectra for detector effects (unfolding) is described in Chapters 14-15, including migration matrix studies, acceptance correction determination as well as the regularized unfolding procedure itself. Final differential cross sections are presented in Chapter 16 with the main results in Figures 16.19-16.20. Summary and discussion close the main analysis part in Chapter 17, supplemented by appendices on the wealthy of analysis control plots of the t$$\\bar{t}$$ → ℓ + jets channel, selected D0 event displays and finally the list of publications and references. Preliminary results of this analysis have been documented in D0 internal notes [UnfoldTop], [p17Top], [p14Top]; as well as presented at conferences [APS08], [APS05]. The author has also been a co-author of more than 135 D0 collaboration publications since 2005. The author has taken part in the jet energy scale calibration efforts performing final closure tests and deriving a correction to jet energy offset due to the suppression of the calorimeter signal. The author has also co-performed the Φ-intercalibration of the hadronic calorimeter and co-supervised the electromagnetic Φ-intercalibration; recently has also been involved in maintaining the jet identification efficiencies measurement as a JetID convener. During the years in Fermilab, many events have taken place in the course of the analysis in persuasion, including more than 170 shifts served for the D0 detector with or without the beam, 168 talks presented with mixed results and reactions; and tens of thousands of code lines in C (and sometimes perhaps even really C++) written while terabytes of data were processed, analyzed, and sometimes also lost. It has been a long but profoundly enriching chapter of my life.« less

Protein unfolding versus β-sheet separation in spider silk nanocrystals

NASA Astrophysics Data System (ADS)

Alam, Parvez

2014-03-01

In this communication a mechanism for spider silk strain hardening is proposed. Shear failure of β-sheet nanocrystals is the first failure mode that gives rise to the creation of smaller nanocrystals, which are of higher strength and stiffness. β-sheet unfolding requires more energy than nanocrystal separation in a shear mode of failure. As a result, unfolding occurs after the nanocrystals separate in shear. β-sheet unfolding yields a secondary strain hardening effect once the β-sheet conformation is geometrically stable and acts like a unidirectional fibre in a fibre reinforced composite. The mechanism suggested herein is based on molecular dynamics calculations of residual inter-β-sheet separation strengths against residual intra-β-sheet unfolding strengths.

Comparative Study of the Mechanical Unfolding Pathways of α- and β-Peptides.

PubMed

Uribe, Lalita; Gauss, Jürgen; Diezemann, Gregor

2015-07-02

Using molecular simulations, we analyze the unfolding pathways of various peptides. We compare the mechanical unfolding of a β-alanine's octamer (β-HAla8) and an α-alanine's decamer (α-Ala10). Using force-probe molecular-dynamics simulations, to induce unfolding, we show that the 3(14)-helix formed by β-HAla8 is mechanically more stable than the α-helix formed by α-Ala10, although both structures are stabilized by six hydrogen bonds. Additionally, computations of the potential of mean force validate this result and show that also the thermal stability of the 3(14)-helix is higher. It is demonstrated that β-HAla8 unfolds in a two-step fashion with a stable intermediate. This is contrasted with the known single-step scenario of the unfolding of α-Ala10. Furthermore, we present a study of the chain-length dependence of the mechanical unfolding pathway of the 3(14)-helix. The calculation of the dynamic strength for oligomers with chain lengths ranging from 6 to 18 monomers shows that the unfolding pathway of helices with an integer and noninteger number of turns has m + 1 and m energy barriers, respectively, with m being the number of complete turns. The additional barrier for helices with an integer number of turns is shown to be related to the breaking of the N-terminus' hydrogen bond.

Resolution of the unfolded state.

NASA Astrophysics Data System (ADS)

Beaucage, Gregory

2008-03-01

The unfolded states in proteins and nucleic acids remain weakly understood despite their importance to protein folding; misfolding diseases (Parkinson's & Alzheimer's); natively unfolded proteins (˜ 30% of eukaryotic proteins); and to understanding ribozymes. Research has been hindered by the inability to quantify the residual (native) structure present in an unfolded protein or nucleic acid. Here, a scaling model is proposed to quantify the degree of folding and the unfolded state (Beaucage, 2004, 2007). The model takes a global view of protein structure and can be applied to a number of analytic methods and to simulations. Three examples are given of application to small-angle scattering from pressure induced unfolding of SNase (Panick, 1998), from acid unfolded Cyt c (Kataoka, 1993) and from folding of Azoarcus ribozyme (Perez-Salas, 2004). These examples quantitatively show 3 characteristic unfolded states for proteins, the statistical nature of a folding pathway and the relationship between extent of folding and chain size during folding for charge driven folding in RNA. Beaucage, G., Biophys. J., in press (2007). Beaucage, G., Phys. Rev. E. 70, 031401 (2004). Kataoka, M., Y. Hagihara, K. Mihara, Y. Goto J. Mol. Biol. 229, 591 (1993). Panick, G., R. Malessa, R. Winter, G. Rapp, K. J. Frye, C. A. Royer J. Mol. Biol. 275, 389 (1998). Perez-Salas U. A., P. Rangan, S. Krueger, R. M. Briber, D. Thirumalai, S. A. Woodson, Biochemistry 43 1746 (2004).

TFE-induced local unfolding and fibrillation of SOD1: bridging the experiment and simulation studies.

PubMed

Kumar, Vijay; Prakash, Amresh; Pandey, Preeti; Lynn, Andrew M; Hassan, Md Imtaiyaz

2018-05-18

Misfolding and aggregation of Cu, Zn Superoxide dismutase (SOD1) is involved in the neurodegenerative disease, amyotrophic lateral sclerosis. Many studies have shown that metal-depleted, monomeric form of SOD1 displays substantial local unfolding dynamics and is the precursor for aggregation. Here, we have studied the structure and dynamics of different apo monomeric SOD1 variants associated with unfolding and aggregation in aqueous trifluoroethanol (TFE) through experiments and simulation. TFE induces partially unfolded β-sheet-rich extended conformations in these SOD1 variants, which subsequently develops aggregates with fibril-like characteristics. Fibrillation was achieved more easily in disulfide-reduced monomeric SOD1 when compared with wild-type and mutant monomeric SOD1. At higher concentrations of TFE, a native-like structure with the increase in α-helical content was observed. The molecular dynamics simulation results illustrate distinct structural dynamics for different regions of SOD1 variants and show uniform local unfolding of β-strands. The strands protected by the zinc-binding and electrostatic loops were found to unfold first in 20% (v/v) TFE, leading to a partial unfolding of β-strands 4, 5, and 6 which are prone to aggregation. Our results thus shed light on the role of local unfolding and conformational dynamics in SOD1 misfolding and aggregation. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Enzyme activity of α-chymotrypsin: Deactivation by gold nano-cluster and reactivation by glutathione.

PubMed

Ghosh, Catherine; Mondal, Tridib; Bhattacharyya, Kankan

2017-05-15

Effect of gold nanoclusters (Au-NCs) on the circular dichroism (CD) spectra and enzymatic activity of α-chymotrypsin (ChT) (towards hydrolysis of a substrate, N-succinyl-l-phenylalanine p-nitroanilide) are studied. The CD spectra indicate that on binding to Au-NC, ChT is completely unfolded, resulting in nearly zero ellipticity. α-chymotrypsin (ChT) coated gold nano-clusters exhibit almost no enzymatic activity. Addition of glutathione (GSH) or oxidized glutathione (GSSG) restore the enzyme activity of α-chymotrypsin by 30-45%. ChT coated Au-NC exhibits two emission maxima-one at 480nm (corresponding to Au 10 ) and one at 640nm (Au 25 ). On addition of glutathione (GSH) or oxidized glutathione (GSSG) the emission peak at 640nm vanishes and only one peak at 480nm (Au 10 ) remains. MALDI mass spectrometry studies suggest addition of glutathione (GSH) to α-chymotrypsin capped Au-NCs results in the formation of glutathione-capped Au-NCs and α-chymotrypsin is released from Au-NCs. CD spectroscopy indicates that the conformation of the released α-chymotrypsin is different from that of the native α-chymotrypsin. Copyright © 2017 Elsevier Inc. All rights reserved.

Use of a priori spectral information in the measurement of x-ray flux with filtered diode arrays

DOE PAGES

Marrs, R. E.; Widmann, K.; Brown, G. V.; ...

2015-10-29

Filtered x-ray diode (XRD) arrays are often used to measure x-ray spectra vs. time from spectrally continuous x-ray sources such as hohlraums. A priori models of the incident x-ray spectrum enable a more accurate unfolding of the x-ray flux as compared to the standard technique of modifying a thermal Planckian with spectral peaks or dips at the response energy of each filtered XRD channel. A model x-ray spectrum consisting of a thermal Planckian, a Gaussian at higher energy, and (in some cases) a high energy background provides an excellent fit to XRD-array measurements of x-ray emission from laser heated hohlraums.more » If high-resolution measurements of part of the x-ray emission spectrum are available, that information can be included in the a priori model. In cases where the x-ray emission spectrum is not Planckian, candidate x-ray spectra can be allowed or excluded by fitting them to measured XRD voltages. Here, examples are presented from the filtered XRD arrays, named Dante, at the National Ignition Facility and the Laboratory for Laser Energetics.« less

A Simulation Program for Dynamic Infrared (IR) Spectra

ERIC Educational Resources Information Center

Zoerb, Matthew C.; Harris, Charles B.

2013-01-01

A free program for the simulation of dynamic infrared (IR) spectra is presented. The program simulates the spectrum of two exchanging IR peaks based on simple input parameters. Larger systems can be simulated with minor modifications. The program is available as an executable program for PCs or can be run in MATLAB on any operating system. Source…

Probing the folded state and mechanical unfolding pathways of T4 lysozyme using all-atom and coarse-grained molecular simulation

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

Zheng, Wenjun, E-mail: wjzheng@buffalo.edu; Glenn, Paul

2015-01-21

The Bacteriophage T4 Lysozyme (T4L) is a prototype modular protein comprised of an N-terminal and a C-domain domain, which was extensively studied to understand the folding/unfolding mechanism of modular proteins. To offer detailed structural and dynamic insights to the folded-state stability and the mechanical unfolding behaviors of T4L, we have performed extensive equilibrium and steered molecular dynamics simulations of both the wild-type (WT) and a circular permutation (CP) variant of T4L using all-atom and coarse-grained force fields. Our all-atom and coarse-grained simulations of the folded state have consistently found greater stability of the C-domain than the N-domain in isolation, whichmore » is in agreement with past thermostatic studies of T4L. While the all-atom simulation cannot fully explain the mechanical unfolding behaviors of the WT and the CP variant observed in an optical tweezers study, the coarse-grained simulations based on the Go model or a modified elastic network model (mENM) are in qualitative agreement with the experimental finding of greater unfolding cooperativity in the WT than the CP variant. Interestingly, the two coarse-grained models predict different structural mechanisms for the observed change in cooperativity between the WT and the CP variant—while the Go model predicts minor modification of the unfolding pathways by circular permutation (i.e., preserving the general order that the N-domain unfolds before the C-domain), the mENM predicts a dramatic change in unfolding pathways (e.g., different order of N/C-domain unfolding in the WT and the CP variant). Based on our simulations, we have analyzed the limitations of and the key differences between these models and offered testable predictions for future experiments to resolve the structural mechanism for cooperative folding/unfolding of T4L.« less

A new atomic force microscope force ramp technique using digital force feedback control reveals mechanically weak protein unfolding events.

PubMed

Kawakami, M; Smith, D A

2008-12-10

We have developed a new force ramp modification of the atomic force microscope (AFM) which can control multiple unfolding events of a multi-modular protein using software-based digital force feedback control. With this feedback the force loading rate can be kept constant regardless the length of soft elastic linkage or number of unfolded polypeptide domains. An unfolding event is detected as a sudden drop in force, immediately after which the feedback control reduces the applied force to a low value of a few pN by lowering the force set point. Hence the remaining folded domains can relax and the subsequent force ramp is applied to relaxed protein domains identically in each case. We have applied this technique to determine the kinetic parameters x(u), which is the distance between the native state and transition state, and α(0), which is the unfolding rate constant at zero force, for the mechanical unfolding of a pentamer of I27 domains of titin. In each force ramp the unfolding probability depends on the number of folded domains remaining in the system and we had to take account of this effect in the analysis of unfolding force data. We obtained values of x(u) and α(0) to be 0.28 nm and 1.02 × 10(-3) s(-1), which are in good agreement with those obtained from conventional constant velocity experiments. This method reveals unfolding data at low forces that are not seen in constant velocity experiments and corrects for the change in stiffness that occurs with most mechanical systems throughout the unfolding process to allow constant force ramp experiments to be carried out. In addition, a mechanically weak structure was detected, which formed from the fully extended polypeptide chain during a force quench. This indicates that the new technique will allow studies of the folding kinetics of previously hidden, mechanically weak species.

The energy spectrum of neutrons from 7Li(d,n)8Be reaction at deuteron energy 2.9 MeV

NASA Astrophysics Data System (ADS)

Mitrofanov, Konstantin V.; Piksaikin, Vladimir M.; Zolotarev, Konstantin I.; Egorov, Andrey S.; Gremyachkin, Dmitrii E.

2017-09-01

The neutron beams generated at the electrostatic accelerators using nuclear reactions T(p,n)3He, D(d,n)3He, 7Li(p,n)7Be, T(d,n)4He, 7Li(d,n)8Be, 9Be(d,n)10B are widely used in neutron physics and in many practical applications. Among these reactions the least studied reactions are 7Li(d,n)8Be and 9Be(d,n)10B. The present work is devoted to the measurement of the neutron spectrum from 7Li(d,n)8Be reaction at 0∘ angle to the deuteron beam axis on the electrostatic accelerator Tandetron (JSC "SSC RF - IPPE") using activation method and a stilbene crystal scintillation detector. The first time ever 7Li(d,n)8Be reaction was measured by activation method. The target was a thick lithium layer on metallic backing. The energy of the incident deuteron was 2.9 MeV. As activation detectors a wide range of nuclear reactions were used: 27Al(n,p)27Mg, 27Al(n,α)24Na, 113In(n,n')113mIn, 115In(n,n')115mIn, 115In(n,γ)116mIn, 58Ni(n,p)58mCo, 58Ni(n,2n)57Ni, 197Au(n,γ)198Au, 197Au(n,2n)196Au, 59Co(n,p)59Fe, 59Co(n,2n)58m+gCo, 59Co (n,g)60Co. Measurement of the induced gamma-activity was carried out using HPGe detector Canberra GX5019 [1]. The up-to-date evaluations of the cross sections for these reactions were used in processing of the data. The program STAYSL was used to unfold the energy spectra. The neutron spectra obtained by activation detectors is consistent with the corresponding data measured by a stilbene crystal scintillation detector within their uncertainties.

OPERATOR NORM INEQUALITIES BETWEEN TENSOR UNFOLDINGS ON THE PARTITION LATTICE

PubMed Central

Wang, Miaoyan; Duc, Khanh Dao; Fischer, Jonathan; Song, Yun S.

2017-01-01

Interest in higher-order tensors has recently surged in data-intensive fields, with a wide range of applications including image processing, blind source separation, community detection, and feature extraction. A common paradigm in tensor-related algorithms advocates unfolding (or flattening) the tensor into a matrix and applying classical methods developed for matrices. Despite the popularity of such techniques, how the functional properties of a tensor changes upon unfolding is currently not well understood. In contrast to the body of existing work which has focused almost exclusively on matricizations, we here consider all possible unfoldings of an order-k tensor, which are in one-to-one correspondence with the set of partitions of {1, …, k}. We derive general inequalities between the lp-norms of arbitrary unfoldings defined on the partition lattice. In particular, we demonstrate how the spectral norm (p = 2) of a tensor is bounded by that of its unfoldings, and obtain an improved upper bound on the ratio of the Frobenius norm to the spectral norm of an arbitrary tensor. For specially-structured tensors satisfying a generalized definition of orthogonal decomposability, we prove that the spectral norm remains invariant under specific subsets of unfolding operations. PMID:28286347

(Un)Folding Mechanisms of the FBP28 WW Domain in Explicit Solvent Revealed by Multiple Rare Event Simulation Methods

PubMed Central

Juraszek, Jarek; Bolhuis, Peter G.

2010-01-01

Abstract We report a numerical study of the (un)folding routes of the truncated FBP28 WW domain at ambient conditions using a combination of four advanced rare event molecular simulation techniques. We explore the free energy landscape of the native state, the unfolded state, and possible intermediates, with replica exchange molecular dynamics. Subsequent application of bias-exchange metadynamics yields three tentative unfolding pathways at room temperature. Using these paths to initiate a transition path sampling simulation reveals the existence of two major folding routes, differing in the formation order of the two main hairpins, and in hydrophobic side-chain interactions. Having established that the hairpin strand separation distances can act as reasonable reaction coordinates, we employ metadynamics to compute the unfolding barriers and find that the barrier with the lowest free energy corresponds with the most likely pathway found by transition path sampling. The unfolding barrier at 300 K is ∼17 kBT ≈ 42 kJ/mol, in agreement with the experimental unfolding rate constant. This work shows that combining several powerful simulation techniques provides a more complete understanding of the kinetic mechanism of protein folding. PMID:20159161

Verification of unfold error estimates in the UFO code

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

Fehl, D.L.; Biggs, F.

Spectral unfolding is an inverse mathematical operation which attempts to obtain spectral source information from a set of tabulated response functions and data measurements. Several unfold algorithms have appeared over the past 30 years; among them is the UFO (UnFold Operator) code. In addition to an unfolded spectrum, UFO also estimates the unfold uncertainty (error) induced by running the code in a Monte Carlo fashion with prescribed data distributions (Gaussian deviates). In the problem studied, data were simulated from an arbitrarily chosen blackbody spectrum (10 keV) and a set of overlapping response functions. The data were assumed to have anmore » imprecision of 5% (standard deviation). 100 random data sets were generated. The built-in estimate of unfold uncertainty agreed with the Monte Carlo estimate to within the statistical resolution of this relatively small sample size (95% confidence level). A possible 10% bias between the two methods was unresolved. The Monte Carlo technique is also useful in underdetemined problems, for which the error matrix method does not apply. UFO has been applied to the diagnosis of low energy x rays emitted by Z-Pinch and ion-beam driven hohlraums.« less

Cooperative unfolding of distinctive mechanoreceptor domains transduces force into signals

PubMed Central

Ju, Lining; Chen, Yunfeng; Xue, Lingzhou; Du, Xiaoping; Zhu, Cheng

2016-01-01

How cells sense their mechanical environment and transduce forces into biochemical signals is a crucial yet unresolved question in mechanobiology. Platelets use receptor glycoprotein Ib (GPIb), specifically its α subunit (GPIbα), to signal as they tether and translocate on von Willebrand factor (VWF) of injured arterial surfaces against blood flow. Force elicits catch bonds to slow VWF–GPIbα dissociation and unfolds the GPIbα leucine-rich repeat domain (LRRD) and juxtamembrane mechanosensitive domain (MSD). How these mechanical processes trigger biochemical signals remains unknown. Here we analyze these extracellular events and the resulting intracellular Ca2+ on a single platelet in real time, revealing that LRRD unfolding intensifies Ca2+ signal whereas MSD unfolding affects the type of Ca2+ signal. Therefore, LRRD and MSD are analog and digital force transducers, respectively. The >30 nm macroglycopeptide separating the two domains transmits force on the VWF–GPIbα bond (whose lifetime is prolonged by LRRD unfolding) to the MSD to enhance its unfolding, resulting in unfolding cooperativity at an optimal force. These elements may provide design principles for a generic mechanosensory protein machine. DOI: http://dx.doi.org/10.7554/eLife.15447.001 PMID:27434669

OPERATOR NORM INEQUALITIES BETWEEN TENSOR UNFOLDINGS ON THE PARTITION LATTICE.

PubMed

Wang, Miaoyan; Duc, Khanh Dao; Fischer, Jonathan; Song, Yun S

2017-05-01

Interest in higher-order tensors has recently surged in data-intensive fields, with a wide range of applications including image processing, blind source separation, community detection, and feature extraction. A common paradigm in tensor-related algorithms advocates unfolding (or flattening) the tensor into a matrix and applying classical methods developed for matrices. Despite the popularity of such techniques, how the functional properties of a tensor changes upon unfolding is currently not well understood. In contrast to the body of existing work which has focused almost exclusively on matricizations, we here consider all possible unfoldings of an order- k tensor, which are in one-to-one correspondence with the set of partitions of {1, …, k }. We derive general inequalities between the l p -norms of arbitrary unfoldings defined on the partition lattice. In particular, we demonstrate how the spectral norm ( p = 2) of a tensor is bounded by that of its unfoldings, and obtain an improved upper bound on the ratio of the Frobenius norm to the spectral norm of an arbitrary tensor. For specially-structured tensors satisfying a generalized definition of orthogonal decomposability, we prove that the spectral norm remains invariant under specific subsets of unfolding operations.

Thermodynamics of the Trp-cage Miniprotein Unfolding in Urea

PubMed Central

Wafer, Lucas N. R.; Streicher, Werner W.; Makhatadze, George I.

2010-01-01

The thermodynamic properties of unfolding of the Trp-cage mini protein in the presence of various concentrations of urea have been characterized using temperature-induced unfolding monitored by far-UV circular dichroism spectroscopy. Analysis of the data using a two-state model allowed the calculation of the Gibbs energy of unfolding at 25°C as a function of urea concentration. This in turn was analyzed by the linear extrapolation model that yielded the dependence of Gibbs energy on urea concentration, i.e. the m-value for Trp-cage unfolding. The m-value obtained from the experimental data, as well as the experimental heat capacity change upon unfolding, were correlated with the structural parameters derived from the three dimensional structure of Trp-cage. It is shown that the m-value can be predicted well using a transfer model, while the heat capacity changes are in very good agreement with the empirical models based on model compounds studies. These results provide direct evidence that Trp-cage, despite its small size, is an excellent model for studies of protein unfolding and provide thermodynamic data that can be used to compare with atomistic computer simulations. PMID:20112418

An artificial solar spectrum substantially alters plant development compared with usual climate room irradiance spectra.

PubMed

Hogewoning, Sander W; Douwstra, Peter; Trouwborst, Govert; van Ieperen, Wim; Harbinson, Jeremy

2010-03-01

Plant responses to the light spectrum under which plants are grown affect their developmental characteristics in a complicated manner. Lamps widely used to provide growth irradiance emit spectra which are very different from natural daylight spectra. Whereas specific responses of plants to a spectrum differing from natural daylight may sometimes be predictable, the overall plant response is generally difficult to predict due to the complicated interaction of the many different responses. So far studies on plant responses to spectra either use no daylight control or, if a natural daylight control is used, it will fluctuate in intensity and spectrum. An artificial solar (AS) spectrum which closely resembles a sunlight spectrum has been engineered, and growth, morphogenesis, and photosynthetic characteristics of cucumber plants grown for 13 d under this spectrum have been compared with their performance under fluorescent tubes (FTs) and a high pressure sodium lamp (HPS). The total dry weight of the AS-grown plants was 2.3 and 1.6 times greater than that of the FT and HPS plants, respectively, and the height of the AS plants was 4-5 times greater. This striking difference appeared to be related to a more efficient light interception by the AS plants, characterized by longer petioles, a greater leaf unfolding rate, and a lower investment in leaf mass relative to leaf area. Photosynthesis per leaf area was not greater for the AS plants. The extreme differences in plant response to the AS spectrum compared with the widely used protected cultivation light sources tested highlights the importance of a more natural spectrum, such as the AS spectrum, if the aim is to produce plants representative of field conditions.

The acidic pH-induced structural changes in Pin1 as revealed by spectral methodologies

NASA Astrophysics Data System (ADS)

Wang, Jing-Zhang; Xi, Lei; Zhu, Guo-Fei; Han, Yong-Guang; Luo, Yue; Wang, Mei; Du, Lin-Fang

2012-12-01

Pin1 is closely associated with the pathogenesis of cancers and Alzheimer's disease (AD). Previously, we have shown the characteristics of the thermal denaturation of Pin1. Herein, the acid-induced denaturation of Pin1 was determined by means of fluorescence emission, synchronous fluorescence, far-UV CD, ANS fluorescence and RLS spectroscopies. The fluorescence emission spectra and the synchronous fluorescence spectra suggested the partially reversible unfolding (approximately from pH 7.0 to 4.0) and refolding (approximately from pH 4.0 to 1.0) of the structures around the chromophores in Pin1, apparently with an intermediate state at about pH 4.0-4.5. The far-UV CD spectra indicated that acidic pH (below pH 4.0) induced the structural transition from α-helix and random coils to β-sheet in Pin1. The ANS fluorescence and the RLS spectra further suggested the exposure of the hydrophobic side-chains of Pin1 and the aggregation of it especially below pH 2.3, and the aggregation possibly resulted in the formation of extra intermolecular β-sheet. The present work primarily shows that acidic pH can induce kinds of irreversible structural changes in Pin1, such as the exposure of the hydrophobic side-chains, the transition from α-helix to β-sheet and the aggregation of Pin1, and also explains why Pin1 loses most of its activity below pH 5.0. The results emphasize the important role of decreased pH in the pathogenesis of some Pin1-related diseases, and support the therapeutic approach for them by targeting acidosis and modifying the intracellular pH gradients.

Preferential role of iron in heme degradation of hemoglobin upon gamma irradiation.

PubMed

Rafiei, Javad; Yavari, Kamal; Moosavi-Movahedi, Ali A

2017-10-01

It is usually believed that γ-ray interaction with biomolecules is intermediately performed by reactive oxygen species (ROS) produced from radiolysis of water. Hemoglobin (Hb) as one of the most abundant biomolecule in blood and well-studied endogenously affected by ROS, was a good candidate for study. Adult human Hb was extracted and irradiated using four distinct 20, 60, 90 and 170Gy doses from Co-60 γ-ray source. UV-vis, fluorescence and FT-IR spectroscopies were used to study the whole conformational changes and partial degradation of heme. Hb species calculated using Benesch equations indicated that the concentration of oxy-Hb was decreased from 9.97μM to 6.56μM, while the total metastable met and deoxy-Hb concentration were just increased 2.39μM and about 8.4% of total heme was diminished. Heme degradation was studied using fluorescence spectra at two 321 and 460nm excitation wavelengths as fully and partially degradation of heme respectively. Inverse behavior of these two fluorescence spectra suggested a new mechanism of heme degradation in which γ-ray preferably absorbed by heme without any intermediary effects of water. It was confirmed by FT-IR spectra at 900-1000cm -1 where the FeN and NH of porphyrin indicate their own stretching vibrational bands. Thermal stability justified that the gamma radiation induced the conformational changes of Hb which is appeared during thermal unfolding. First derivative of thermal spectra indicated that the Tm of 170Gy dose irradiated sample is 2°C lowered and total concentration of Hb was decreased 14%. Copyright © 2017 Elsevier B.V. All rights reserved.

Stereochemistry and solvent role in protein folding: nuclear magnetic resonance and molecular dynamics studies of poly-L and alternating-L,D homopolypeptides in dimethyl sulfoxide.

PubMed

Srivastava, Kinshuk Raj; Kumar, Anil; Goyal, Bhupesh; Durani, Susheel

2011-05-26

The competing interactions folding and unfolding protein structure remain obscure. Using homopolypeptides, we ask if poly-L structure may have a role. We mutate the structure to alternating-L,D stereochemistry and substitute water as the fold-promoting solvent with methanol and dimethyl sulfoxide (DMSO) as the fold-denaturing solvents. Circular dichroism and molecular dynamics established previously that, while both isomers were folded in water, the poly-L isomer was unfolded and alternating-L,D isomer folded in methanol. Nuclear magnetic resonance and molecular dynamics establish now that both isomers are unfolded in DMSO. We calculated energetics of folding-unfolding equilibrium with water and methanol as solvents. We have now calculated interactions of unfolded polypeptide structures with DMSO as solvent. Methanol was found to unfold and water fold poly-L structure as a dielectric. DMSO has now been found to unfold both poly-L and alternating-L,D structures by strong solvation of peptides to disrupt their hydrogen bonds. Accordingly, we propose that while linked peptides fold protein structure with hydrogen bonds they unfold the structure electrostatically due to the stereochemical effect of the poly-L structure. Protein folding to ordering of peptide hydrogen bonds with water as canonical solvent may thus involve two specific and independent solvent effects-one, strong screening of electrostatics of poly-L linked peptides, and two, weak dipolar solvation of peptides. Correspondingly, protein denaturation may involve two independent solvent effects-one, weak dielectric to unfold poly-L structure electrostatically, and two, strong polarity to disrupt peptide hydrogen bonds by solvation of peptides.

As Simple As Possible, but Not Simpler: Exploring the Fidelity of Coarse-Grained Protein Models for Simulated Force Spectroscopy

PubMed Central

Rottler, Jörg; Plotkin, Steven S.

2016-01-01

Mechanical unfolding of a single domain of loop-truncated superoxide dismutase protein has been simulated via force spectroscopy techniques with both all-atom (AA) models and several coarse-grained models having different levels of resolution: A Gō model containing all heavy atoms in the protein (HA-Gō), the associative memory, water mediated, structure and energy model (AWSEM) which has 3 interaction sites per amino acid, and a Gō model containing only one interaction site per amino acid at the Cα position (Cα-Gō). To systematically compare results across models, the scales of time, energy, and force had to be suitably renormalized in each model. Surprisingly, the HA-Gō model gives the softest protein, exhibiting much smaller force peaks than all other models after the above renormalization. Clustering to render a structural taxonomy as the protein unfolds showed that the AA, HA-Gō, and Cα-Gō models exhibit a single pathway for early unfolding, which eventually bifurcates repeatedly to multiple branches only after the protein is about half-unfolded. The AWSEM model shows a single dominant unfolding pathway over the whole range of unfolding, in contrast to all other models. TM alignment, clustering analysis, and native contact maps show that the AWSEM pathway has however the most structural similarity to the AA model at high nativeness, but the least structural similarity to the AA model at low nativeness. In comparison to the AA model, the sequence of native contact breakage is best predicted by the HA-Gō model. All models consistently predict a similar unfolding mechanism for early force-induced unfolding events, but diverge in their predictions for late stage unfolding events when the protein is more significantly disordered. PMID:27898663

Unfolding of Ubiquitin Studied by Picosecond Time-Resolved Fluorescence of the Tyrosine Residue

PubMed Central

Noronha, Melinda; Lima, João C.; Bastos, Margarida; Santos, Helena; Maçanita, António L.

2004-01-01

The photophysics of the single tyrosine in bovine ubiquitin (UBQ) was studied by picosecond time-resolved fluorescence spectroscopy, as a function of pH and along thermal and chemical unfolding, with the following results: First, at room temperature (25°C) and below pH 1.5, native UBQ shows single-exponential decays. From pH 2 to 7, triple-exponential decays were observed and the three decay times were attributed to the presence of tyrosine, a tyrosine-carboxylate hydrogen-bonded complex, and excited-state tyrosinate. Second, at pH 1.5, the water-exposed tyrosine of either thermally or chemically unfolded UBQ decays as a sum of two exponentials. The double-exponential decays were interpreted and analyzed in terms of excited-state intramolecular electron transfer from the phenol to the amide moiety, occurring in one of the three rotamers of tyrosine in UBQ. The values of the rate constants indicate the presence of different unfolded states and an increase in the mobility of the tyrosine residue during unfolding. Finally, from the pre-exponential coefficients of the fluorescence decays, the unfolding equilibrium constants (KU) were calculated, as a function of temperature or denaturant concentration. Despite the presence of different unfolded states, both thermal and chemical unfolding data of UBQ could be fitted to a two-state model. The thermodynamic parameters Tm = 54.6°C, ΔHTm = 56.5 kcal/mol, and ΔCp = 890 cal/mol//K, were determined from the unfolding equilibrium constants calculated accordingly, and compared to values obtained by differential scanning calorimetry also under the assumption of a two-state transition, Tm = 57.0°C, ΔHm= 51.4 kcal/mol, and ΔCp = 730 cal/mol//K. PMID:15454455

As Simple As Possible, but Not Simpler: Exploring the Fidelity of Coarse-Grained Protein Models for Simulated Force Spectroscopy.

PubMed

Habibi, Mona; Rottler, Jörg; Plotkin, Steven S

2016-11-01

Mechanical unfolding of a single domain of loop-truncated superoxide dismutase protein has been simulated via force spectroscopy techniques with both all-atom (AA) models and several coarse-grained models having different levels of resolution: A Gō model containing all heavy atoms in the protein (HA-Gō), the associative memory, water mediated, structure and energy model (AWSEM) which has 3 interaction sites per amino acid, and a Gō model containing only one interaction site per amino acid at the Cα position (Cα-Gō). To systematically compare results across models, the scales of time, energy, and force had to be suitably renormalized in each model. Surprisingly, the HA-Gō model gives the softest protein, exhibiting much smaller force peaks than all other models after the above renormalization. Clustering to render a structural taxonomy as the protein unfolds showed that the AA, HA-Gō, and Cα-Gō models exhibit a single pathway for early unfolding, which eventually bifurcates repeatedly to multiple branches only after the protein is about half-unfolded. The AWSEM model shows a single dominant unfolding pathway over the whole range of unfolding, in contrast to all other models. TM alignment, clustering analysis, and native contact maps show that the AWSEM pathway has however the most structural similarity to the AA model at high nativeness, but the least structural similarity to the AA model at low nativeness. In comparison to the AA model, the sequence of native contact breakage is best predicted by the HA-Gō model. All models consistently predict a similar unfolding mechanism for early force-induced unfolding events, but diverge in their predictions for late stage unfolding events when the protein is more significantly disordered.

Structure of a AAA+ unfoldase in the process of unfolding substrate

PubMed Central

Ripstein, Zev A; Huang, Rui; Augustyniak, Rafal; Kay, Lewis E; Rubinstein, John L

2017-01-01

AAA+ unfoldases are thought to unfold substrate through the central pore of their hexameric structures, but how this process occurs is not known. VAT, the Thermoplasma acidophilum homologue of eukaryotic CDC48/p97, works in conjunction with the proteasome to degrade misfolded or damaged proteins. We show that in the presence of ATP, VAT with its regulatory N-terminal domains removed unfolds other VAT complexes as substrate. We captured images of this transient process by electron cryomicroscopy (cryo-EM) to reveal the structure of the substrate-bound intermediate. Substrate binding breaks the six-fold symmetry of the complex, allowing five of the six VAT subunits to constrict into a tight helix that grips an ~80 Å stretch of unfolded protein. The structure suggests a processive hand-over-hand unfolding mechanism, where each VAT subunit releases the substrate in turn before re-engaging further along the target protein, thereby unfolding it. DOI: http://dx.doi.org/10.7554/eLife.25754.001 PMID:28390173

Conformational dynamics of a protein in the folded and the unfolded state

NASA Astrophysics Data System (ADS)

Fitter, Jörg

2003-08-01

In a quasielastic neutron scattering experiment, the picosecond dynamics of α-amylase was investigated for the folded and the unfolded state of the protein. In order to ensure a reasonable interpretation of the internal protein dynamics, the protein was measured in D 2O-buffer solution. The much higher structural flexibility of the pH induced unfolded state as compared to the native folded state was quantified using a simple analytical model, describing a local diffusion inside a sphere. In terms of this model the conformational volume, which is explored mainly by confined protein side-chain movements, is parameterized by the radius of a sphere (folded state, r=1.2 Å; unfolded state, 1.8 Å). Differences in conformational dynamics between the folded and the unfolded state of a protein are of fundamental interest in the field of protein science, because they are assumed to play an important role for the thermodynamics of folding/unfolding transition and for protein stability.

Common features in the unfolding and misfolding of PDZ domains and beyond: the modulatory effect of domain swapping and extra-elements.

PubMed

Murciano-Calles, Javier; Güell-Bosch, Jofre; Villegas, Sandra; Martinez, Jose C

2016-01-12

PDZ domains are protein-protein interaction modules sharing the same structural arrangement. To discern whether they display common features in their unfolding/misfolding behaviour we have analyzed in this work the unfolding thermodynamics, together with the misfolding kinetics, of the PDZ fold using three archetypical examples: the second and third PDZ domains of the PSD95 protein and the Erbin PDZ domain. Results showed that all domains passed through a common intermediate, which populated upon unfolding, and that this in turn drove the misfolding towards worm-like fibrillar structures. Thus, the unfolding/misfolding behaviour appears to be shared within these domains. We have also analyzed how this landscape can be modified upon the inclusion of extra-elements, as it is in the nNOS PDZ domain, or the organization of swapped species, as happens in the second PDZ domain of the ZO2 protein. Although the intermediates still formed upon thermal unfolding, the misfolding was prevented to varying degrees.

Anomalous Diffusion Reports on the Interaction of Misfolded Proteins with the Quality Control Machinery in the Endoplasmic Reticulum

PubMed Central

Malchus, Nina; Weiss, Matthias

2010-01-01

A multitude of transmembrane proteins enters the endoplasmic reticulum (ER) as unfolded polypeptide chains. During their folding process, they interact repetitively with the ER's quality control machinery. Here, we have used fluorescence correlation spectroscopy to probe these interactions for a prototypical transmembrane protein, VSVG ts045, in vivo. While both folded and unfolded VSVG ts045 showed anomalous diffusion, the unfolded protein had a significantly stronger anomaly. This difference subsided when unfolded VSVG ts045 was in a complex with its chaperone calnexin, or when a mutant form of VSVG ts045 with only one glycan was used. Our experimental data and accompanying simulations suggest that the folding sensor of the quality control (UGT1) oligomerizes unfolded VSVG ts045, leading to a more anomalous/obstructed diffusion. In contrast, calnexin dissolves the oligomers, rendering unfolded VSVG ts045 more mobile, and hence prevents poisoning of the ER. PMID:20713018

Protein unfolding as a switch from self-recognition to high-affinity client binding

PubMed Central

Groitl, Bastian; Horowitz, Scott; Makepeace, Karl A. T.; Petrotchenko, Evgeniy V.; Borchers, Christoph H.; Reichmann, Dana; Bardwell, James C. A.; Jakob, Ursula

2016-01-01

Stress-specific activation of the chaperone Hsp33 requires the unfolding of a central linker region. This activation mechanism suggests an intriguing functional relationship between the chaperone's own partial unfolding and its ability to bind other partially folded client proteins. However, identifying where Hsp33 binds its clients has remained a major gap in our understanding of Hsp33's working mechanism. By using site-specific Fluorine-19 nuclear magnetic resonance experiments guided by in vivo crosslinking studies, we now reveal that the partial unfolding of Hsp33's linker region facilitates client binding to an amphipathic docking surface on Hsp33. Furthermore, our results provide experimental evidence for the direct involvement of conditionally disordered regions in unfolded protein binding. The observed structural similarities between Hsp33's own metastable linker region and client proteins present a possible model for how Hsp33 uses protein unfolding as a switch from self-recognition to high-affinity client binding. PMID:26787517

Native aggregation as a cause of origin of temporary cellular structures needed for all forms of cellular activity, signaling and transformations.

PubMed

Matveev, Vladimir V

2010-06-09

According to the hypothesis explored in this paper, native aggregation is genetically controlled (programmed) reversible aggregation that occurs when interacting proteins form new temporary structures through highly specific interactions. It is assumed that Anfinsen's dogma may be extended to protein aggregation: composition and amino acid sequence determine not only the secondary and tertiary structure of single protein, but also the structure of protein aggregates (associates). Cell function is considered as a transition between two states (two states model), the resting state and state of activity (this applies to the cell as a whole and to its individual structures). In the resting state, the key proteins are found in the following inactive forms: natively unfolded and globular. When the cell is activated, secondary structures appear in natively unfolded proteins (including unfolded regions in other proteins), and globular proteins begin to melt and their secondary structures become available for interaction with the secondary structures of other proteins. These temporary secondary structures provide a means for highly specific interactions between proteins. As a result, native aggregation creates temporary structures necessary for cell activity."One of the principal objects of theoretical research in any department of knowledge is to find the point of view from which the subject appears in its greatest simplicity."Josiah Willard Gibbs (1839-1903).

Determination of thermodynamics and kinetics of RNA reactions by force

PubMed Central

Tinoco, Ignacio; Li, Pan T. X.; Bustamante, Carlos

2008-01-01

Single-molecule methods have made it possible to apply force to an individual RNA molecule. Two beads are attached to the RNA; one is on a micropipette, the other is in a laser trap. The force on the RNA and the distance between the beads are measured. Force can change the equilibrium and the rate of any reaction in which the product has a different extension from the reactant. This review describes use of laser tweezers to measure thermodynamics and kinetics of unfolding/refolding RNA. For a reversible reaction the work directly provides the free energy; for irreversible reactions the free energy is obtained from the distribution of work values. The rate constants for the folding and unfolding reactions can be measured by several methods. The effect of pulling rate on the distribution of force-unfolding values leads to rate constants for unfolding. Hopping of the RNA between folded and unfolded states at constant force provides both unfolding and folding rates. Force-jumps and force-drops, similar to the temperature jump method, provide direct measurement of reaction rates over a wide range of forces. The advantages of applying force and using single-molecule methods are discussed. These methods, for example, allow reactions to be studied in non-denaturing solvents at physiological temperatures; they also simplify analysis of kinetic mechanisms because only one intermediate at a time is present. Unfolding of RNA in biological cells by helicases, or ribosomes, has similarities to unfolding by force. PMID:17040613

Force-Induced Unfolding of Fibronectin in the Extracellular Matrix of Living Cells

PubMed Central

Smith, Michael L; Gourdon, Delphine; Little, William C; Kubow, Kristopher E; Eguiluz, R. Andresen; Luna-Morris, Sheila; Vogel, Viola

2007-01-01

Whether mechanically unfolded fibronectin (Fn) is present within native extracellular matrix fibrils is controversial. Fn extensibility under the influence of cell traction forces has been proposed to originate either from the force-induced lengthening of an initially compact, folded quaternary structure as is found in solution (quaternary structure model, where the dimeric arms of Fn cross each other), or from the force-induced unfolding of type III modules (unfolding model). Clarification of this issue is central to our understanding of the structural arrangement of Fn within fibrils, the mechanism of fibrillogenesis, and whether cryptic sites, which are exposed by partial protein unfolding, can be exposed by cell-derived force. In order to differentiate between these two models, two fluorescence resonance energy transfer schemes to label plasma Fn were applied, with sensitivity to either compact-to-extended conformation (arm separation) without loss of secondary structure or compact-to-unfolded conformation. Fluorescence resonance energy transfer studies revealed that a significant fraction of fibrillar Fn within a three-dimensional human fibroblast matrix is partially unfolded. Complete relaxation of Fn fibrils led to a refolding of Fn. The compactly folded quaternary structure with crossed Fn arms, however, was never detected within extracellular matrix fibrils. We conclude that the resting state of Fn fibrils does not contain Fn molecules with crossed-over arms, and that the several-fold extensibility of Fn fibrils involves the unfolding of type III modules. This could imply that Fn might play a significant role in mechanotransduction processes. PMID:17914904

How long does it take to equilibrate the unfolded state of a protein?

PubMed Central

Levy, Ronald M; Dai, Wei; Deng, Nan-Jie; Makarov, Dmitrii E

2013-01-01

How long does it take to equilibrate the unfolded state of a protein? The answer to this question has important implications for our understanding of why many small proteins fold with two state kinetics. When the equilibration within the unfolded state U is much faster than the folding, the folding kinetics will be two state even if there are many folding pathways with different barriers. Yet the mean first passage times (MFPTs) between different regions of the unfolded state can be much longer than the folding time. This seems to imply that the equilibration within U is much slower than the folding. In this communication we resolve this paradox. We present a formula for estimating the time to equilibrate the unfolded state of a protein. We also present a formula for the MFPT to any state within U, which is proportional to the average lifetime of that state divided by the state population. This relation is valid when the equilibration within U is very fast as compared with folding as it often is for small proteins. To illustrate the concepts, we apply the formulas to estimate the time to equilibrate the unfolded state of Trp-cage and MFPTs within the unfolded state based on a Markov State Model using an ultra-long 208 microsecond trajectory of the miniprotein to parameterize the model. The time to equilibrate the unfolded state of Trp-cage is ∼100 ns while the typical MFPTs within U are tens of microseconds or longer. PMID:23963761

Limiting Charged Particle Flux Spectrum at the Heliopause and Beyond

NASA Astrophysics Data System (ADS)

Cooper, J. F.

2009-04-01

Ongoing Voyager 1 and 2 measurements show proton and heavier ion flux spectra unfolding upwards at MeV energies and in time with presumably decreasing distance to the heliopause interface of the heliosheath and local interstellar medium (LISM) plasma environments. Despite large spatial separation between the two spacecraft, the respective flux measurements are converging to a common spectrum consistent with a source beyond both spacecraft. This trend may conceivably reverse in response to increasing solar modulation of the new sunspot cycle but otherwise it must approach some limiting form of the plasma and energetic particle spectra near and beyond the heliopause. If an outer heliosheath region is bounded outwards by a postulated heliospheric bow shock, there could be an intermediate spectrum of shock-accelerated particles, but otherwise the limiting spectrum is that of the LISM. As reported earlier, a simple power-law extrapolation from known LISM plasma distributions at eV energies to the relatively unmodulated fluxes of galactic protons at GeV energies yields the "universal" stochastic cascade spectrum of Fisk and Gloeckler. Although the heliopause interface of the inner heliosheath and LISM plasma flow environments is usually visualized as laminar with little flow across the interface, boundary instabilities and charge exchange processes at a more chaotic and realistic boundary could enable interpenetrating flows. The limiting heliosheath spectrum now being approached by measurements from both spacecraft is suggested to be the LISM spectrum. Lack of significant and sustained spectral changes in response to increasing solar modulation within the supersonic heliosphere, and continuity of the unfolded spectral form for future measurements across the heliopause, would support direct LISM and/or outer heliosheath origins for the suprathermal ions of the inner heliosheath. This could further require modification of source and transport models for the so-called "anomalous component" ions at higher MeV energies. Unlimited extension of the presently observed suprathermal ion spectrum into the neutral gas environment beyond the heliopause, e.g. within the hydrogen wall region, would also impact energy and directional distributions of energetic neutral atoms now being measured from Earth orbit by the Interstellar Boundary Explorer (IBEX) mission.

Research on Spectroscopy, Opacity, and Atmospheres

NASA Technical Reports Server (NTRS)

Oliversen, Ronald (Technical Monitor); Kurucz, Robert L.

2004-01-01

I propose to continue providing observers with basic data for interpreting spectra from stars, novas, supernovas, clusters, and galaxies. These data will include allowed forbidden line lists both laboratory and computed, for the first five to ten ions of all atoms and for all relevant diatomic molecules. I will eventually expend to all ions of the first thirty elements to treat far UV end X-ray spectra, and for envelope opacities. I also include triatomic molecules providing by other researchers. I have made CDs with Partridge and Schwanke's water data for work on M stars.The luna data also serve as input to my model atmosphere and synthesis programs that generated energy distributions, photometry, limb darkening, and spectra that can be used for planning observations and for fitting observed spectra. The spectrum synthesis programs produce detailed plots with the line identified. Grids of stellar spectra can be used for radial velocity-, rotation-, or abundance templates and for population synthesis. I am fitting spectra of bright stars to test the data and to produce atlases to guide observer. For each star the whole spectrum is computed from the UV to the far IR. The line data, opacities, models, spectra, and programs are freely distributed on CDs and on my web site and represent a unique resource for many NASA programs.

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.

Hydrophobic core malleability of a de novo designed three-helix bundle protein.

PubMed

Walsh, S T; Sukharev, V I; Betz, S F; Vekshin, N L; DeGrado, W F

2001-01-12

De novo protein design provides a tool for testing the principles that stabilize the structures of proteins. Recently, we described the design and structure determination of alpha(3)D, a three-helix bundle protein with a well-packed hydrophobic core. Here, we test the malleability and adaptability of this protein's structure by mutating a small, Ala residue (A60) in its core to larger, hydrophobic side-chains, Leu and Ile. Such changes introduce strain into the structures of natural proteins, and therefore generally destabilize the native state. By contrast, these mutations were slightly stabilizing ( approximately 1.5 kcal mol(-1)) to the tertiary structure of alpha(3)D. The value of DeltaC(p) for unfolding of these mutants was not greatly affected relative to wild-type, indicating that the change in solvent accessibility for unfolding was similar. However, two-dimensional heteronuclear single quantum coherence spectra indicate that the protein adjusts to the introduction of steric bulk in different ways. A60L-alpha(3)D showed serious erosion in the dispersion of both the amide backbone as well as the side-chain methyl chemical shifts. By contrast, A60I-alpha(3)D showed excellent dispersion of the backbone resonances, and selective changes in dispersion of the aliphatic side-chains proximal to the site of mutation. Together, these data suggest that alpha(3)D, although folded into a unique three-dimensional structure, is nevertheless more malleable and flexible than most natural, native proteins. Copyright 2001 Academic Press.

A Latent Class Unfolding Model for Analyzing Single Stimulus Preference Ratings.

ERIC Educational Resources Information Center

De Soete, Geert; Heiser, Willem J.

1993-01-01

A latent class unfolding model is developed for single stimulus preference ratings. One advantage is the possibility of testing the spatial unfolding model against the unconstrained latent class model for rating data. The model is applied to data about party preferences of members of the Dutch parliament. (SLD)

XBP1, Unfolded Protein Response, and Endocrine Responsiveness

DTIC Science & Technology

2011-05-01

initially modeled in yeast cells (21, 22). Components of the unfolded protein response (UPR) are also conserved across species and these include...response in tumors. Mol Cancer Res 2005;3:597–605. 38. Sriburi R, Jackowski S, Mori K, Brewer JW. XBP1: a link between the unfolded protein response

Unfolding Case-Based Practicum Curriculum Infusing Crisis, Trauma, and Disaster Preparation

ERIC Educational Resources Information Center

Greene, Catie A.; Williams, Amy E.; Harris, Pamela N.; Travis, Sterling P.; Kim, Sharon Y.

2016-01-01

The authors evaluated an unfolding case-based approach to a practicum in counseling course infusing crisis, trauma, and disaster preparation for changes in students' crisis self-efficacy across a semester. The course, informed by constructivist-developmental pedagogy and centered on the unfolding case, resulted in significant increases in…

Molecular Dynamics Simulations of the Temperature Induced Unfolding of Crambin Follow the Arrhenius Equation.

PubMed

Dalby, Andrew; Shamsir, Mohd Shahir

2015-01-01

Molecular dynamics simulations have been used extensively to model the folding and unfolding of proteins. The rates of folding and unfolding should follow the Arrhenius equation over a limited range of temperatures. This study shows that molecular dynamic simulations of the unfolding of crambin between 500K and 560K do follow the Arrhenius equation. They also show that while there is a large amount of variation between the simulations the average values for the rate show a very high degree of correlation.

Molecular Dynamics Simulations of the Temperature Induced Unfolding of Crambin Follow the Arrhenius Equation.

PubMed Central

Dalby, Andrew; Shamsir, Mohd Shahir

2015-01-01

Molecular dynamics simulations have been used extensively to model the folding and unfolding of proteins. The rates of folding and unfolding should follow the Arrhenius equation over a limited range of temperatures. This study shows that molecular dynamic simulations of the unfolding of crambin between 500K and 560K do follow the Arrhenius equation. They also show that while there is a large amount of variation between the simulations the average values for the rate show a very high degree of correlation. PMID:26539292

Proof-of-principle to unfold an angle-energy dependent source from forward and adjoint calculations

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

Pace, J.V. III

For many years there has existed a discrepancy between the measured and calculated responses from the Little Boy weapon in Hiroshima. A myriad of solutions have been proposed, but to no avail. If one can rationalize to himself that it does not really matter exactly what happened with the weapon when it exploded, and if sufficient information exist about the measurements, one should be able to unfold the source. Moreover, if a source can be unfolded in a controlled environment, then it should be possible to unfold a more complicated source, for example, the Little Boy source. This report recordsmore » the findings of a proof-of-principle test to unfold a source in the controlled environment.« less

Unfolding mechanism of lysozyme in various urea solutions: Insights from fluorescence spectroscopy

NASA Astrophysics Data System (ADS)

Chen, Bang; Zhang, Hongjia; Xi, Wenying; Zhao, Liqing; Liang, Li; Chen, Yantao

2014-11-01

Fluorescence spectroscopic technique is very popular in exploring the folding/unfolding process of proteins. In this paper, unfolding process of hen egg-white lysozyme was investigated in various denaturing solutions. Firstly, polymer solution theory was employed to comprehend the dependence of fluorescence quenching effect on protein concentration, and dynamic contact concentration was suggested as a critical value for related fluorescence experiment. Secondly, it was found that urea alone could not completely unfold lysozyme but did when together with DTT or HCl. Lysozyme was destabilized in concentrated urea solution, but still could maintain its spatial structure. Phase diagram of fluorescence intensities revealed that HCl could enhance the denaturing capacity of urea, resulting in the emergence of intermediate state in the thermodynamic unfolding process of lysozyme.

Nonparametric density estimation and optimal bandwidth selection for protein unfolding and unbinding data

NASA Astrophysics Data System (ADS)

Bura, E.; Zhmurov, A.; Barsegov, V.

2009-01-01

Dynamic force spectroscopy and steered molecular simulations have become powerful tools for analyzing the mechanical properties of proteins, and the strength of protein-protein complexes and aggregates. Probability density functions of the unfolding forces and unfolding times for proteins, and rupture forces and bond lifetimes for protein-protein complexes allow quantification of the forced unfolding and unbinding transitions, and mapping the biomolecular free energy landscape. The inference of the unknown probability distribution functions from the experimental and simulated forced unfolding and unbinding data, as well as the assessment of analytically tractable models of the protein unfolding and unbinding requires the use of a bandwidth. The choice of this quantity is typically subjective as it draws heavily on the investigator's intuition and past experience. We describe several approaches for selecting the "optimal bandwidth" for nonparametric density estimators, such as the traditionally used histogram and the more advanced kernel density estimators. The performance of these methods is tested on unimodal and multimodal skewed, long-tailed distributed data, as typically observed in force spectroscopy experiments and in molecular pulling simulations. The results of these studies can serve as a guideline for selecting the optimal bandwidth to resolve the underlying distributions from the forced unfolding and unbinding data for proteins.

Multistage unfolding of an SH3 domain: an initial urea-filled dry molten globule precedes a wet molten globule with non-native structure.

PubMed

Dasgupta, Amrita; Udgaonkar, Jayant B; Das, Payel

2014-06-19

The unfolding of the SH3 domain of the PI3 kinase in aqueous urea has been studied using a synergistic experiment-simulation approach. The experimental observation of a transient wet molten globule intermediate, IU, with an unusual non-native burial of the sole Trp residue, W53, provides the benchmark for the unfolding simulations performed (eight in total, each at least 0.5 μs long). The simulations reveal that the partially unfolded IU ensemble is preceded by an early native-like molten globule intermediate ensemble I*. In the very initial stage of unfolding, dry globule conformations with the protein core filled with urea instead of water are transiently observed within the I* ensemble. Water penetration into the urea-filled core of dry globule conformations is frequently accompanied by very transient burial of W53. Later during gradual unfolding, W53 is seen to again become transiently buried in the IU ensemble for a much longer time. In the structurally heterogeneous IU ensemble, conformational flexibility of the C-terminal β-strands enables W53 burial by the formation of non-native, tertiary contacts with hydrophobic residues, which could serve to protect the protein from aggregation during unfolding.

Characterization of two distinct beta2-microglobulin unfolding intermediates that may lead to amyloid fibrils of different morphology.

PubMed

Armen, Roger S; Daggett, Valerie

2005-12-13

The self-assembly of beta(2)-microglobulin into fibrils leads to dialysis-related amyloidosis. pH-mediated partial unfolding is required for the formation of the amyloidogenic intermediate that then self-assembles into amyloid fibrils. Two partially folded intermediates of beta(2)-microglobulin have been identified experimentally and linked to the formation of fibrils of distinct morphology, yet it remains difficult to characterize these partially unfolded states at high resolution using experimental approaches. Consequently, we have performed molecular dynamics simulations at neutral and low pH to determine the structures of these partially unfolded amyloidogenic intermediates. In the low-pH simulations, we observed the formation of alpha-sheet structure, which was first proposed by Pauling and Corey. Multiple simulations were performed, and two distinct intermediate state ensembles were identified that may account for the different fibril morphologies. The predominant early unfolding intermediate was nativelike in structure, in agreement with previous NMR studies. The late unfolding intermediate was significantly disordered, but it maintained an extended elongated structure, with hydrophobic clusters and residual alpha-extended chain strands in specific regions of the sequence that map to amyloidogenic peptides. We propose that the formation of alpha-sheet facilitates self-assembly into partially unfolded prefibrillar amyloidogenic intermediates.

Branches of Triangulated Origami Near the Unfolded State

NASA Astrophysics Data System (ADS)

Chen, Bryan Gin-ge; Santangelo, Christian D.

2018-01-01

Origami structures are characterized by a network of folds and vertices joining unbendable plates. For applications to mechanical design and self-folding structures, it is essential to understand the interplay between the set of folds in the unfolded origami and the possible 3D folded configurations. When deforming a structure that has been folded, one can often linearize the geometric constraints, but the degeneracy of the unfolded state makes a linear approach impossible there. We derive a theory for the second-order infinitesimal rigidity of an initially unfolded triangulated origami structure and use it to study the set of nearly unfolded configurations of origami with four boundary vertices. We find that locally, this set consists of a number of distinct "branches" which intersect at the unfolded state, and that the number of these branches is exponential in the number of vertices. We find numerical and analytical evidence that suggests that the branches are characterized by choosing each internal vertex to either "pop up" or "pop down." The large number of pathways along which one can fold an initially unfolded origami structure strongly indicates that a generic structure is likely to become trapped in a "misfolded" state. Thus, new techniques for creating self-folding origami are likely necessary; controlling the popping state of the vertices may be one possibility.

Studies on the Dissociation and Urea-Induced Unfolding of FtsZ Support the Dimer Nucleus Polymerization Mechanism

PubMed Central

Montecinos-Franjola, Felipe; Ross, Justin A.; Sánchez, Susana A.; Brunet, Juan E.; Lagos, Rosalba; Jameson, David M.; Monasterio, Octavio

2012-01-01

FtsZ is a major protein in bacterial cytokinesis that polymerizes into single filaments. A dimer has been proposed to be the nucleating species in FtsZ polymerization. To investigate the influence of the self-assembly of FtsZ on its unfolding pathway, we characterized its oligomerization and unfolding thermodynamics. We studied the assembly using size-exclusion chromatography and fluorescence spectroscopy, and the unfolding using circular dichroism and two-photon fluorescence correlation spectroscopy. The chromatographic analysis demonstrated the presence of monomers, dimers, and tetramers with populations dependent on protein concentration. Dilution experiments using fluorescent conjugates revealed dimer-to-monomer and tetramer-to-dimer dissociation constants in the micromolar range. Measurements of fluorescence lifetimes and rotational correlation times of the conjugates supported the presence of tetramers at high protein concentrations and monomers at low protein concentrations. The unfolding study demonstrated that the three-state unfolding of FtsZ was due to the mainly dimeric state of the protein, and that the monomer unfolds through a two-state mechanism. The monomer-to-dimer equilibrium characterized here (Kd = 9 μM) indicates a significant fraction (∼10%) of stable dimers at the critical concentration for polymerization, supporting a role of the dimeric species in the first steps of FtsZ polymerization. PMID:22824282

Thermal, Chemical and pH Induced Denaturation of a Multimeric β-Galactosidase Reveals Multiple Unfolding Pathways

PubMed Central

Kishore, Devesh; Kundu, Suman; Kayastha, Arvind M.

2012-01-01

Background In this case study, we analysed the properties of unfolded states and pathways leading to complete denaturation of a multimeric chick pea β-galactosidase (CpGAL), as obtained from treatment with guanidium hydrochloride, urea, elevated temperature and extreme pH. Methodology/Principal Findings CpGAL, a heterodimeric protein with native molecular mass of 85 kDa, belongs to α+β class of protein. The conformational stability and thermodynamic parameters of CpGAL unfolding in different states were estimated and interpreted using circular dichroism and fluorescence spectroscopic measurements. The enzyme was found to be structurally and functionally stable in the entire pH range and upto 50°C temperature. Further increase in temperature induces unfolding followed by aggregation. Chemical induced denaturation was found to be cooperative and transitions were irreversible, non-coincidental and sigmoidal. Free energy of protein unfolding (ΔG0) and unfolding constant (Kobs) were also calculated for chemically denatured CpGAL. Significance The protein seems to use different pathways for unfolding in different environments and is a classical example of how the environment dictates the path a protein might take to fold while its amino acid sequence only defines its final three-dimensional conformation. The knowledge accumulated could be of immense biotechnological significance as well. PMID:23185611

First Passage Times, Lifetimes, and Relaxation Times of Unfolded Proteins

NASA Astrophysics Data System (ADS)

Dai, Wei; Sengupta, Anirvan M.; Levy, Ronald M.

2015-07-01

The dynamics of proteins in the unfolded state can be quantified in computer simulations by calculating a spectrum of relaxation times which describes the time scales over which the population fluctuations decay to equilibrium. If the unfolded state space is discretized, we can evaluate the relaxation time of each state. We derive a simple relation that shows the mean first passage time to any state is equal to the relaxation time of that state divided by the equilibrium population. This explains why mean first passage times from state to state within the unfolded ensemble can be very long but the energy landscape can still be smooth (minimally frustrated). In fact, when the folding kinetics is two-state, all of the unfolded state relaxation times within the unfolded free energy basin are faster than the folding time. This result supports the well-established funnel energy landscape picture and resolves an apparent contradiction between this model and the recently proposed kinetic hub model of protein folding. We validate these concepts by analyzing a Markov state model of the kinetics in the unfolded state and folding of the miniprotein NTL9 (where NTL9 is the N -terminal domain of the ribosomal protein L9), constructed from a 2.9 ms simulation provided by D. E. Shaw Research.

First Passage Times, Lifetimes, and Relaxation Times of Unfolded Proteins.

PubMed

Dai, Wei; Sengupta, Anirvan M; Levy, Ronald M

2015-07-24

The dynamics of proteins in the unfolded state can be quantified in computer simulations by calculating a spectrum of relaxation times which describes the time scales over which the population fluctuations decay to equilibrium. If the unfolded state space is discretized, we can evaluate the relaxation time of each state. We derive a simple relation that shows the mean first passage time to any state is equal to the relaxation time of that state divided by the equilibrium population. This explains why mean first passage times from state to state within the unfolded ensemble can be very long but the energy landscape can still be smooth (minimally frustrated). In fact, when the folding kinetics is two-state, all of the unfolded state relaxation times within the unfolded free energy basin are faster than the folding time. This result supports the well-established funnel energy landscape picture and resolves an apparent contradiction between this model and the recently proposed kinetic hub model of protein folding. We validate these concepts by analyzing a Markov state model of the kinetics in the unfolded state and folding of the miniprotein NTL9 (where NTL9 is the N-terminal domain of the ribosomal protein L9), constructed from a 2.9 ms simulation provided by D. E. Shaw Research.

Isothermal chemical denaturation of large proteins: Path-dependence and irreversibility.

PubMed

Wafer, Lucas; Kloczewiak, Marek; Polleck, Sharon M; Luo, Yin

2017-12-15

State functions (e.g., ΔG) are path independent and quantitatively describe the equilibrium states of a thermodynamic system. Isothermal chemical denaturation (ICD) is often used to extrapolate state function parameters for protein unfolding in native buffer conditions. The approach is prudent when the unfolding/refolding processes are path independent and reversible, but may lead to erroneous results if the processes are not reversible. The reversibility was demonstrated in several early studies for smaller proteins, but was assumed in some reports for large proteins with complex structures. In this work, the unfolding/refolding of several proteins were systematically studied using an automated ICD instrument. It is shown that: (i) the apparent unfolding mechanism and conformational stability of large proteins can be denaturant-dependent, (ii) equilibration times for large proteins are non-trivial and may introduce significant error into calculations of ΔG, (iii) fluorescence emission spectroscopy may not correspond to other methods, such as circular dichroism, when used to measure protein unfolding, and (iv) irreversible unfolding and hysteresis can occur in the absence of aggregation. These results suggest that thorough confirmation of the state functions by, for example, performing refolding experiments or using additional denaturants, is needed when quantitatively studying the thermodynamics of protein unfolding using ICD. Copyright © 2017 Elsevier Inc. All rights reserved.

High Temperature Unfolding and Low Temperature Refolding Pathway of Chymotrypsin Inhibitor 2 Using Molecular Dynamics Simulation

NASA Astrophysics Data System (ADS)

Malau, N. D.; Sumaryada, T.

2016-01-01

The mechanism that explains the unfolding/refolding process of the protein is still a major problem that has not been fully understood. In this paper we present our study on the unfolding and refolding pathway of Chymotrypsin Inhibitor 2 (CI2) protein through a molecular dynamics simulation technique. The high temperature unfolding simulation were performed at 500 K for 35 ns. While the low temperature refolding simulation performed at 200 K for 35 ns. The unfolding and refolding pathway of protein were analysed by looking at the dynamics of root mean squared deviation (RMSD) and secondary structure profiles. The signatures of unfolding were observed from significant increase of RMSD within the time span of 10 ns to 35 ns. For the refolding process, the initial structure was prepared from the structure of unfolding protein at t=15 ns and T=500 K. Analysis have shown that some of the secondary structures of CI2 protein that have been damaged at high temperature can be refolded back to its initial structure at low temperature simulation. Our results suggest that most of α-helix structure of CI2 protein can be refolded back to its initial state, while only half beta-sheet structure can be reformed.

Engineered Bi-Histidine Metal Chelation Sites Map the Structure of the Mechanical Unfolding Transition State of an Elastomeric Protein Domain GB1

PubMed Central

Shen, Tao; Cao, Yi; Zhuang, Shulin; Li, Hongbin

2012-01-01

Determining the structure of the transition state is critical for elucidating the mechanism behind how proteins fold and unfold. Due to its high free energy, however, the transition state generally cannot be trapped and studied directly using traditional structural biology methods. Thus, characterizing the structure of the transition state that occurs as proteins fold and unfold remains a major challenge. Here, we report a novel (to our knowledge) method that uses engineered bi-histidine (bi-His) metal-binding sites to directly map the structure of the mechanical unfolding transition state of proteins. This method is adapted from the traditional ψ-value analysis, which uses engineered bi-His metal chelation sites to probe chemical (un)folding transition-state structure. The ϕM2+U-value is defined as ΔΔG‡-N/ΔΔGU-N, which is the energetic effects of metal chelation by the bi-His site on the unfolding energy barrier (ΔG‡-N) relative to its thermodynamic stability (ΔGU-N) and can be used to obtain information about the transition state in the mutational site. As a proof of principle, we used the small protein GB1 as a model system and set out to map its mechanical unfolding transition-state structure. Using single-molecule atomic force microscopy and spectrofluorimetry, we directly quantified the effect of divalent metal ion binding on the mechanical unfolding free energy and thermodynamic stability of GB1, which allowed us to quantify ϕM2+U-values for different sites in GB1. Our results enabled us to map the structure of the mechanical unfolding transition state of GB1. Within GB1’s mechanical unfolding transition state, the interface between force-bearing β-strands 1 and 4 is largely disrupted, and the first β-hairpin is partially disordered while the second β-hairpin and the α-helix remain structured. Our results demonstrate the unique application of ψ-value analysis in elucidating the structure of the transition state that occurs during the mechanical unfolding process, offering a potentially powerful new method for investigating the design of novel elastomeric proteins. PMID:22947942

Engineered bi-histidine metal chelation sites map the structure of the mechanical unfolding transition state of an elastomeric protein domain GB1.

PubMed

Shen, Tao; Cao, Yi; Zhuang, Shulin; Li, Hongbin

2012-08-22

Determining the structure of the transition state is critical for elucidating the mechanism behind how proteins fold and unfold. Due to its high free energy, however, the transition state generally cannot be trapped and studied directly using traditional structural biology methods. Thus, characterizing the structure of the transition state that occurs as proteins fold and unfold remains a major challenge. Here, we report a novel (to our knowledge) method that uses engineered bi-histidine (bi-His) metal-binding sites to directly map the structure of the mechanical unfolding transition state of proteins. This method is adapted from the traditional ψ-value analysis, which uses engineered bi-His metal chelation sites to probe chemical (un)folding transition-state structure. The φ(M2+)(U)-value is defined as ΔΔG(‡-N)/ΔΔG(U-N), which is the energetic effects of metal chelation by the bi-His site on the unfolding energy barrier (ΔG(‡-N)) relative to its thermodynamic stability (ΔG(U-N)) and can be used to obtain information about the transition state in the mutational site. As a proof of principle, we used the small protein GB1 as a model system and set out to map its mechanical unfolding transition-state structure. Using single-molecule atomic force microscopy and spectrofluorimetry, we directly quantified the effect of divalent metal ion binding on the mechanical unfolding free energy and thermodynamic stability of GB1, which allowed us to quantify φ(M2+)(U)-values for different sites in GB1. Our results enabled us to map the structure of the mechanical unfolding transition state of GB1. Within GB1's mechanical unfolding transition state, the interface between force-bearing β-strands 1 and 4 is largely disrupted, and the first β-hairpin is partially disordered while the second β-hairpin and the α-helix remain structured. Our results demonstrate the unique application of ψ-value analysis in elucidating the structure of the transition state that occurs during the mechanical unfolding process, offering a potentially powerful new method for investigating the design of novel elastomeric proteins. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Sequential protein unfolding through a carbon nanotube pore

NASA Astrophysics Data System (ADS)

Xu, Zhonghe; Zhang, Shuang; Weber, Jeffrey K.; Luan, Binquan; Zhou, Ruhong; Li, Jingyuan

2016-06-01

An assortment of biological processes, like protein degradation and the transport of proteins across membranes, depend on protein unfolding events mediated by nanopore interfaces. In this work, we exploit fully atomistic simulations of an artificial, CNT-based nanopore to investigate the nature of ubiquitin unfolding. With one end of the protein subjected to an external force, we observe non-canonical unfolding behaviour as ubiquitin is pulled through the pore opening. Secondary structural elements are sequentially detached from the protein and threaded into the nanotube, interestingly, the remaining part maintains native-like characteristics. The constraints of the nanopore interface thus facilitate the formation of stable ``unfoldon'' motifs above the nanotube aperture that can exist in the absence of specific native contacts with the other secondary structure. Destruction of these unfoldons gives rise to distinct force peaks in our simulations, providing us with a sensitive probe for studying the kinetics of serial unfolding events. Our detailed analysis of nanopore-mediated protein unfolding events not only provides insight into how related processes might proceed in the cell, but also serves to deepen our understanding of structural arrangements which form the basis for protein conformational stability.An assortment of biological processes, like protein degradation and the transport of proteins across membranes, depend on protein unfolding events mediated by nanopore interfaces. In this work, we exploit fully atomistic simulations of an artificial, CNT-based nanopore to investigate the nature of ubiquitin unfolding. With one end of the protein subjected to an external force, we observe non-canonical unfolding behaviour as ubiquitin is pulled through the pore opening. Secondary structural elements are sequentially detached from the protein and threaded into the nanotube, interestingly, the remaining part maintains native-like characteristics. The constraints of the nanopore interface thus facilitate the formation of stable ``unfoldon'' motifs above the nanotube aperture that can exist in the absence of specific native contacts with the other secondary structure. Destruction of these unfoldons gives rise to distinct force peaks in our simulations, providing us with a sensitive probe for studying the kinetics of serial unfolding events. Our detailed analysis of nanopore-mediated protein unfolding events not only provides insight into how related processes might proceed in the cell, but also serves to deepen our understanding of structural arrangements which form the basis for protein conformational stability. Electronic supplementary information (ESI) available. See DOI: 10.1039/c6nr00410e

Predictors of natively unfolded proteins: unanimous consensus score to detect a twilight zone between order and disorder in generic datasets.

PubMed

Deiana, Antonio; Giansanti, Andrea

2010-04-21

Natively unfolded proteins lack a well defined three dimensional structure but have important biological functions, suggesting a re-assignment of the structure-function paradigm. To assess that a given protein is natively unfolded requires laborious experimental investigations, then reliable sequence-only methods for predicting whether a sequence corresponds to a folded or to an unfolded protein are of interest in fundamental and applicative studies. Many proteins have amino acidic compositions compatible both with the folded and unfolded status, and belong to a twilight zone between order and disorder. This makes difficult a dichotomic classification of protein sequences into folded and natively unfolded ones. In this work we propose an operational method to identify proteins belonging to the twilight zone by combining into a consensus score good performing single predictors of folding. In this methodological paper dichotomic folding indexes are considered: hydrophobicity-charge, mean packing, mean pairwise energy, Poodle-W and a new global index, that is called here gVSL2, based on the local disorder predictor VSL2. The performance of these indexes is evaluated on different datasets, in particular on a new dataset composed by 2369 folded and 81 natively unfolded proteins. Poodle-W, gVSL2 and mean pairwise energy have good performance and stability in all the datasets considered and are combined into a strictly unanimous combination score SSU, that leaves proteins unclassified when the consensus of all combined indexes is not reached. The unclassified proteins: i) belong to an overlap region in the vector space of amino acidic compositions occupied by both folded and unfolded proteins; ii) are composed by approximately the same number of order-promoting and disorder-promoting amino acids; iii) have a mean flexibility intermediate between that of folded and that of unfolded proteins. Our results show that proteins unclassified by SSU belong to a twilight zone. Proteins left unclassified by the consensus score SSU have physical properties intermediate between those of folded and those of natively unfolded proteins and their structural properties and evolutionary history are worth to be investigated.

Predictors of natively unfolded proteins: unanimous consensus score to detect a twilight zone between order and disorder in generic datasets

PubMed Central

2010-01-01

Background Natively unfolded proteins lack a well defined three dimensional structure but have important biological functions, suggesting a re-assignment of the structure-function paradigm. To assess that a given protein is natively unfolded requires laborious experimental investigations, then reliable sequence-only methods for predicting whether a sequence corresponds to a folded or to an unfolded protein are of interest in fundamental and applicative studies. Many proteins have amino acidic compositions compatible both with the folded and unfolded status, and belong to a twilight zone between order and disorder. This makes difficult a dichotomic classification of protein sequences into folded and natively unfolded ones. In this work we propose an operational method to identify proteins belonging to the twilight zone by combining into a consensus score good performing single predictors of folding. Results In this methodological paper dichotomic folding indexes are considered: hydrophobicity-charge, mean packing, mean pairwise energy, Poodle-W and a new global index, that is called here gVSL2, based on the local disorder predictor VSL2. The performance of these indexes is evaluated on different datasets, in particular on a new dataset composed by 2369 folded and 81 natively unfolded proteins. Poodle-W, gVSL2 and mean pairwise energy have good performance and stability in all the datasets considered and are combined into a strictly unanimous combination score SSU, that leaves proteins unclassified when the consensus of all combined indexes is not reached. The unclassified proteins: i) belong to an overlap region in the vector space of amino acidic compositions occupied by both folded and unfolded proteins; ii) are composed by approximately the same number of order-promoting and disorder-promoting amino acids; iii) have a mean flexibility intermediate between that of folded and that of unfolded proteins. Conclusions Our results show that proteins unclassified by SSU belong to a twilight zone. Proteins left unclassified by the consensus score SSU have physical properties intermediate between those of folded and those of natively unfolded proteins and their structural properties and evolutionary history are worth to be investigated. PMID:20409339

A Simple Spreadsheet Program to Simulate and Analyze the Far-UV Circular Dichroism Spectra of Proteins

ERIC Educational Resources Information Center

Abriata, Luciano A.

2011-01-01

A simple algorithm was implemented in a spreadsheet program to simulate the circular dichroism spectra of proteins from their secondary structure content and to fit [alpha]-helix, [beta]-sheet, and random coil contents from experimental far-UV circular dichroism spectra. The physical basis of the method is briefly reviewed within the context of…

An investigation of a mathematical model for atmospheric absorption spectra

NASA Technical Reports Server (NTRS)

Niple, E. R.

1979-01-01

A computer program that calculates absorption spectra for slant paths through the atmosphere is described. The program uses an efficient convolution technique (Romberg integration) to simulate instrument resolution effects. A brief information analysis is performed on a set of calculated spectra to illustrate how such techniques may be used to explore the quality of the information in a spectrum.

Measuring neutron spectra in radiotherapy using the nested neutron spectrometer.

PubMed

Maglieri, Robert; Licea, Angel; Evans, Michael; Seuntjens, Jan; Kildea, John

2015-11-01

Out-of-field neutron doses resulting from photonuclear interactions in the head of a linear accelerator pose an iatrogenic risk to patients and an occupational risk to personnel during radiotherapy. To quantify neutron production, in-room measurements have traditionally been carried out using Bonner sphere systems (BSS) with activation foils and TLDs. In this work, a recently developed active detector, the nested neutron spectrometer (NNS), was tested in radiotherapy bunkers. The NNS is designed for easy handling and is more practical than the traditional BSS. Operated in current-mode, the problem of pulse pileup due to high dose-rates is overcome by measuring current, similar to an ionization chamber. In a bunker housing a Varian Clinac 21EX, the performance of the NNS was evaluated in terms of reproducibility, linearity, and dose-rate effects. Using a custom maximum-likelihood expectation-maximization algorithm, measured neutron spectra at various locations inside the bunker were then compared to Monte Carlo simulations of an identical setup. In terms of dose, neutron ambient dose equivalents were calculated from the measured spectra and compared to bubble detector neutron dose equivalent measurements. The NNS-measured spectra for neutrons at various locations in a treatment room were found to be consistent with expectations for both relative shape and absolute magnitude. Neutron fluence-rate decreased with distance from the source and the shape of the spectrum changed from a dominant fast neutron peak near the Linac head to a dominant thermal neutron peak in the moderating conditions of the maze. Monte Carlo data and NNS-measured spectra agreed within 30% at all locations except in the maze where the deviation was a maximum of 40%. Neutron ambient dose equivalents calculated from the authors' measured spectra were consistent (one standard deviation) with bubble detector measurements in the treatment room. The NNS may be used to reliably measure the neutron spectrum of a radiotherapy beam in less than 1 h, including setup and data unfolding. This work thus represents a new, fast, and practical method for neutron spectral measurements in radiotherapy.

pH dependent unfolding characteristics of DLC8 dimer: Residue level details from NMR.

PubMed

Mohan, P M Krishna; Hosur, Ramakrishna V

2008-11-01

Environment dependence of folding and unfolding of a protein is central to its function. In the same vein, knowledge of pH dependence of stability and folding/unfolding is crucial for many biophysical equilibrium and kinetic studies designed to understand protein folding mechanisms. In the present study we investigated the guanidine induced unfolding transition of dynein light chain protein (DLC8), a cargo adaptor of the dynein complex in the pH range 7-10. It is observed that while the protein remains a dimer in the entire pH range, its stability is somewhat reduced at alkaline pH. Global unfolding features monitored using fluorescence spectroscopy revealed that the unfolding transition of DLC8 at pH 7 is best described by a three-state model, whereas, that at pH 10 is best described by a two-state model. Chemical shift perturbations due to pH change provided insights into the corresponding residue level structural perturbations in the DLC8 dimer. Likewise, backbone (15)N relaxation measurements threw light on the corresponding motional changes in the dimeric protein. These observations have been rationalized on the basis of expected changes with increasing pH in the protonation states of the titratable residues on the structure of the protein. These, in turn provide an explanation for the change from three-state to two-state guanidine induced unfolding transition as the pH is increased from 7 to 10. All these results exemplify and highlight the role of environment vis-à-vis the sequence and structure of a given protein in dictating its folding/unfolding characteristics.

The role of sequence in altering the unfolding pathway of an RNA pseudoknot: a steered molecular dynamics study.

PubMed

Gupta, Asmita; Bansal, Manju

2016-10-19

Mechanical unfolding studies on Ribonucleic Acid (RNA) structures are a subject of tremendous interest as they shed light on the principles of higher order assembly of these structures. Pseudoknotting is one of the most elementary ways in which this higher order assembly is achieved as discrete secondary structural units in RNA are brought in close proximity to form a tertiary structure. Using steered molecular dynamics (SMD) simulations, we have studied the unfolding of five RNA pseudoknot structures that differ from each other either by base substitutions in helices or loops. Our SMD simulations reveal the manner in which a biologically functional RNA pseudoknot unfolds and the effect of changes in the primary structure on this unfolding pathway, providing necessary insights into the driving forces behind the functioning of these structures. We observed that an A → C mutation in the loop sequence makes the pseudoknot far more resistant against force induced disruption relative to its wild type structure. In contrast to this, a base-pair substitution GC → AU near the pseudoknot junction region renders it more vulnerable to this disruption. The quantitative estimation of differences in the unfolding paths was carried out using force extension curves, potential of mean force profiles, and the opening of different Watson-Crick and non-Watson-Crick interactions. The results provide a quantified view in which the unfolding paths of the small RNA structures can be used for investigating the programmability of RNA chains for designing RNA switches and aptamers as their biological folding and unfolding could be assessed and manipulated.

Equilibrium unfolding of A. niger RNase: pH dependence of chemical and thermal denaturation.

PubMed

Kumar, Gundampati Ravi; Sharma, Anurag; Kumari, Moni; Jagannadham, Medicherla V; Debnath, Mira

2011-08-01

Equilibrium unfolding of A. niger RNase with chemical denaturants, for example GuHCl and urea, and thermal unfolding have been studied as a function of pH using fluorescence, far-UV, near-UV, and absorbance spectroscopy. Because of their ability to affect electrostatic interactions, pH and chemical denaturants have a marked effect on the stability, structure, and function of many globular proteins. ANS binding studies have been conducted to enable understanding of the folding mechanism of the protein in the presence of the denaturants. Spectroscopic studies by absorbance, fluorescence, and circular dichroism and use of K2D software revealed that the enzyme has α + β type secondary structure with approximately 29% α-helix, 24% β-sheet, and 47% random coil. Under neutral conditions the enzyme is stable in urea whereas GuHCl-induced equilibrium unfolding was cooperative. A. niger RNase has little ANS binding even under neutral conditions. Multiple intermediates were populated during the pH-induced unfolding of A. niger RNase. Urea and temperature-induced unfolding of A. niger RNase into the molten globule-like state is non-cooperative, in contrast to the cooperativity seen with the native protein, suggesting the presence of two parts/domains, in the molecular structure of A. niger RNase, with different stability that unfolds in steps. Interestingly, the GuHCl-induced unfolding of the A state (molten globule state) of A. niger RNase is unique, because a low concentration of denaturant not only induces structural change but also facilitates transition from one molten globule like state (A(MG1)) into another (I(MG2)).

Protein denaturation in vacuo: intrinsic unfolding pathways associated with the native tertiary structure of lysozyme

NASA Astrophysics Data System (ADS)

Arteca, Gustavo A.; Tapia, O.

Using computer-simulated molecular dynamics, we study the effect of sequence mutation on the unfolding mechanism of a native fold. The system considered is the native fold of hen egg-white lysozyme, exposed to centrifugal unfolding in vacuo. This unfolding bias elicits configurational transitions that imitate the behaviour of anhydrous proteins diffusing after electrospraying from neutral-pH solutions. By changing the sequences threaded onto the native fold of lysozyme, we probe the role of disulfide bridges and the effect of a global mutation. We find that the initial denaturing steps share common characteristics for the tested sequences. Recurrent features are: (i) the presence of dumbbell conformers with significant residual secondary structure, (ii) the ubiquitous formation of hairpins and two-stranded β-sheets regardless of disulfide bridges, and (iii) an unfolding pattern where the reduction in folding complexity is highly correlated with the decrease in chain compactness. These findings appear to be intrinsic to the shape of the native fold, suggesting that similar unfolding pathways may be accessible to many protein sequences.

Characteristics and application of spherical-type activation detectors in neutron spectrum measurements at a boron neutron capture therapy (BNCT) facility

NASA Astrophysics Data System (ADS)

Lin, Heng-Xiao; Chen, Wei-Lin; Liu, Yuan-Hao; Sheu, Rong-Jiun

2016-03-01

A set of spherical-type activation detectors was developed aiming to provide better determination of the neutron spectrum at the Tsing Hua Open-pool Reactor (THOR) BNCT facility. An activation foil embedded in a specially designed spherical holder exhibits three advantages: (1) minimizing the effect of neutron angular dependence, (2) creating response functions with broadened coverage of neutron energies by introducing additional moderators or absorbers to the central activation foil, and (3) reducing irradiation time because of improved detection efficiencies to epithermal neutron beam. This paper presents the design concept and the calculated response functions of new detectors. Theoretical and experimental demonstrations of the performance of the detectors are provided through comparisons of the unfolded neutron spectra determined using this method and conventional multiple-foil activation techniques.

Observations of Interchange Between Acceleration and Thermalization Processes in Auroral Electrons. Ph.D. Thesis

NASA Technical Reports Server (NTRS)

Pongratz, M. B.

1972-01-01

The results of high time-resolution measurements of energetic electrons in an auroral break up are presented. Electrons with energies from 500 eV to over 100 keV and pitch angles from 0 to 150 deg were detected with two detectors onboard sounding rocket 18:63 UE. Complete energy spectra were taken every 0.1 seconds. The procedure for cleaning and activating the BeCu dynodes of a small, rugged, high gain electron multiplier is described. A theoretical study of the energy-angular response of a spherical plate electrostatic analyzer is compared to experimental results. An energy spectrum unfolding technique which does not require the assumption of a histogram-type energy spectrum is presented. A method of determining sounding rocket orientation from the output of a single magnetometer is described.

Multidimensional free energy surface of unfolding of HP-36: Microscopic origin of ruggedness

NASA Astrophysics Data System (ADS)

Ghosh, Rikhia; Roy, Susmita; Bagchi, Biman

2014-10-01

The protein folding funnel paradigm suggests that folding and unfolding proceed as directed diffusion in a multidimensional free energy surface where a multitude of pathways can be traversed during the protein's sojourn from initial to final state. However, finding even a single pathway, with the detail chronicling of intermediates, is an arduous task. In this work we explore the free energy surface of unfolding pathway through umbrella sampling, for a small globular α-helical protein chicken-villin headpiece (HP-36) when the melting of secondary structures is induced by adding DMSO in aqueous solution. We find that the unfolding proceeds through the initial separation or melting of aggregated hydrophobic core that comprises of three phenylalanine residues (Phe7, Phe11, and Phe18). This separation is accompanied by simultaneous melting of the second helix. Unfolding is found to be a multistage process involving crossing of three consecutive minima and two barriers at the initial stage. At a molecular level, Phe18 is observed to reorient itself towards other hydrophobic grooves to stabilize the intermediate states. We identify the configuration of the intermediates and correlate the intermediates with those obtained in our previous works. We also give an estimate of the barriers for different transition states and observe the softening of the barriers with increasing DMSO concentration. We show that higher concentration of DMSO tunes the unfolding pathway by destabilizing the third minimum and stabilizing the second one, indicating the development of a solvent modified, less rugged pathway. The prime outcome of this work is the demonstration that mixed solvents can profoundly transform the nature of the energy landscape and induce unfolding via a modified route. A successful application of Kramer's rate equation correlating the free energy simulation results shows faster rate of unfolding with increasing DMSO concentration. This work perhaps presents the first systematic theoretical study of the effect of a chemical denaturant on the microscopic free energy surface and rates of unfolding of HP-36.

Mechanical unfolding reveals stable 3-helix intermediates in talin and α-catenin

PubMed Central

2018-01-01

Mechanical stability is a key feature in the regulation of structural scaffolding proteins and their functions. Despite the abundance of α-helical structures among the human proteome and their undisputed importance in health and disease, the fundamental principles of their behavior under mechanical load are poorly understood. Talin and α-catenin are two key molecules in focal adhesions and adherens junctions, respectively. In this study, we used a combination of atomistic steered molecular dynamics (SMD) simulations, polyprotein engineering, and single-molecule atomic force microscopy (smAFM) to investigate unfolding of these proteins. SMD simulations revealed that talin rod α-helix bundles as well as α-catenin α-helix domains unfold through stable 3-helix intermediates. While the 5-helix bundles were found to be mechanically stable, a second stable conformation corresponding to the 3-helix state was revealed. Mechanically weaker 4-helix bundles easily unfolded into a stable 3-helix conformation. The results of smAFM experiments were in agreement with the findings of the computational simulations. The disulfide clamp mutants, designed to protect the stable state, support the 3-helix intermediate model in both experimental and computational setups. As a result, multiple discrete unfolding intermediate states in the talin and α-catenin unfolding pathway were discovered. Better understanding of the mechanical unfolding mechanism of α-helix proteins is a key step towards comprehensive models describing the mechanoregulation of proteins. PMID:29698481

The Graded Unfolding Model: A Unidimensional Item Response Model for Unfolding Graded Responses.

ERIC Educational Resources Information Center

Roberts, James S.; Laughlin, James E.

Binary or graded disagree-agree responses to attitude items are often collected for the purpose of attitude measurement. Although such data are sometimes analyzed with cumulative measurement models, recent investigations suggest that unfolding models are more appropriate (J. S. Roberts, 1995; W. H. Van Schuur and H. A. L. Kiers, 1994). Advances in…

A Unidimensional Item Response Model for Unfolding Responses from a Graded Disagree-Agree Response Scale.

ERIC Educational Resources Information Center

Roberts, James S.; Laughlin, James E.

1996-01-01

A parametric item response theory model for unfolding binary or graded responses is developed. The graded unfolding model (GUM) is a generalization of the hyperbolic cosine model for binary data of D. Andrich and G. Luo (1993). Applicability of the GUM to attitude testing is illustrated with real data. (SLD)

Immobilized unfolded cytochrome c acts as a catalyst for dioxygen reduction.

PubMed

Tavagnacco, Claudio; Monari, Stefano; Ranieri, Antonio; Bortolotti, Carlo Augusto; Peressini, Silvia; Borsari, Marco

2011-10-21

Unfolding turns immobilized cytochrome c into a His-His ligated form endowed with catalytic activity towards O(2), which is absent in the native protein. Dioxygen could be used by naturally occurring unfolded cytochrome c as a substrate for the production of partially reduced oxygen species (PROS) contributing to the cell oxidative stress.

Using Data Augmentation and Markov Chain Monte Carlo for the Estimation of Unfolding Response Models

ERIC Educational Resources Information Center

Johnson, Matthew S.; Junker, Brian W.

2003-01-01

Unfolding response models, a class of item response theory (IRT) models that assume a unimodal item response function (IRF), are often used for the measurement of attitudes. Verhelst and Verstralen (1993)and Andrich and Luo (1993) independently developed unfolding response models by relating the observed responses to a more common monotone IRT…

Multidimensional Unfolding by Nonmetric Multidimensional Scaling of Spearman Distances in the Extended Permutation Polytope

ERIC Educational Resources Information Center

Van Deun, Katrijn; Heiser, Willem J.; Delbeke, Luc

2007-01-01

A multidimensional unfolding technique that is not prone to degenerate solutions and is based on multidimensional scaling of a complete data matrix is proposed: distance information about the unfolding data and about the distances both among judges and among objects is included in the complete matrix. The latter information is derived from the…

Modified Likelihood-Based Item Fit Statistics for the Generalized Graded Unfolding Model

ERIC Educational Resources Information Center

Roberts, James S.

2008-01-01

Orlando and Thissen (2000) developed an item fit statistic for binary item response theory (IRT) models known as S-X[superscript 2]. This article generalizes their statistic to polytomous unfolding models. Four alternative formulations of S-X[superscript 2] are developed for the generalized graded unfolding model (GGUM). The GGUM is a…

The Random-Threshold Generalized Unfolding Model and Its Application of Computerized Adaptive Testing

ERIC Educational Resources Information Center

Wang, Wen-Chung; Liu, Chen-Wei; Wu, Shiu-Lien

2013-01-01

The random-threshold generalized unfolding model (RTGUM) was developed by treating the thresholds in the generalized unfolding model as random effects rather than fixed effects to account for the subjective nature of the selection of categories in Likert items. The parameters of the new model can be estimated with the JAGS (Just Another Gibbs…

Forced-Unfolding and Force-Quench Refolding of RNA Hairpins

PubMed Central

Hyeon, Changbong; Thirumalai, D.

2006-01-01

Nanomanipulation of individual RNA molecules, using laser optical tweezers, has made it possible to infer the major features of their energy landscape. Time-dependent mechanical unfolding trajectories, measured at a constant stretching force (fS) of simple RNA structures (hairpins and three-helix junctions) sandwiched between RNA/DNA hybrid handles show that they unfold in a reversible all-or-none manner. To provide a molecular interpretation of the experiments we use a general coarse-grained off-lattice Gō-like model, in which each nucleotide is represented using three interaction sites. Using the coarse-grained model we have explored forced-unfolding of RNA hairpin as a function of fS and the loading rate (rf). The simulations and theoretical analysis have been done both with and without the handles that are explicitly modeled by semiflexible polymer chains. The mechanisms and timescales for denaturation by temperature jump and mechanical unfolding are vastly different. The directed perturbation of the native state by fS results in a sequential unfolding of the hairpin starting from their ends, whereas thermal denaturation occurs stochastically. From the dependence of the unfolding rates on rf and fS we show that the position of the unfolding transition state is not a constant but moves dramatically as either rf or fS is changed. The transition-state movements are interpreted by adopting the Hammond postulate for forced-unfolding. Forced-unfolding simulations of RNA, with handles attached to the two ends, show that the value of the unfolding force increases (especially at high pulling speeds) as the length of the handles increases. The pathways for refolding of RNA from stretched initial conformation, upon quenching fS to the quench force fQ, are highly heterogeneous. The refolding times, upon force-quench, are at least an order-of-magnitude greater than those obtained by temperature-quench. The long fQ-dependent refolding times starting from fully stretched states are analyzed using a model that accounts for the microscopic steps in the rate-limiting step, which involves the trans to gauche transitions of the dihedral angles in the GAAA tetraloop. The simulations with explicit molecular model for the handles show that the dynamics of force-quench refolding is strongly dependent on the interplay of their contour length and persistence length and the RNA persistence length. Using the generality of our results, we also make a number of precise experimentally testable predictions. PMID:16473903

Unfolding single RNA molecules: bridging the gap between equilibrium and non-equilibrium statistical thermodynamics.

PubMed

Bustamante, Carlos

2005-11-01

During the last 15 years, scientists have developed methods that permit the direct mechanical manipulation of individual molecules. Using this approach, they have begun to investigate the effect of force and torque in chemical and biochemical reactions. These studies span from the study of the mechanical properties of macromolecules, to the characterization of molecular motors, to the mechanical unfolding of individual proteins and RNA. Here I present a review of some of our most recent results using mechanical force to unfold individual molecules of RNA. These studies make it possible to follow in real time the trajectory of each molecule as it unfolds and characterize the various intermediates of the reaction. Moreover, if the process takes place reversibly it is possible to extract both kinetic and thermodynamic information from these experiments at the same time that we characterize the forces that maintain the three-dimensional structure of the molecule in solution. These studies bring us closer to the biological unfolding processes in the cell as they simulate in vitro, the mechanical unfolding of RNAs carried out in the cell by helicases. If the unfolding process occurs irreversibly, I show here that single-molecule experiments can still provide equilibrium, thermodynamic information from non-equilibrium data by using recently discovered fluctuation theorems. Such theorems represent a bridge between equilibrium and non-equilibrium statistical mechanics. In fact, first derived in 1997, the first experimental demonstration of the validity of fluctuation theorems was obtained by unfolding mechanically a single molecule of RNA. It is perhaps a sign of the times that important physical results are these days used to extract information about biological systems and that biological systems are being used to test and confirm fundamental new laws in physics.

Investigating the structural transitions of proteins during dissolution by mass spectrometry.

PubMed

Gong, Xiaoyun; Xiong, Xingchuang; Qi, Lin; Fang, Xiang

2017-03-01

An appropriate solvent environment is essential for the implementation of biological functions of proteins. Interactions between protein residues and solvent molecules are of great importance for proteins to maintain their active structure and catalyze biochemical reactions. In this study, we investigated such interactions and studied the structural transitions of proteins during their dissolution process. Our previously developed technique, namely solvent assisted electric field induced desorption/ionization, was used for the dissolution and immediate ionization of proteins. Different solvents and proteins were involved in the investigation. According to the results, cytochrome c underwent significant unfolding during dissolution in the most commonly used NH 4 Ac buffer. The unfolding got more serious when the concentration of NH 4 Ac was further increased. Extending the dissolution time resulted in the re-folding of cytochrome c. In comparison, no unfolding was observed if cytochrome c was pre-dissolved in NH 4 Ac buffer and detected by nano-ESI. Furthermore, no unfolding was observed during the dissolution process of cytochrome c in water. Interactions between the residues of cytochrome c and the solute of NH 4 Ac might be the reason for the unfolding phenomenon. Similar unfolding phenomenon was observed on holo-myoglobin. However, the observed dissolution feature of insulin was different. No unfolding was observed on insulin during dissolution in NH 4 Ac buffers. Insulin underwent observable unfolding when water was used for dissolution. This might be due to the structural difference between different proteins. The obtained results in the present study furthered our insights into the interactions between proteins and the solvents during the phase transition of dissolution. Copyright © 2016 Elsevier B.V. All rights reserved.

High stability of a ferredoxin from the hyperthermophilic archaeon A. ambivalens: Involvement of electrostatic interactions and cofactors

PubMed Central

Moczygemba, Charmaine; Guidry, Jesse; Jones, Kathryn L.; Gomes, Cláudio M.; Teixeira, Miguel; Wittung-Stafshede, Pernilla

2001-01-01

The ferredoxin from the thermophilic archaeon Acidianus ambivalens is a small monomeric seven-iron protein with a thermal midpoint (Tm) of 122°C (pH 7). To gain insight into the basis of its thermostability, we have characterized unfolding reactions induced chemically and thermally at various pHs. Thermal unfolding of this ferredoxin, in the presence of various guanidine hydrochloride (GuHCl) concentrations, yields a linear correlation between unfolding enthalpies (ΔH[Tm]) and Tm from which an upper limit for the heat capacity of unfolding (ΔCP) was determined to be 3.15 ± 0.1 kJ/(mole • K). Only by the use of the stronger denaturant guanidine thiocyanate (GuSCN) is unfolding of A. ambivalens ferredoxin at pH 7 (20°C) observed ([GuSCN]1/2 = 3.1 M; ΔGU[H2O] = 79 ± 8 kJ/mole). The protein is, however, less stable at low pH: At pH 2.5, Tm is 64 ± 1°C, and GuHCl-induced unfolding shows a midpoint at 2.3 M (ΔGU[H2O] = 20 ± 1 kJ/mole). These results support that electrostatic interactions contribute significantly to the stability. Analysis of the three-dimensional molecular model of the protein shows that there are several possible ion pairs on the surface. In addition, ferredoxin incorporates two iron–sulfur clusters and a zinc ion that all coordinate deprotonated side chains. The zinc remains bound in the unfolded state whereas the iron–sulfur clusters transiently form linear three-iron species (in pH range 2.5 to 10), which are associated with the unfolded polypeptide, before their complete degradation. PMID:11468351

Microscopic dynamics of water around unfolded structures of barstar at room temperature

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

Pal, Somedatta; Chakraborty, Kaushik; Khatua, Prabir

2015-02-07

The breaking of the native structure of a protein and its influences on the dynamic response of the surrounding solvent is an important issue in protein folding. In this work, we have carried out atomistic molecular dynamics simulations to unfold the protein barstar at two different temperatures (400 K and 450 K). The two unfolded forms obtained at such high temperatures are further studied at room temperature to explore the effects of nonuniform unfolding of the protein secondary structures along two different pathways on the microscopic dynamical properties of the surface water molecules. It is demonstrated that though the structuralmore » transition of the protein in general results in less restricted water motions around its segments, but there are evidences of formation of new conformational motifs upon unfolding with increasingly confined environment around them, thereby resulting in further restricted water mobility in their hydration layers. Moreover, it is noticed that the effects of nonuniform unfolding of the protein segments on the relaxation times of the protein–water (PW) and the water–water (WW) hydrogen bonds are correlated with hindered hydration water motions. However, the kinetics of breaking and reformation of such hydrogen bonds are found to be influenced differently at the interface. It is observed that while the effects of unfolding on the PW hydrogen bond kinetics seem to be minimum, but the kinetics involving the WW hydrogen bonds around the protein segments exhibit noticeably heterogeneous characteristics. We believe that this is an important observation, which can provide valuable insights on the origin of heterogeneous influence of unfolding of a protein on the microscopic properties of its hydration water.« less

Unfolding single- and multilayers

NASA Astrophysics Data System (ADS)

Llorens, Maria-Gema; Bons, Paul D.; Griera, Albert; Gomez-Rivas, Enrique

2014-05-01

When planar structures (e.g. sedimentary layers, veins, dykes, cleavages, etc.) are subjected to deformation, they have about equal chances to be shortened or stretched. The most common shortening and stretching structures are folds and boudinage, respectively. However, boudinage requires additional deformation mechanisms apart from viscous flow, like formation of fractures or strain localization. When folded layers are subjected to extension, they could potentially unfold back to straight layers. Although probably not uncommon, this would be difficult to recognize. Open questions are whether folded layers can unfold, what determines their mechanical behaviour and how we can recognize them in the field. In order to approach these questions, we present a series of numerical experiments that simulate stretching of previously folded single- and multi-layers in simple shear, using the two dimensional numerical modelling platform ELLE, including the finite element module BASIL that calculates viscous deformation. We investigate the parameters that affect a fold train once it rotates into the extensional field. The results show that the unfolding process strongly depends on the viscosity contrast between the layer and matrix (Llorens et al., 2013). Layers do not completely unfold when they experience softening before or during the stretching process or when other neighbouring competent layers prevent them from unfolding. The foliation refraction patterns are the main indicators of unfolded folds. Additionally, intrafolial folds and cusp-like folds adjacent to straight layers, as well as variations in fold amplitudes and limb lengths of irregular folds can also be used as indicators of stretching of a layer after shortening and folding. References: Llorens, M-.G., Bons, P.D., Griera, A. and Gomez-Rivas, E. 2013. When do folds unfold during progressive shear?. Geology, 41, 563-566.

PGOPHER: A program for simulating rotational, vibrational and electronic spectra

NASA Astrophysics Data System (ADS)

Western, Colin M.

2017-01-01

The PGOPHER program is a general purpose program for simulating and fitting molecular spectra, particularly the rotational structure. The current version can handle linear molecules, symmetric tops and asymmetric tops and many possible transitions, both allowed and forbidden, including multiphoton and Raman spectra in addition to the common electric dipole absorptions. Many different interactions can be included in the calculation, including those arising from electron and nuclear spin, and external electric and magnetic fields. Multiple states and interactions between them can also be accounted for, limited only by available memory. Fitting of experimental data can be to line positions (in many common formats), intensities or band contours and the parameters determined can be level populations as well as rotational constants. PGOPHER is provided with a powerful and flexible graphical user interface to simplify many of the tasks required in simulating, understanding and fitting molecular spectra, including Fortrat diagrams and energy level plots in addition to overlaying experimental and simulated spectra. The program is open source, and can be compiled with open source tools. This paper provides a formal description of the operation of version 9.1.

The fluorescence intensities ratio is not a reliable parameter for evaluation of protein unfolding transitions.

PubMed

Žoldák, Gabriel; Jancura, Daniel; Sedlák, Erik

2017-06-01

Monitoring the fluorescence of proteins, particularly the fluorescence of intrinsic tryptophan residues, is a popular method often used in the analysis of unfolding transitions (induced by temperature, chemical denaturant, and pH) in proteins. The tryptophan fluorescence provides several suitable parameters, such as steady-state fluorescence intensity, apparent quantum yield, mean fluorescence lifetime, position of emission maximum that are often utilized for the observation of the conformational/unfolding transitions of proteins. In addition, the fluorescence intensities ratio at different wavelengths (usually at 330 nm and 350 nm) is becoming an increasingly popular parameter for the evaluation of thermal transitions. We show that, under certain conditions, the use of this parameter for the analysis of unfolding transitions leads to the incorrect determination of thermodynamic parameters characterizing unfolding transitions in proteins (e.g., melting temperature) and, hence, can compromise the hit identification during high-throughput drug screening campaigns. © 2017 The Protein Society.

GroEL stimulates protein folding through forced unfolding

PubMed Central

Lin, Zong; Madan, Damian; Rye, Hays S

2013-01-01

Many proteins cannot fold without the assistance of chaperonin machines like GroEL and GroES. The nature of this assistance, however, remains poorly understood. Here we demonstrate that unfolding of a substrate protein by GroEL enhances protein folding. We first show that capture of a protein on the open ring of a GroEL–ADP–GroES complex, GroEL’s physiological acceptor state for non-native proteins in vivo, leaves the substrate protein in an unexpectedly compact state. Subsequent binding of ATP to the same GroEL ring causes rapid, forced unfolding of the substrate protein. Notably, the fraction of the substrate protein that commits to the native state following GroES binding and protein release into the GroEL–GroES cavity is proportional to the extent of substrate-protein unfolding. Forced protein unfolding is thus a central component of the multilayered stimulatory mechanism used by GroEL to drive protein folding. PMID:18311152

Toward an atomistic description of the urea-denatured state of proteins.

PubMed

Candotti, Michela; Esteban-Martín, Santiago; Salvatella, Xavier; Orozco, Modesto

2013-04-09

We present here the characterization of the structural, dynamics, and energetics of properties of the urea-denatured state of ubiquitin, a small prototypical soluble protein. By combining state-of-the-art molecular dynamics simulations with NMR and small-angle X-ray scattering data, we were able to: (i) define the unfolded state ensemble, (ii) understand the energetics stabilizing unfolded structures in urea, (iii) describe the dedifferential nature of the interactions of the fully unfolded proteins with urea and water, and (iv) characterize the early stages of protein refolding when chemically denatured proteins are transferred to native conditions. The results presented herein are unique in providing a complete picture of the chemically unfolded state of proteins and contribute to deciphering the mechanisms that stabilize the native state of proteins, as well as those that maintain them unfolded in the presence of urea.

Toward an atomistic description of the urea-denatured state of proteins

PubMed Central

Candotti, Michela; Esteban-Martín, Santiago; Salvatella, Xavier; Orozco, Modesto

2013-01-01

We present here the characterization of the structural, dynamics, and energetics of properties of the urea-denatured state of ubiquitin, a small prototypical soluble protein. By combining state-of-the-art molecular dynamics simulations with NMR and small-angle X-ray scattering data, we were able to: (i) define the unfolded state ensemble, (ii) understand the energetics stabilizing unfolded structures in urea, (iii) describe the dedifferential nature of the interactions of the fully unfolded proteins with urea and water, and (iv) characterize the early stages of protein refolding when chemically denatured proteins are transferred to native conditions. The results presented herein are unique in providing a complete picture of the chemically unfolded state of proteins and contribute to deciphering the mechanisms that stabilize the native state of proteins, as well as those that maintain them unfolded in the presence of urea. PMID:23536295

Structural basis for the unfolding of anthrax lethal factor by protective antigen oligomers

PubMed Central

Feld, Geoffrey K.; Thoren, Katie L.; Kintzer, Alexander F.; Sterling, Harry J.; Tang, Iok I.; Greenberg, Shoshana G.; Williams, Evan R.; Krantz, Bryan A.

2011-01-01

The protein transporter, anthrax lethal toxin, is comprised of protective antigen (PA), a transmembrane translocase, and lethal factor (LF), a cytotoxic enzyme. Following assembly into holotoxin complexes, PA forms an oligomeric channel that unfolds LF and translocates it into the host cell. We report the crystal structure of the core of a lethal toxin complex to 3.1-Å resolution; the structure contains a PA octamer bound to four LF PA-binding domains (LFN). The first α helix and β strand of each LFN unfold and dock into a deep amphipathic cleft on the surface of the PA octamer, which we call the α clamp. The α clamp possesses nonspecific polypeptide binding activity and is functionally relevant to efficient holotoxin assembly, PA octamer formation, and LF unfolding and translocation. This structure provides insight on the mechanism of translocation-coupled protein unfolding. PMID:21037566

Signatures of unfolding in the early stages of protein denaturation

NASA Astrophysics Data System (ADS)

Gray, Harry B.; Winkler, Jay R.; Kozak, John J.

2012-04-01

A comparative study of the early stages of unfolding of five proteins: cyt c, c-b 562, cyt c‧, azurin, and lysozyme is reported. From crystallographic data, helical regions and intervening non-helical (or 'turning') regions are identified in each. Exploiting a previously introduced geometrical model, the paper describes quantitatively the stepwise extension of a polypeptide chain subject to the geometrical constraint that the spatial relationship among the residues of each triplet is fixed by native-state crystallographic data. Despite differences among the above-cited proteins, remarkable universality of behavior is found in the early stages of unfolding. At the very earliest stages, internal residues in each helical region have a common unfolding history; the terminal residues, however, are extraordinarily sensitive to structural perturbations. Residues in non-helical sections of the polypeptide unfold after residues in the internal helical regions, but with increasing steric perturbation playing a dominant role in advancing denaturation.

Development and Application of a High Throughput Protein Unfolding Kinetic Assay

PubMed Central

Wang, Qiang; Waterhouse, Nicklas; Feyijinmi, Olusegun; Dominguez, Matthew J.; Martinez, Lisa M.; Sharp, Zoey; Service, Rachel; Bothe, Jameson R.; Stollar, Elliott J.

2016-01-01

The kinetics of folding and unfolding underlie protein stability and quantification of these rates provides important insights into the folding process. Here, we present a simple high throughput protein unfolding kinetic assay using a plate reader that is applicable to the studies of the majority of 2-state folding proteins. We validate the assay by measuring kinetic unfolding data for the SH3 (Src Homology 3) domain from Actin Binding Protein 1 (AbpSH3) and its stabilized mutants. The results of our approach are in excellent agreement with published values. We further combine our kinetic assay with a plate reader equilibrium assay, to obtain indirect estimates of folding rates and use these approaches to characterize an AbpSH3-peptide hybrid. Our high throughput protein unfolding kinetic assays allow accurate screening of libraries of mutants by providing both kinetic and equilibrium measurements and provide a means for in-depth ϕ-value analyses. PMID:26745729

Expansion and internal friction in unfolded protein chain.

PubMed

Yasin, U Mahammad; Sashi, Pulikallu; Bhuyan, Abani K

2013-10-10

Similarities in global properties of homopolymers and unfolded proteins provide approaches to mechanistic description of protein folding. Here, hydrodynamic properties and relaxation rates of the unfolded state of carbonmonoxide-liganded cytochrome c (cyt-CO) have been measured using nuclear magnetic resonance and laser photolysis methods. Hydrodynamic radius of the unfolded chain gradually increases as the solvent turns increasingly better, consistent with theory. Curiously, however, the rate of intrachain contact formation also increases with an increasing denaturant concentration, which, by Szabo, Schulten, and Schulten theory for the rate of intramolecular contact formation in a Gaussian polymer, indicates growing intramolecular diffusion. It is argued that diminishing nonbonded atom interactions with increasing denaturant reduces internal friction and, thus, increases the rate of polypeptide relaxation. Qualitative scaling of the extent of unfolding with nonbonded repulsions allows for description of internal friction by a phenomenological model. The degree of nonbonded atom interactions largely determines the extent of internal friction.

Soft Vibrational Modes Predict Breaking Events during Force-Induced Protein Unfolding.

PubMed

Habibi, Mona; Plotkin, Steven S; Rottler, Jörg

2018-02-06

We investigate the correlation between soft vibrational modes and unfolding events in simulated force spectroscopy of proteins. Unfolding trajectories are obtained from molecular dynamics simulations of a Gō model of a monomer of a mutant of superoxide dismutase 1 protein containing all heavy atoms in the protein, and a normal mode analysis is performed based on the anisotropic network model. We show that a softness map constructed from the superposition of the amplitudes of localized soft modes correlates with unfolding events at different stages of the unfolding process. Soft residues are up to eight times more likely to undergo disruption of native structure than the average amino acid. The memory of the softness map is retained for extensions of up to several nanometers, but decorrelates more rapidly during force drops. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

KSC-99pp1244

NASA Image and Video Library

1999-10-22

KSC volunteers for Days of Caring '99 unfold protective materials before getting ready to paint at Baxley Manor, an apartment building for senior citizens on Merritt Island. Coordinated by the KSC Community Relations Council, Days of Caring provides an opportunity for employees to volunteer their services in projects such as painting, planting flowers, reading to school children, and more. Organizations accepting volunteers include The Embers, Yellow Umbrella, Serene Harbor, Domestic Violence Program, the YMCA of Brevard County, and others

Unfolding mechanism of thrombin-binding aptamer revealed by molecular dynamics simulation and Markov State Model

NASA Astrophysics Data System (ADS)

Zeng, Xiaojun; Zhang, Liyun; Xiao, Xiuchan; Jiang, Yuanyuan; Guo, Yanzhi; Yu, Xinyan; Pu, Xuemei; Li, Menglong

2016-04-01

Thrombin-binding aptamer (TBA) with the sequence 5‧GGTTGGTGTGGTTGG3‧ could fold into G-quadruplex, which correlates with functionally important genomic regionsis. However, unfolding mechanism involved in the structural stability of G-quadruplex has not been satisfactorily elucidated on experiments so far. Herein, we studied the unfolding pathway of TBA by a combination of molecular dynamics simulation (MD) and Markov State Model (MSM). Our results revealed that the unfolding of TBA is not a simple two-state process but proceeds along multiple pathways with multistate intermediates. One high flux confirms some observations from NMR experiment. Another high flux exhibits a different and simpler unfolding pathway with less intermediates. Two important intermediate states were identified. One is similar to the G-triplex reported in the folding of G-quadruplex, but lack of H-bonding between guanines in the upper plane. More importantly, another intermediate state acting as a connector to link the folding region and the unfolding one, was the first time identified, which exhibits higher population and stability than the G-triplex-like intermediate. These results will provide valuable information for extending our understanding the folding landscape of G-quadruplex formation.

Characterization of the Protein Unfolding Processes Induced by Urea and Temperature

PubMed Central

Rocco, Alessandro Guerini; Mollica, Luca; Ricchiuto, Piero; Baptista, António M.; Gianazza, Elisabetta; Eberini, Ivano

2008-01-01

Correct folding is critical for the biological activities of proteins. As a contribution to a better understanding of the protein (un)folding problem, we studied the effect of temperature and of urea on peptostreptococcal Protein L destructuration. We performed standard molecular dynamics simulations at 300 K, 350 K, 400 K, and 480 K, both in 10 M urea and in water. Protein L followed at least two alternative unfolding pathways. Urea caused the loss of secondary structure acting preferentially on the β-sheets, while leaving the α-helices almost intact; on the contrary, high temperature preserved the β-sheets and led to a complete loss of the α-helices. These data suggest that urea and high temperature act through different unfolding mechanisms, and protein secondary motives reveal a differential sensitivity to various denaturant treatments. As further validation of our results, replica-exchange molecular dynamics simulations of the temperature-induced unfolding process in the presence of urea were performed. This set of simulations allowed us to compute the thermodynamical parameters of the process and confirmed that, in the configurational space of Protein L unfolding, both of the above pathways are accessible, although to a different relative extent. PMID:18065481

The Role of Binding Site on the Mechanical Unfolding Mechanism of Ubiquitin

NASA Astrophysics Data System (ADS)

Cao, Penghui; Yoon, Gwonchan; Tao, Weiwei; Eom, Kilho; Park, Harold S.

2015-03-01

We apply novel atomistic simulations based on potential energy surface exploration to investigate the constant force-induced unfolding of ubiquitin. At the experimentally-studied force clamping level of 100 pN, we find a new unfolding mechanism starting with the detachment between β5 and β3 involving the binding site of ubiquitin, the Ile44 residue. This new unfolding pathway leads to the discovery of new intermediate configurations, which correspond to the end-to-end extensions previously seen experimentally. More importantly, it demonstrates the novel finding that the binding site of ubiquitin can be responsible not only for its biological functions, but also its unfolding dynamics. We also report in contrast to previous single molecule constant force experiments that when the clamping force becomes smaller than about 300 pN, the number of intermediate configurations increases dramatically, where almost all unfolding events at 100 pN involve an intermediate configuration. By directly calculating the life times of the intermediate configurations from the height of the barriers that were crossed on the potential energy surface, we demonstrate that these intermediate states were likely not observed experimentally due to their lifetimes typically being about two orders of magnitude smaller than the experimental temporal resolution.

Assessment of cardiac time intervals using high temporal resolution real-time spiral phase contrast with UNFOLDed-SENSE.

PubMed

Kowalik, Grzegorz T; Knight, Daniel S; Steeden, Jennifer A; Tann, Oliver; Odille, Freddy; Atkinson, David; Taylor, Andrew; Muthurangu, Vivek

2015-02-01

To develop a real-time phase contrast MR sequence with high enough temporal resolution to assess cardiac time intervals. The sequence utilized spiral trajectories with an acquisition strategy that allowed a combination of temporal encoding (Unaliasing by fourier-encoding the overlaps using the temporal dimension; UNFOLD) and parallel imaging (Sensitivity encoding; SENSE) to be used (UNFOLDed-SENSE). An in silico experiment was performed to determine the optimum UNFOLD filter. In vitro experiments were carried out to validate the accuracy of time intervals calculation and peak mean velocity quantification. In addition, 15 healthy volunteers were imaged with the new sequence, and cardiac time intervals were compared to reference standard Doppler echocardiography measures. For comparison, in silico, in vitro, and in vivo experiments were also carried out using sliding window reconstructions. The in vitro experiments demonstrated good agreement between real-time spiral UNFOLDed-SENSE phase contrast MR and the reference standard measurements of velocity and time intervals. The protocol was successfully performed in all volunteers. Subsequent measurement of time intervals produced values in keeping with literature values and good agreement with the gold standard echocardiography. Importantly, the proposed UNFOLDed-SENSE sequence outperformed the sliding window reconstructions. Cardiac time intervals can be successfully assessed with UNFOLDed-SENSE real-time spiral phase contrast. Real-time MR assessment of cardiac time intervals may be beneficial in assessment of patients with cardiac conditions such as diastolic dysfunction. © 2014 Wiley Periodicals, Inc.

Declining global warming effects on the phenology of spring leaf unfolding.

PubMed

Fu, Yongshuo H; Zhao, Hongfang; Piao, Shilong; Peaucelle, Marc; Peng, Shushi; Zhou, Guiyun; Ciais, Philippe; Huang, Mengtian; Menzel, Annette; Peñuelas, Josep; Song, Yang; Vitasse, Yann; Zeng, Zhenzhong; Janssens, Ivan A

2015-10-01

Earlier spring leaf unfolding is a frequently observed response of plants to climate warming. Many deciduous tree species require chilling for dormancy release, and warming-related reductions in chilling may counteract the advance of leaf unfolding in response to warming. Empirical evidence for this, however, is limited to saplings or twigs in climate-controlled chambers. Using long-term in situ observations of leaf unfolding for seven dominant European tree species at 1,245 sites, here we show that the apparent response of leaf unfolding to climate warming (ST, expressed in days advance of leaf unfolding per °C warming) has significantly decreased from 1980 to 2013 in all monitored tree species. Averaged across all species and sites, ST decreased by 40% from 4.0 ± 1.8 days °C(-1) during 1980-1994 to 2.3 ± 1.6 days °C(-1) during 1999-2013. The declining ST was also simulated by chilling-based phenology models, albeit with a weaker decline (24-30%) than observed in situ. The reduction in ST is likely to be partly attributable to reduced chilling. Nonetheless, other mechanisms may also have a role, such as 'photoperiod limitation' mechanisms that may become ultimately limiting when leaf unfolding dates occur too early in the season. Our results provide empirical evidence for a declining ST, but also suggest that the predicted strong winter warming in the future may further reduce ST and therefore result in a slowdown in the advance of tree spring phenology.

Investigations on landmine detection by neutron-based techniques.

PubMed

Csikai, J; Dóczi, R; Király, B

2004-07-01

Principles and techniques of some neutron-based methods used to identify the antipersonnel landmines (APMs) are discussed. New results have been achieved in the field of neutron reflection, transmission, scattering and reaction techniques. Some conclusions are as follows: The neutron hand-held detector is suitable for the observation of anomaly caused by a DLM2-like sample in different soils with a scanning speed of 1m(2)/1.5 min; the reflection cross section of thermal neutrons rendered the determination of equivalent thickness of different soil components possible; a simple method was developed for the determination of the thermal neutron flux perturbation factor needed for multi-elemental analysis of bulky samples; unfolded spectra of elastically backscattered neutrons using broad-spectrum sources render the identification of APMs possible; the knowledge of leakage spectra of different source neutrons is indispensable for the determination of the differential and integrated reaction rates and through it the dimension of the interrogated volume; the precise determination of the C/O atom fraction requires the investigations on the angular distribution of the 6.13MeV gamma-ray emitted in the (16)O(n,n'gamma) reaction. These results, in addition to the identification of landmines, render the improvement of the non-intrusive neutron methods possible.

Effects of ultrasound and ultrasound assisted alkaline pretreatments on the enzymolysis and structural characteristics of rice protein.

PubMed

Li, Suyun; Yang, Xue; Zhang, Yanyan; Ma, Haile; Liang, Qiufang; Qu, Wenjuan; He, Ronghai; Zhou, Cunshan; Mahunu, Gustav Komla

2016-07-01

The objectives of this study were to investigate the effects of multi-frequency energy-gathered ultrasound (MFEGU) and MFEGU assisted alkaline pretreatments on the enzymolysis and the mechanism of two pretreatments accelerating the rice protein (RP) proteolysis process. The results showed that MFEGU and MFEGU assisted alkaline pretreatments improved significantly (P<0.05) the degree of hydrolysis (DH) and the protein elution amount of RP. Furthermore under the same DH conditions, ultrasound and ultrasound assisted alkaline pretreatments were more save the enzymolysis time than the unpretreatment. The changes in UV-vis spectra, fluorescence emission spectra indicated unfolding and destruction of RP by MFEGU and MFEGU assisted alkaline pretreatments. The circular dichroism analysis showed that both pretreatments decreased α-helix but increased β-sheet and random coil of RP. Amino acid composition revealed that MFEGU and MFEGU assisted alkaline pretreatments could increase the protein elution amount and the ratio of hydrophobic amino acids. Atomic force microscopy (AFM) indicated that both pretreatments destroyed the microstructures and reduced the particle size of RP. Therefore, MFEGU and MFEGU assisted alkaline pretreatments are beneficial to improving the degree of hydrolysis due to its sonochemistry effect on the molecular conformation as well as on the microstructure of protein. Copyright © 2015 Elsevier B.V. All rights reserved.

Effects of multi-frequency power ultrasound on the enzymolysis and structural characteristics of corn gluten meal.

PubMed

Jin, Jian; Ma, Haile; Wang, Kai; Yagoub, Abu El-Gasim A; Owusu, John; Qu, Wenjuan; He, Ronghai; Zhou, Cunshan; Ye, Xiaofei

2015-05-01

The aim of this study was to investigate the effect of multi-frequency power ultrasound (sweeping frequency and pulsed ultrasound (SFPU) and sequential dual frequency ultrasound (SDFU)) on the enzymolysis of corn gluten meal (CGM) and on the structures of the major protein fractions (zein, glutelin) of CGM. The results showed that multi-frequency power ultrasound pretreatments improved significantly (P<0.05) the degree of hydrolysis and conversion rate of CGM. The changes in UV-Vis spectra, fluorescence emission spectra, surface hydrophobicity (H0), and the content of SH and SS groups indicated unfolding of zein and glutelin by ultrasound. The circular dichroism analysis showed that both pretreatments decreased α-helix and increased β-sheet of glutelin. The SFPU pretreatment had little impact on the secondary structure of zein, while the SDFU increased the α-helix and decreased the β-sheet remarkably. Scanning electron microscope indicated that both pretreatments destroyed the microstructures of glutelin and CGM, reduced the particle size of zein despite that the SDFU induced aggregation was observed. In conclusion, multi-frequency power ultrasound pretreatment is an efficient method in protein proteolysis due to its sonochemistry effect on the molecular conformation as well as on the microstructure of protein. Copyright © 2014 Elsevier B.V. All rights reserved.

Development and Characterization of a High Sensitivity Segmented Fast Neutron Spectrometer (FaNS-2)

PubMed Central

Langford, T.J.; Beise, E.J.; Breuer, H.; Heimbach, C.R.; Ji, G.; Nico, J.S.

2016-01-01

We present the development of a segmented fast neutron spectrometer (FaNS-2) based upon plastic scintillator and 3He proportional counters. It was designed to measure both the flux and spectrum of fast neutrons in the energy range of few MeV to 1 GeV. FaNS-2 utilizes capture-gated spectroscopy to identify neutron events and reject backgrounds. Neutrons deposit energy in the plastic scintillator before capturing on a 3He nucleus in the proportional counters. Segmentation improves neutron energy reconstruction while the large volume of scintillator increases sensitivity to low neutron fluxes. A main goal of its design is to study comparatively low neutron fluxes, such as cosmogenic neutrons at the Earth's surface, in an underground environment, or from low-activity neutron sources. In this paper, we present details of its design and construction as well as its characterization with a calibrated 252Cf source and monoenergetic neutron fields of 2.5 MeV and 14 MeV. Detected monoenergetic neutron spectra are unfolded using a Singular Value Decomposition method, demonstrating a 5% energy resolution at 14 MeV. Finally, we discuss plans for measuring the surface and underground cosmogenic neutron spectra with FaNS-2. PMID:27226807

Structural origins of pH and ionic strength effects on protein stability. Acid denaturation of sperm whale apomyoglobin.

PubMed

Yang, A S; Honig, B

1994-04-15

A recently developed approach to calculate the pH dependence of protein stability from three-dimensional structure information is applied to the analysis of acid denaturation of sperm whale apomyoglobin. The finite difference Poisson-Boltzmann method is used to calculate pKa values and these are used to obtain titration curves for the folded protein as well as for compact intermediates. The total electrostatic free energy change involved in apomyoglobin unfolding is then evaluated. Calculations are carried out of the unfolding free energy of the native (N) and the compact intermediate (I) of apomyoglobin relative to the unfolded state (U) over a range of pH at various ionic strengths. The contributions from key ionizable groups to the unfolding process are discussed. For the acid-induced partial unfolding of apomyoglobin near pH 5, the transition from N to I is found to be driven by three histidines that are exposed when the B, C, D and E helices unfold. Similarly, the unfolding of the compact intermediate I consisting of the A, G and H helices is driven primarily by a few carboxylic acids with low pKa values in the compact state. This picture is in contrast to the view which attributes acid denaturation to electrostatic repulsion resulting from the build up of positive charge. In fact, charge-charge interactions in myoglobin are found to be attractive at all pH values where the protein unfolds. pH-dependent changes in these interactions contribute to acid denaturation but other electrostatic effects, such as hydrogen bonding and solvation, are important as well. The effect of increasing ionic strength on unfolding is attributed to the decrease of attractive charge-charge interactions which destabilize the N state relative to I, but stabilize the I state relative to U by reducing the pKa shifts of a few critical carboxylic acids. The I state is found to be more stable than U at neutral pH thus accounting for its presence as an intermediate on the protein folding pathway. Our results have implications for the origins of compact intermediates or "molten globule" states.

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

Characterization of extended range Bonner Sphere Spectrometers in the CERF high-energy broad neutron field at CERN

NASA Astrophysics Data System (ADS)

Agosteo, S.; Bedogni, R.; Caresana, M.; Charitonidis, N.; Chiti, M.; Esposito, A.; Ferrarini, M.; Severino, C.; Silari, M.

2012-12-01

The accurate determination of the ambient dose equivalent in the mixed neutron-photon fields encountered around high-energy particle accelerators still represents a challenging task. The main complexity arises from the extreme variability of the neutron energy, which spans over 10 orders of magnitude or more. Operational survey instruments, which response function attempts to mimic the fluence-to-ambient dose equivalent conversion coefficient up to GeV neutrons, are available on the market, but their response is not fully reliable over the entire energy range. Extended range rem counters (ERRC) do not require the exact knowledge of the energy distribution of the neutron field and the calibration can be done with a source spectrum. If the actual neutron field has an energy distribution different from the calibration spectrum, the measurement is affected by an added uncertainty related to the partial overlap of the fluence-to-ambient dose equivalent conversion curve and the response function. For this reason their operational use should always be preceded by an "in-field" calibration, i.e. a calibration made against a reference instrument exposed in the same field where the survey-meter will be employed. In practice the extended-range Bonner Sphere Spectrometer (ERBSS) is the only device which can serve as reference instrument in these fields, because of its wide energy range and the possibility to assess the neutron fluence and the ambient dose equivalent (H*(10)) values with the appropriate accuracy. Nevertheless, the experience gained by a number of experimental groups suggests that mandatory conditions for obtaining accurate results in workplaces are: (1) the use of a well-established response matrix, thus implying validation campaigns in reference monochromatic neutrons fields, (2) the expert and critical use of suitable unfolding codes, and (3) the performance test of the whole system (experimental set-up, elaboration and unfolding procedures) in a well controlled workplace field. The CERF (CERN-EU high-energy reference field) facility is a unique example of such a field, where a number of experimental campaigns and Monte Carlo simulations have been performed over the past years. With the aim of performing this kind of workplace performance test, four different ERBSS with different degrees of validation, operated by three groups (CERN, INFN-LNF and Politecnico of Milano), were exposed in two fixed positions at CERF. Using different unfolding codes (MAXED, GRAVEL, FRUIT and FRUIT SGM), the experimental data were analyzed to provide the neutron spectra and the related dosimetric quantities. The results allow assessing the overall performance of each ERBSS and of the unfolding codes, as well as comparing the performance of three ERRCs when used in a neutron field with energy distribution different from the calibration spectrum.

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

Norton, R.S.; Cossins, A.I.; Kem, W.R.

The solution properties of the polypeptide neurotoxin I from the sea anemone Stichodactyla helianthus (Sh I) have been investigated by high-resolution H nuclear magnetic resonance (NMR) spectroscopy at 300 MHz. The pH dependence of the spectra has been examined over the range 1.1-12.2 at 27{degree}C. Individual pK{sub a} values have been obtained for the {alpha}-ammonium group of Ala-1 (8.6) and the side chains of Glu-8 (3.7), Tyr-36 (10.9), and Tyr-37 (10.8). For the remaining seven carboxyl groups in the molecule, four pK{sub a} values can be clearly identified. The five Lys residues titrate in the range 10.5-11, but individual pK{submore » a} values could not be obtained because of peak overlap. Conformational changes associated with the protonation of carboxylates occur below pH 4, while in the alkaline pH range major unfolding occurs above pH 10. The molecule also unfolds at elevated temperatures. Exchange of the backbone amide protons has been monitored at various values of pH and temperature in the ranges pH 4-5 and 12-27{degree}C. Comparison of these properties of Sh I in solution with those of the related polypeptides anthopleurin A and Anemonia sulcata toxins I and II indicates that Sh I is less stable thermally and that there are some significant differences in the ionic interactions that maintain the tertiary structure. The solvent accessibility of aromatic residues has been probed with photochemically induced dynamic nuclear polarization NMR at 360 MHz.« less

Application of principal component analysis in protein unfolding: an all-atom molecular dynamics simulation study.

PubMed

Das, Atanu; Mukhopadhyay, Chaitali

2007-10-28

We have performed molecular dynamics (MD) simulation of the thermal denaturation of one protein and one peptide-ubiquitin and melittin. To identify the correlation in dynamics among various secondary structural fragments and also the individual contribution of different residues towards thermal unfolding, principal component analysis method was applied in order to give a new insight to protein dynamics by analyzing the contribution of coefficients of principal components. The cross-correlation matrix obtained from MD simulation trajectory provided important information regarding the anisotropy of backbone dynamics that leads to unfolding. Unfolding of ubiquitin was found to be a three-state process, while that of melittin, though smaller and mostly helical, is more complicated.

Application of principal component analysis in protein unfolding: An all-atom molecular dynamics simulation study

NASA Astrophysics Data System (ADS)

Das, Atanu; Mukhopadhyay, Chaitali

2007-10-01

We have performed molecular dynamics (MD) simulation of the thermal denaturation of one protein and one peptide—ubiquitin and melittin. To identify the correlation in dynamics among various secondary structural fragments and also the individual contribution of different residues towards thermal unfolding, principal component analysis method was applied in order to give a new insight to protein dynamics by analyzing the contribution of coefficients of principal components. The cross-correlation matrix obtained from MD simulation trajectory provided important information regarding the anisotropy of backbone dynamics that leads to unfolding. Unfolding of ubiquitin was found to be a three-state process, while that of melittin, though smaller and mostly helical, is more complicated.

Odijk excluded volume interactions during the unfolding of DNA confined in a nanochannel.

PubMed

Reifenberger, Jeffrey G; Cao, Han; Dorfman, Kevin D

2018-02-13

We report experimental data on the unfolding of human and E. coli genomic DNA molecules shortly after injection into a 45 nm nanochannel. The unfolding dynamics are deterministic, consistent with previous experiments and modeling in larger channels, and do not depend on the biological origin of the DNA. The measured entropic unfolding force per friction per unit contour length agrees with that predicted by combining the Odijk excluded volume with numerical calculations of the Kirkwood diffusivity of confined DNA. The time scale emerging from our analysis has implications for genome mapping in nanochannels, especially as the technology moves towards longer DNA, by setting a lower bound for the delay time before making a measurement.

Exploiting oleuropein for inhibiting collagen fibril formation.

PubMed

Bharathy, H; Fathima, N Nishad

2017-08-01

Collagen fibrils accumulate in excessive amounts and impair the normal functioning of the organ; therefore it stimulates the interest for identifying the compounds that could prevent the formation of fibrils. Herein, inhibition of self-assembly of collagen using oleuropein has been studied. The changes in the physico-chemical characteristics of collagen on interaction with increasing concentration of oleuropein has been studied using techniques like viscosity, UV-vis, CD and FT-IR. The inhibitory effect of oleuropein on fibril formation of collagen was proved using SEM. Circular dichroism and FT-IR spectra elucidates the alterations in the secondary structure of collagen suggesting non-covalent interactions between oleuropein and collagen. The decreased rate of collagen fibril formation also confirms the inhibition in the self-assembly of collagen. Hence, our study suggests that inhibition of the self-assembly process using oleuropein may unfold new avenues to treat fibrotic diseases. Copyright © 2017 Elsevier B.V. All rights reserved.

Conformational rearrangements in the transmembrane domain of CNGA1 channels revealed by single-molecule force spectroscopy

NASA Astrophysics Data System (ADS)

Maity, Sourav; Mazzolini, Monica; Arcangeletti, Manuel; Valbuena, Alejandro; Fabris, Paolo; Lazzarino, Marco; Torre, Vincent

2015-05-01

Cyclic nucleotide-gated (CNG) channels are activated by binding of cyclic nucleotides. Although structural studies have identified the channel pore and selectivity filter, conformation changes associated with gating remain poorly understood. Here we combine single-molecule force spectroscopy (SMFS) with mutagenesis, bioinformatics and electrophysiology to study conformational changes associated with gating. By expressing functional channels with SMFS fingerprints in Xenopus laevis oocytes, we were able to investigate gating of CNGA1 in a physiological-like membrane. Force spectra determined that the S4 transmembrane domain is mechanically coupled to S5 in the closed state, but S3 in the open state. We also show there are multiple pathways for the unfolding of the transmembrane domains, probably caused by a different degree of α-helix folding. This approach demonstrates that CNG transmembrane domains have dynamic structure and establishes SMFS as a tool for probing conformational change in ion channels.

Single-molecule protein unfolding and translocation by an ATP-fueled proteolytic machine

PubMed Central

Aubin-Tam, Marie-Eve; Olivares, Adrian O.; Sauer, Robert T.; Baker, Tania A.; Lang, Matthew J.

2011-01-01

All cells employ ATP-powered proteases for protein-quality control and regulation. In the ClpXP protease, ClpX is a AAA+ machine that recognizes specific protein substrates, unfolds these molecules, and then translocates the denatured polypeptide through a central pore and into ClpP for degradation. Here, we use optical-trapping nanometry to probe the mechanics of enzymatic unfolding and translocation of single molecules of a multidomain substrate. Our experiments demonstrate the capacity of ClpXP and ClpX to perform mechanical work under load, reveal very fast and highly cooperative unfolding of individual substrate domains, suggest a translocation step size of 5–8 amino acids, and support a power-stroke model of denaturation in which successful enzyme-mediated unfolding of stable domains requires coincidence between mechanical pulling by the enzyme and a transient stochastic reduction in protein stability. We anticipate that single-molecule studies of the mechanical properties of other AAA+ proteolytic machines will reveal many shared features with ClpXP. PMID:21496645

Estimating Parameters in the Generalized Graded Unfolding Model: Sensitivity to the Prior Distribution Assumption and the Number of Quadrature Points Used.

ERIC Educational Resources Information Center

Roberts, James S.; Donoghue, John R.; Laughlin, James E.

The generalized graded unfolding model (J. Roberts, J. Donoghue, and J. Laughlin, 1998, 1999) is an item response theory model designed to unfold polytomous responses. The model is based on a proximity relation that postulates higher levels of expected agreement with a given statement to the extent that a respondent is located close to the…

Mechanisms of m-cresol induced protein aggregation studied using a model protein cytochrome c†

PubMed Central

Singh, Surinder M.; Hutchings, Regina L.; Mallela, Krishna M.G.

2014-01-01

Multi-dose protein formulations require an effective antimicrobial preservative (AP) to inhibit microbial growth during long-term storage of unused formulations. m-cresol is one such AP, but has been shown to cause protein aggregation. However, the fundamental physical mechanisms underlying such AP-induced protein aggregation are not understood. In this study, we used a model protein cytochrome c to identify the protein unfolding that triggers protein aggregation. m-cresol induced cytochrome c aggregation at preservative concentrations that are commonly used to inhibit microbial growth. Addition of m-cresol decreased the temperature at which the protein aggregated and increased the aggregation rate. However, m-cresol did not perturb the tertiary or secondary structure of cytochrome c. Instead, it populated an “invisible” partially unfolded intermediate where a local protein region around the methionine residue at position 80 was unfolded. Stabilizing the Met80 region drastically decreased the protein aggregation, which conclusively shows that this local protein region acts as an aggregation “hot-spot”. Based on these results, we propose that APs induce protein aggregation by partial rather than global unfolding. Because of the availability of site-specific probes to monitor different levels of protein unfolding, cytochrome c provided a unique advantage in characterizing the partial protein unfolding that triggers protein aggregation. PMID:21229618

Temperature-induced unfolding of epidermal growth factor (EGF): insight from molecular dynamics simulation

PubMed Central

Yan, Chunli; Pattani, Varun; Tunnell, James W.; Ren, Pengyu

2010-01-01

Thermal disruption of protein structure and function is a potentially powerful therapeutic vehicle. With the emerging nanoparticle-targeting and femtosecond laser technology, it is possible to deliver heating locally to specific molecules. It is therefore important to understand how fast a protein can unfold or lose its function at high temperatures, such as near the water boiling point. In this study, the thermal damage of EGF was investigated by combining the replica exchange (136 replicas) and conventional molecular dynamics simulations. The REMD simulation was employed to rigorously explore the free energy landscape of EGF unfolding. Interestingly, besides the native and unfolded states, we also observed a distinct molten globule (MG) state that retained substantial amount of native contacts. Based on the understanding that which the unfolding of EGF is a three-state process, we have examined the unfolding kinetics of EGF (N→ MG→h multiple 20-ns conventional MD simulations. The Arrhenius prefactors and activation energy barriers determined from the simulation are within the range of previously studied proteins. In contrast to the thermal damage of cells and tissues which take place on the time scale of seconds to hours at relatively low temperatures, the denaturation of proteins occur in nanoseconds when the temperature of heat bath approaches the boiling point. PMID:20466569

Microsecond Unfolding Kinetics of Sheep Prion Protein Reveals an Intermediate that Correlates with Susceptibility to Classical Scrapie

PubMed Central

Chen, Kai-Chun; Xu, Ming; Wedemeyer, William J.; Roder, Heinrich

2011-01-01

The microsecond folding and unfolding kinetics of ovine prion proteins (ovPrP) were measured under various solution conditions. A fragment comprising residues 94–233 of the full-length ovPrP was studied for four variants with differing susceptibilities to classical scrapie in sheep. The observed biexponential unfolding kinetics of ovPrP provides evidence for an intermediate species. However, in contrast to previous results for human PrP, there is no evidence for an intermediate under refolding conditions. Global analysis of the kinetic data, based on a sequential three-state mechanism, quantitatively accounts for all folding and unfolding data as a function of denaturant concentration. The simulations predict that an intermediate accumulates under both folding and unfolding conditions, but is observable only in unfolding experiments because the intermediate is optically indistinguishable from the native state. The relative population of intermediates in two ovPrP variants, both transiently and under destabilizing equilibrium conditions, correlates with their propensities for classical scrapie. The variant susceptible to classical scrapie has a larger population of the intermediate state than the resistant variant. Thus, the susceptible variant should be favored to undergo the PrPC to PrPSc conversion and oligomerization. PMID:21889460

Computer Series, 36: Bits and Pieces, 13.

ERIC Educational Resources Information Center

Moore, John W.

1983-01-01

Eleven computer/calculator programs (most are available from authors) are described. Topics include visualizing molecular vibrations, dynamic nuclear magnetic resonance spectra of two-spin systems, programming utilities for Apple II Plus, gas chromatography simulation for TRS-80, infrared spectra analysis on a calculator, naming chemical…

A hypothesis to reconcile the physical and chemical unfolding of proteins

PubMed Central

de Oliveira, Guilherme A. P.; Silva, Jerson L.

2015-01-01

High pressure (HP) or urea is commonly used to disturb folding species. Pressure favors the reversible unfolding of proteins by causing changes in the volumetric properties of the protein–solvent system. However, no mechanistic model has fully elucidated the effects of urea on structure unfolding, even though protein–urea interactions are considered to be crucial. Here, we provide NMR spectroscopy and 3D reconstructions from X-ray scattering to develop the “push-and-pull” hypothesis, which helps to explain the initial mechanism of chemical unfolding in light of the physical events triggered by HP. In studying MpNep2 from Moniliophthora perniciosa, we tracked two cooperative units using HP-NMR as MpNep2 moved uphill in the energy landscape; this process contrasts with the overall structural unfolding that occurs upon reaching a threshold concentration of urea. At subdenaturing concentrations of urea, we were able to trap a state in which urea is preferentially bound to the protein (as determined by NMR intensities and chemical shifts); this state is still folded and not additionally exposed to solvent [fluorescence and small-angle X-ray scattering (SAXS)]. This state has a higher susceptibility to pressure denaturation (lower p1/2 and larger ΔVu); thus, urea and HP share concomitant effects of urea binding and pulling and water-inducing pushing, respectively. These observations explain the differences between the molecular mechanisms that control the physical and chemical unfolding of proteins, thus opening up new possibilities for the study of protein folding and providing an interpretation of the nature of cooperativity in the folding and unfolding processes. PMID:25964355

Thermal unfolding of tetrameric melittin: comparison with the molten globule state of cytochrome c.

PubMed Central

Hagihara, Y.; Oobatake, M.; Goto, Y.

1994-01-01

Whereas melittin at micromolar concentrations is unfolded under conditions of low salt at neutral pH, it transforms to a tetrameric alpha-helical structure under several conditions, such as high peptide concentration, high anion concentration, or alkaline pH. The anion- and pH-dependent stabilization of the tetrameric structure is similar to that of the molten globule state of several acid-denatured proteins, suggesting that tetrameric melittin might be a state similar to the molten globule state. To test this possibility, we studied the thermal unfolding of tetrameric melittin using far-UV CD and differential scanning calorimetry. The latter technique revealed a broad but distinct heat absorption peak. The heat absorption curves were consistent with the unfolding transition observed by CD and were explainable by a 2-state transition mechanism between the tetrameric alpha-helical state and the monomeric unfolded state. From the peptide or salt-concentration dependence of unfolding, the heat capacity change upon unfolding was estimated to be 5 kJ (mol of tetramer)-1 K-1 at 30 degrees C and decreased with increasing temperature. The observed change in heat capacity was much smaller than that predicted from the crystallographic structure (9.2 kJ (mol of tetramer)-1 K-1), suggesting that the hydrophobic residues of tetrameric melittin in solution are exposed in comparison with the crystallographic structure. However, the results also indicate that the structure is more ordered than that of a typical molten globule state. We consider that the conformation is intermediate between the molten globule state and the native state of globular proteins. PMID:7833804

Bench to Bedside: Understanding Symptom Response to Acupuncture Treatment and Designing a Successful Acupuncture Treatment Program

DTIC Science & Technology

2017-09-01

analysis. Along the way we learned that the Chinese Medicine information will be difficult to integrate into the main data set. At this point only a poster... psychological health, social context, physical function. We completed and submitted for review a simpler analysis of this relationship in The Importance and...the unfolding integration of complementary medical practices into regular medicine. It will assist acupuncturists and VA officials in determining if

Acoustic Multipurpose Cargo Transfer Bag

NASA Technical Reports Server (NTRS)

Baccus, Shelley

2015-01-01

The Logistics Reduction (LR) project within the Advanced Exploration Systems (AES) program is tasked with reducing logistical mass and repurposing logistical items. Multipurpose Cargo Transfer Bags (MCTB) are designed to be the same external volume as a regular cargo transfer bag, the common logistics carrier for the International Space Station. After use as a cargo bag, the MCTB can be unzipped and unfolded to be reused. This Acoustic MCTBs transform into acoustic blankets after the initial logistics carrying objective is complete.

Dynameomics: design of a computational lab workflow and scientific data repository for protein simulations.

PubMed

Simms, Andrew M; Toofanny, Rudesh D; Kehl, Catherine; Benson, Noah C; Daggett, Valerie

2008-06-01

Dynameomics is a project to investigate and catalog the native-state dynamics and thermal unfolding pathways of representatives of all protein folds using solvated molecular dynamics simulations, as described in the preceding paper. Here we introduce the design of the molecular dynamics data warehouse, a scalable, reliable repository that houses simulation data that vastly simplifies management and access. In the succeeding paper, we describe the development of a complementary multidimensional database. A single protein unfolding or native-state simulation can take weeks to months to complete, and produces gigabytes of coordinate and analysis data. Mining information from over 3000 completed simulations is complicated and time-consuming. Even the simplest queries involve writing intricate programs that must be built from low-level file system access primitives and include significant logic to correctly locate and parse data of interest. As a result, programs to answer questions that require data from hundreds of simulations are very difficult to write. Thus, organization and access to simulation data have been major obstacles to the discovery of new knowledge in the Dynameomics project. This repository is used internally and is the foundation of the Dynameomics portal site http://www.dynameomics.org. By organizing simulation data into a scalable, manageable and accessible form, we can begin to address substantial questions that move us closer to solving biomedical and bioengineering problems.

A neutron spectrum unfolding computer code based on artificial neural networks

NASA Astrophysics Data System (ADS)

Ortiz-Rodríguez, J. M.; Reyes Alfaro, A.; Reyes Haro, A.; Cervantes Viramontes, J. M.; Vega-Carrillo, H. R.

2014-02-01

The Bonner Spheres Spectrometer consists of a thermal neutron sensor placed at the center of a number of moderating polyethylene spheres of different diameters. From the measured readings, information can be derived about the spectrum of the neutron field where measurements were made. Disadvantages of the Bonner system are the weight associated with each sphere and the need to sequentially irradiate the spheres, requiring long exposure periods. Provided a well-established response matrix and adequate irradiation conditions, the most delicate part of neutron spectrometry, is the unfolding process. The derivation of the spectral information is not simple because the unknown is not given directly as a result of the measurements. The drawbacks associated with traditional unfolding procedures have motivated the need of complementary approaches. Novel methods based on Artificial Intelligence, mainly Artificial Neural Networks, have been widely investigated. In this work, a neutron spectrum unfolding code based on neural nets technology is presented. This code is called Neutron Spectrometry and Dosimetry with Artificial Neural networks unfolding code that was designed in a graphical interface. The core of the code is an embedded neural network architecture previously optimized using the robust design of artificial neural networks methodology. The main features of the code are: easy to use, friendly and intuitive to the user. This code was designed for a Bonner Sphere System based on a 6LiI(Eu) neutron detector and a response matrix expressed in 60 energy bins taken from an International Atomic Energy Agency compilation. The main feature of the code is that as entrance data, for unfolding the neutron spectrum, only seven rate counts measured with seven Bonner spheres are required; simultaneously the code calculates 15 dosimetric quantities as well as the total flux for radiation protection purposes. This code generates a full report with all information of the unfolding in the HTML format. NSDann unfolding code is freely available, upon request to the authors.

Spreadsheet-Based Program for Simulating Atomic Emission Spectra

ERIC Educational Resources Information Center

Flannigan, David J.

2014-01-01

A simple Excel spreadsheet-based program for simulating atomic emission spectra from the properties of neutral atoms (e.g., energies and statistical weights of the electronic states, electronic partition functions, transition probabilities, etc.) is described. The contents of the spreadsheet (i.e., input parameters, formulas for calculating…

Use of mutation spectra analysis software.

PubMed

Rogozin, I; Kondrashov, F; Glazko, G

2001-02-01

The study and comparison of mutation(al) spectra is an important problem in molecular biology, because these spectra often reflect on important features of mutations and their fixation. Such features include the interaction of DNA with various mutagens, the function of repair/replication enzymes, and properties of target proteins. It is known that mutability varies significantly along nucleotide sequences, such that mutations often concentrate at certain positions, called "hotspots," in a sequence. In this paper, we discuss in detail two approaches for mutation spectra analysis: the comparison of mutation spectra with a HG-PUBL program, (FTP: sunsite.unc.edu/pub/academic/biology/dna-mutations/hyperg) and hotspot prediction with the CLUSTERM program (www.itba.mi.cnr.it/webmutation; ftp.bionet.nsc.ru/pub/biology/dbms/clusterm.zip). Several other approaches for mutational spectra analysis, such as the analysis of a target protein structure, hotspot context revealing, multiple spectra comparisons, as well as a number of mutation databases are briefly described. Mutation spectra in the lacI gene of E. coli and the human p53 gene are used for illustration of various difficulties of such analysis. Copyright 2001 Wiley-Liss, Inc.

Unfolding sphere size distributions with a density estimator based on Tikhonov regularization

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

Weese, J.; Korat, E.; Maier, D.

1997-12-01

This report proposes a method for unfolding sphere size distributions given a sample of radii that combines the advantages of a density estimator with those of Tikhonov regularization methods. The following topics are discusses in this report to achieve this method: the relation between the profile and the sphere size distribution; the method for unfolding sphere size distributions; the results based on simulations; and the experimental data comparison.

Spontaneous Unfolding-Refolding of Fibronectin Type III Domains Assayed by Thiol Exchange

PubMed Central

Shah, Riddhi; Ohashi, Tomoo; Erickson, Harold P.; Oas, Terrence G.

2017-01-01

Globular proteins are not permanently folded but spontaneously unfold and refold on time scales that can span orders of magnitude for different proteins. A longstanding debate in the protein-folding field is whether unfolding rates or folding rates correlate to the stability of a protein. In the present study, we have determined the unfolding and folding kinetics of 10 FNIII domains. FNIII domains are one of the most common protein folds and are present in 2% of animal proteins. FNIII domains are ideal for this study because they have an identical seven-strand β-sandwich structure, but they vary widely in sequence and thermodynamic stability. We assayed thermodynamic stability of each domain by equilibrium denaturation in urea. We then assayed the kinetics of domain opening and closing by a technique known as thiol exchange. For this we introduced a buried Cys at the identical location in each FNIII domain and measured the kinetics of labeling with DTNB over a range of urea concentrations. A global fit of the kinetics data gave the kinetics of spontaneous unfolding and refolding in zero urea. We found that the folding rates were relatively similar, ∼0.1–1 s−1, for the different domains. The unfolding rates varied widely and correlated with thermodynamic stability. Our study is the first to address this question using a set of domains that are structurally homologous but evolved with widely varying sequence identity and thermodynamic stability. These data add new evidence that thermodynamic stability correlates primarily with unfolding rate rather than folding rate. The study also has implications for the question of whether opening of FNIII domains contributes to the stretching of fibronectin matrix fibrils. PMID:27909052

Energetics, Ion and Water Binding of the Unfolding of AA/UU Base Pair Stacks and UAU/UAU Base Triplet Stacks in RNA.

PubMed

Carr, Carolyn E; Khutsishvili, Irine; Marky, Luis A

2018-06-22

Triplex formation occurs via interaction of a third strand with the major groove of double stranded nucleic acid, through Hoogsteen hydrogen bonding. In this work, we use a combination of temperature-dependent UV spectroscopy and differential scanning calorimetry to determine complete thermodynamic profiles for the unfolding of poly(rA)•poly(rU) (Duplex) and poly(rA)•2poly(rU) (Triplex). Our thermodynamic results are in good agreement with the much earlier work of Krakauer and Sturtevant using only UV melting techniques. The folding of these two helices yielded an uptake of ions, ΔnNa+ = 0.15 mol Na+/mol base-pair (Duplex) and 0.30 mol Na+/mole base-triplet (Triplex), which are consistent with their polymer behavior and the higher charge density parameter of triple helices. The osmotic stress technique yielded a release of structural water, ΔnW = 2 mol H2O/mol base-pair (Duplex unfolding into single strands) and an uptake of structural water, ΔnW = 2 mol H2O/mole base-pair (Triplex unfolding into Duplex and a single strand). However, an overall release of electrostricted waters is obtained for the unfolding of both complexes from pressure perturbation calorimetric experiments. In total, the ΔV values obtained for the unfolding of Triplex into Duplex and a single strand correspond to an immobilization of two structural waters and a release of three electrostricted waters. The ΔV values obtained for the unfolding of Duplex into two single strands correspond to the release of two structural waters and the immobilization of four electrostricted water molecules.

Unfolding of chondroitinase ABC Ι is dependent on thermodynamic driving force by kinetically rate constant-amplitude compensation: A stopped-flow fluorescence study.

PubMed

Shirdel, S Akram; Khalifeh, Khosrow; Ranjbar, Bijan; Golestani, Abolfazl; Khajeh, Khosro

2016-11-01

We had previously investigated the role of a loop on the activity and conformational stability of chondroitinase ABC Ι (cABC Ι) by constructing some representative mutants in which a network interaction around Asp 689 was manipulated. Here we extended our study by measuring the proteolytic resistance, long term and thermal stability as well as unfolding kinetics of these variants. Long term stability data at 4 and 25°C for 3 weeks indicates that all mutants remain considerably active at 4°C. Thermoinactivation rates for all variants shows that the wild type (WT) enzyme retained 50% of its activity after 2min keeping at 40°C, while L701T, H700N and H700N/L701T as conformationally stabilized variants, have slower inactivation rate. It was also found that compact and thermodynamically stabilized variants are more resistant to tryptolytic digestion. Also, kinetic curves of chemical unfolding of the enzyme variants from stopped-flow fluorescence measurements were best fitted into a three-exponential function with three rate constants and corresponding amplitudes. We found that the energy barrier of the fast unfolding phase is lower in stabilized variants; while the amplitude of this phase to the whole amplitude of the unfolding reaction is lower than that of destabilized variants, indicating more population of stabilized mutants unfold via slower unfolding phase. We concluded that the rate of local conformational change alone is not the same that is expected from global thermodynamic stability; however the corresponding amplitude can compensate the rate constant toward thermodynamic stability. Copyright © 2016 Elsevier Inc. All rights reserved.

Science Teacher Education in Australia: Initiatives and Challenges to Improve the Quality of Teaching

NASA Astrophysics Data System (ADS)

Treagust, David F.; Won, Mihye; Petersen, Jacinta; Wynne, Georgie

2015-02-01

In this article, we describe how teachers in the Australian school system are educated to teach science and the different qualifications that teachers need to enter the profession. The latest comparisons of Australian students in international science assessments have brought about various accountability measures to improve the quality of science teachers at all levels. We discuss the issues and implications of government initiatives in preservice and early career teacher education programs, such as the implementation of national science curriculum, the stricter entry requirements to teacher education programs, an alternative pathway to teaching and the measure of effectiveness of teacher education programs. The politicized discussion and initiatives to improve the quality of science teacher education in Australia are still unfolding as we write in 2014.

A thiol probe for measuring unfolded protein load and proteostasis in cells.

PubMed

Chen, Moore Z; Moily, Nagaraj S; Bridgford, Jessica L; Wood, Rebecca J; Radwan, Mona; Smith, Trevor A; Song, Zhegang; Tang, Ben Zhong; Tilley, Leann; Xu, Xiaohong; Reid, Gavin E; Pouladi, Mahmoud A; Hong, Yuning; Hatters, Danny M

2017-09-07

When proteostasis becomes unbalanced, unfolded proteins can accumulate and aggregate. Here we report that the dye, tetraphenylethene maleimide (TPE-MI) can be used to measure cellular unfolded protein load. TPE-MI fluorescence is activated upon labelling free cysteine thiols, normally buried in the core of globular proteins that are exposed upon unfolding. Crucially TPE-MI does not become fluorescent when conjugated to soluble glutathione. We find that TPE-MI fluorescence is enhanced upon reaction with cellular proteomes under conditions promoting accumulation of unfolded proteins. TPE-MI reactivity can be used to track which proteins expose more cysteine residues under stress through proteomic analysis. We show that TPE-MI can report imbalances in proteostasis in induced pluripotent stem cell models of Huntington disease, as well as cells transfected with mutant Huntington exon 1 before the formation of visible aggregates. TPE-MI also detects protein damage following dihydroartemisinin treatment of the malaria parasites Plasmodium falciparum. TPE-MI therefore holds promise as a tool to probe proteostasis mechanisms in disease.Proteostasis is maintained through a number of molecular mechanisms, some of which function to protect the folded state of proteins. Here the authors demonstrate the use of TPE-MI in a fluorigenic dye assay for the quantitation of unfolded proteins that can be used to assess proteostasis on a cellular or proteome scale.

Comparative analysis of thermal unfolding simulations of RNA recognition motifs (RRMs) of TAR DNA-binding protein 43 (TDP-43).

PubMed

Prakash, Amresh; Kumar, Vijay; Meena, Naveen Kumar; Hassan, Md Imtaiyaz; Lynn, Andrew M

2018-01-10

TAR DNA-binding protein 43 (TDP-43) inclusions have been found in Amyotrophic lateral sclerosis (ALS) and several other neurodegenerative diseases. Many studies suggest the involvement of RNA recognition motifs (RRMs) in TDP-43 proteinopathy. To elucidate the structural stability and the unfolding dynamics of RRMs, we have carried out atomistic molecular dynamics simulations at two different temperatures (300 and 500 K). The simulations results indicate that there are distinct structural differences in the unfolding pathway between the two domains and RRM1 unfolds faster than RRM2 in accordance with the lower thermal stability found experimentally. The unfolding behaviors of secondary structures showed that the α-helix was more stable than β-sheet and structural rearrangements of β-sheets results in formation of additional α-helices. At higher temperature, RRM1 exhibit increased overall flexibility and unfolding than RRM2. The temperature-dependent free energy landscapes consist of multiple metastable states stabilized by non-native contacts and hydrogen bonds in RRM2, thus rendering the RRM2 more prone to misfolding. The structural rearrangements of RRM2 could lead to aberrant protein-protein interactions that may account for enhanced aggregation and toxicity of TDP-43. Our analysis, thus identify the structural and thermodynamic characteristics of the RRMs of TDP-43, which will serve to uncover molecular mechanisms and driving forces in TDP-43 misfolding and aggregation.

How cooperative are protein folding and unfolding transitions?

PubMed Central

Malhotra, Pooja

2016-01-01

Abstract A thermodynamically and kinetically simple picture of protein folding envisages only two states, native (N) and unfolded (U), separated by a single activation free energy barrier, and interconverting by cooperative two‐state transitions. The folding/unfolding transitions of many proteins occur, however, in multiple discrete steps associated with the formation of intermediates, which is indicative of reduced cooperativity. Furthermore, much advancement in experimental and computational approaches has demonstrated entirely non‐cooperative (gradual) transitions via a continuum of states and a multitude of small energetic barriers between the N and U states of some proteins. These findings have been instrumental towards providing a structural rationale for cooperative versus noncooperative transitions, based on the coupling between interaction networks in proteins. The cooperativity inherent in a folding/unfolding reaction appears to be context dependent, and can be tuned via experimental conditions which change the stabilities of N and U. The evolution of cooperativity in protein folding transitions is linked closely to the evolution of function as well as the aggregation propensity of the protein. A large activation energy barrier in a fully cooperative transition can provide the kinetic control required to prevent the accumulation of partially unfolded forms, which may promote aggregation. Nevertheless, increasing evidence for barrier‐less “downhill” folding, as well as for continuous “uphill” unfolding transitions, indicate that gradual non‐cooperative processes may be ubiquitous features on the free energy landscape of protein folding. PMID:27522064

Unfolding the HIV-1 reverse transcriptase RNase H domain – how to lose a molecular tug-of-war

DOE PAGES

Zheng, Xunhai; Pedersen, Lars C.; Gabel, Scott A.; ...

2016-01-14

Formation of the mature HIV-1 reverse transcriptase (RT) p66/p51 heterodimer requires subunit-specific processing of the p66/p66' homodimer precursor. Since the ribonuclease H (RH) domain contains an occult cleavage site located near its center, cleavage must occur either prior to folding or subsequent to unfolding. Recent NMR studies have identified a slow, subunit-specific RH domain unfolding process proposed to result from a residue tug-of-war between the polymerase and RH domains on the functionally inactive, p66' subunit. Here, we describe a structural comparison of the isolated RH domain with a domain swapped RH dimer that reveals several intrinsically destabilizing characteristics of themore » isolated domain that facilitate excursions of Tyr427 from its binding pocket and separation of helices B and D. These studies provide independent support for the subunit-selective RH domain unfolding pathway in which instability of the Tyr427 binding pocket facilitates its release followed by domain transfer, acting as a trigger for further RH domain destabilization and subsequent unfolding. As further support for this pathway, NMR studies demonstrate that addition of an RH active site-directed isoquinolone ligand retards the subunit-selective RH' domain unfolding behavior of the p66/p66' homodimer. As a result, this study demonstrates the feasibility of directly targeting RT maturation with therapeutics.« less

Unfolding the HIV-1 reverse transcriptase RNase H domain – how to lose a molecular tug-of-war

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

Zheng, Xunhai; Pedersen, Lars C.; Gabel, Scott A.

Formation of the mature HIV-1 reverse transcriptase (RT) p66/p51 heterodimer requires subunit-specific processing of the p66/p66' homodimer precursor. Since the ribonuclease H (RH) domain contains an occult cleavage site located near its center, cleavage must occur either prior to folding or subsequent to unfolding. Recent NMR studies have identified a slow, subunit-specific RH domain unfolding process proposed to result from a residue tug-of-war between the polymerase and RH domains on the functionally inactive, p66' subunit. Here, we describe a structural comparison of the isolated RH domain with a domain swapped RH dimer that reveals several intrinsically destabilizing characteristics of themore » isolated domain that facilitate excursions of Tyr427 from its binding pocket and separation of helices B and D. These studies provide independent support for the subunit-selective RH domain unfolding pathway in which instability of the Tyr427 binding pocket facilitates its release followed by domain transfer, acting as a trigger for further RH domain destabilization and subsequent unfolding. As further support for this pathway, NMR studies demonstrate that addition of an RH active site-directed isoquinolone ligand retards the subunit-selective RH' domain unfolding behavior of the p66/p66' homodimer. As a result, this study demonstrates the feasibility of directly targeting RT maturation with therapeutics.« less

Quantifying internal friction in unfolded and intrinsically disordered proteins with single-molecule spectroscopy

PubMed Central

Soranno, Andrea; Buchli, Brigitte; Nettels, Daniel; Cheng, Ryan R.; Müller-Späth, Sonja; Pfeil, Shawn H.; Hoffmann, Armin; Lipman, Everett A.; Makarov, Dmitrii E.; Schuler, Benjamin

2012-01-01

Internal friction, which reflects the “roughness” of the energy landscape, plays an important role for proteins by modulating the dynamics of their folding and other conformational changes. However, the experimental quantification of internal friction and its contribution to folding dynamics has remained challenging. Here we use the combination of single-molecule Förster resonance energy transfer, nanosecond fluorescence correlation spectroscopy, and microfluidic mixing to determine the reconfiguration times of unfolded proteins and investigate the mechanisms of internal friction contributing to their dynamics. Using concepts from polymer dynamics, we determine internal friction with three complementary, largely independent, and consistent approaches as an additive contribution to the reconfiguration time of the unfolded state. We find that the magnitude of internal friction correlates with the compactness of the unfolded protein: its contribution dominates the reconfiguration time of approximately 100 ns of the compact unfolded state of a small cold shock protein under native conditions, but decreases for more expanded chains, and approaches zero both at high denaturant concentrations and in intrinsically disordered proteins that are expanded due to intramolecular charge repulsion. Our results suggest that internal friction in the unfolded state will be particularly relevant for the kinetics of proteins that fold in the microsecond range or faster. The low internal friction in expanded intrinsically disordered proteins may have implications for the dynamics of their interactions with cellular binding partners. PMID:22492978

Quantifying internal friction in unfolded and intrinsically disordered proteins with single-molecule spectroscopy.

PubMed

Soranno, Andrea; Buchli, Brigitte; Nettels, Daniel; Cheng, Ryan R; Müller-Späth, Sonja; Pfeil, Shawn H; Hoffmann, Armin; Lipman, Everett A; Makarov, Dmitrii E; Schuler, Benjamin

2012-10-30

Internal friction, which reflects the "roughness" of the energy landscape, plays an important role for proteins by modulating the dynamics of their folding and other conformational changes. However, the experimental quantification of internal friction and its contribution to folding dynamics has remained challenging. Here we use the combination of single-molecule Förster resonance energy transfer, nanosecond fluorescence correlation spectroscopy, and microfluidic mixing to determine the reconfiguration times of unfolded proteins and investigate the mechanisms of internal friction contributing to their dynamics. Using concepts from polymer dynamics, we determine internal friction with three complementary, largely independent, and consistent approaches as an additive contribution to the reconfiguration time of the unfolded state. We find that the magnitude of internal friction correlates with the compactness of the unfolded protein: its contribution dominates the reconfiguration time of approximately 100 ns of the compact unfolded state of a small cold shock protein under native conditions, but decreases for more expanded chains, and approaches zero both at high denaturant concentrations and in intrinsically disordered proteins that are expanded due to intramolecular charge repulsion. Our results suggest that internal friction in the unfolded state will be particularly relevant for the kinetics of proteins that fold in the microsecond range or faster. The low internal friction in expanded intrinsically disordered proteins may have implications for the dynamics of their interactions with cellular binding partners.

Unfolding Kinetics of β-Lactoglobulin Induced by Surfactant and Denaturant: A Stopped-Flow/Fluorescence Study

PubMed Central

Viseu, Maria Isabel; Melo, Eduardo P.; Carvalho, Teresa Isabel; Correia, Raquel F.; Costa, Sílvia M. B.

2007-01-01

The β→α transition of β-lactoglobulin, a globular protein abundant in the milk of several mammals, is investigated in this work. This transition, induced by the cationic surfactant dodecyltrimethylammonium chloride (DTAC), is accompanied by partial unfolding of the protein. In this work, unfolding of bovine β-lactoglobulin in DTAC is compared with its unfolding induced by the chemical denaturant guanidine hydrochloride (GnHCl). The final protein states attained in the two media have quite different secondary structure: in DTAC the α-helical content increases, leading to the so-called α-state; in GnHCl the amount of ordered secondary-structure decreases, resulting in a random coil-rich final state (denatured, or D, state). To obtain information on both mechanistic routes, in DTAC and GnHCl, and to characterize intermediates, the kinetics of unfolding were investigated in the two media. Equilibrium and kinetic data show the partial accumulation of an on-pathway intermediate in each unfolding route: in DTAC, an intermediate (I1) with mostly native secondary structure but loose tertiary structure appears between the native (β) and α-states; in GnHCl, another intermediate (I2) appears between states β and D. Kinetic rate constants follow a linear Chevron-plot representation in GnHCl, but show a more complex mechanism in DTAC, which acts like a stronger binding species. PMID:17693475

Effect of antimicrobial preservatives on partial protein unfolding and aggregation†

PubMed Central

Hutchings, Regina L.; Singh, Surinder M.; Cabello-Villegas, Javier; Mallela, Krishna M. G.

2014-01-01

One-third of protein formulations are multi-dose. These require antimicrobial preservatives (APs); however, some APs have been shown to cause protein aggregation. Our previous work on a model protein cytochrome c indicated that partial protein unfolding, rather than complete unfolding, triggers aggregation. Here, we examined the relative strength of five commonly used APs on such unfolding and aggregation, and explored whether stabilizing the aggregation “hot-spot” reduces such aggregation. All APs induced protein aggregation in the order m-cresol > phenol > benzyl alcohol > phenoxyethanol > chlorobutanol. All these enhanced the partial protein unfolding that includes a local region which was predicted to be the aggregation “hot-spot”. The extent of destabilization correlated with the extent of aggregation. Further, we show that stabilizing the “hot-spot” reduces aggregation induced by all five APs. These results indicate that m-cresol causes the most protein aggregation, whereas chlorobutanol causes the least protein aggregation. The same protein region acts as the “hot-spot” for aggregation induced by different APs, implying that developing strategies to prevent protein aggregation induced by one AP will also work for others. PMID:23169345

Amino acid substitutions affecting protein dynamics in eglin C do not affect heat capacity change upon unfolding.

PubMed

Gribenko, Alexey V; Keiffer, Timothy R; Makhatadze, George I

2006-08-01

The heat capacity change upon unfolding (deltaC(p)) is a thermodynamic parameter that defines the temperature dependence of the thermodynamic stability of proteins; however, physical basis of the heat capacity change is not completely understood. Although empirical surface area-based calculations can predict heat capacity changes reasonably well, accumulating evidence suggests that changes in hydration of those surfaces is not the only parameter contributing to the observed heat capacity changes upon unfolding. Because packing density in the protein interior is similar to that observed in organic crystals, we hypothesized that changes in protein dynamics resulting in increased rigidity of the protein structure might contribute to the observed heat capacity change upon unfolding. Using differential scanning calorimetry we characterized the thermodynamic behavior of a serine protease inhibitor eglin C and two eglin C variants with altered native state dynamics, as determined by NMR. We found no evidence of changes in deltaC(p) in either of the variants, suggesting that changes in rigidity do not contribute to the heat capacity change upon unfolding in this model system. Copyright 2006 Wiley-Liss, Inc.

Mechanical Unfolding Studies on Single-Domain SUMO and Multi-Domain Periplasmic Binding Proteins

NASA Astrophysics Data System (ADS)

Kotamarthi, Hema Chandra; Ainavarapu, Sri Rama Koti

Protein mechanics is a key component of many cellular and sub-cellular processes. The current review focuses on recent studies from our laboratory that probe the effect of sequence on the mechanical stability of structurally similar proteins and the unfolding mechanisms of multi-domain periplasmic binding proteins. Ubiquitin and small ubiquitin-related modifiers (SUMOs) are structurally similar and possess different mechanical stabilities, ubiquitin being stronger than SUMOs as revealed from their unfolding forces. These differences are plausibly due to the variation in number of inter-residue contacts. The unfolding potential widths determined from the pulling speed-dependent studies revealed that SUMOs are mechanically more flexible than ubiquitin. This flexibility of SUMOs plays a role in ligand binding and our single-molecule studies on SUMO interaction with SUMO binding motifs (SBMs) have shown that ligand binding decreases the SUMO flexibility and increases its mechanical stability. Studies on multi-domain periplasmic binding proteins have revealed that the unfolding energy landscape of these proteins is complex and they follow kinetic partitioning between two-state and multiple three-state pathways.

KSC volunteers help paint Baxley Manor as part of Days of Caring '99

NASA Technical Reports Server (NTRS)

1999-01-01

KSC volunteers for Days of Caring '99 unfold protective materials before getting ready to paint at Baxley Manor, an apartment building for senior citizens on Merritt Island. Coordinated by the KSC Community Relations Council, Days of Caring provides an opportunity for employees to volunteer their services in projects such as painting, planting flowers, reading to school children, and more. Organizations accepting volunteers include The Embers, Yellow Umbrella, Serene Harbor, Domestic Violence Program, the YMCA of Brevard County, and others.

Beyond the glass ceiling. The top 25 women in Healthcare honorees take on key roles as significant change in the industry unfolds.

PubMed

Kutscher, Beth; Landen, Rachel; Zigmond, Jessica

2013-04-08

At a time when a national conversation is focused on the gender imbalance atop most professions, healthcare can boast of having women in some of the most important policy and executive positions. Those honored in ourTop 25 Women in Healthcare program have a few lessons to share. "Be willing to take the job that everyone else isn't migrating to and make it work for you," says Marilyn Tavenner, acting CMS administrator.

Unfolding energetics and stability of banana lectin.

PubMed

Gupta, Garima; Sinha, Sharmistha; Surolia, Avadhesha

2008-08-01

The unfolding pathway of banana lectin from Musa paradisiaca was determined by isothermal denaturation induced by the chaotrope GdnCl. The unfolding was found to be a reversible process. The data obtained by isothermal denaturation provided information on conformational stability of banana lectin. The high values of DeltaG of unfolding at various temperatures indicated the strength of intersubunit interactions. It was found that banana lectin is a very stable and denatures at high chaotrope concentrations only. The basis of the stability may be attributed to strong hydrogen bonds of the order 2.5-3.1 A at the dimeric interface along with the presence of water bridges. This is perhaps very unique example in proteins where subunit association is not a consequence of the predominance of hydrophobic interactions. (c) 2008 Wiley-Liss, Inc.

Advanced Technologies and Instrumentation at the National Science Foundation

NASA Astrophysics Data System (ADS)

Kurczynski, Peter; Neff, James E.

2018-01-01

Over its more than thirty-year history, the Advanced Technologies and Instrumentation (ATI) program within the Division of Astronomical Sciences has provided grants to support the development and deployment of detectors and instrumentation for ground-based astronomy. This program has enabled scientific advances in diverse fields from solar physics to exoplanets to cosmology. ATI has provided instrumentation for both small and large observatories from radio through visible wavebands. It has played a role in the early development of major initiatives such as the Large Synoptic Survey Telescope. Technology development for astronomy unfolds over a longer period than the lifetime of a single grant. This review will consider ATI from an historical perspective to assess its impact on astronomy.

Comment on ``Experimental Free Energy Reconstruction From Single-Molecule Force Spectroscopy Using Jarzynski's Equality''

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

Friddle, R W

2008-01-14

Harris, Song and Kiang [1] (HSK) describe their results on reconstructing the free energy profiles for both the stretch of the titin polymer, and the unfolding of an individual I27 domain. The new finding reported in [1] is the measurement of the free energy barrier (or activation energy) to unfolding the I27 domain. Due to a misinterpretation of the mechanics involved, the free energy surface (and thus the energy barrier) to unfolding the I27 domain was not measured.

Structural changes in cartilage and collagen studied by high temperature Raman spectroscopy.

PubMed

Fields, Mark; Spencer, Nicholas; Dudhia, Jayesh; McMillan, Paul F

2017-06-01

Understanding the high temperature behavior of collagen and collagenous tissue is important for surgical procedures and biomaterials processing for the food, pharmaceutical, and cosmetics industries. One primary event for proteins is thermal denaturation that involves unfolding the polypeptide chains while maintaining the primary structure intact. Collagen in the extracellular matrix of cartilage and other connective tissue is a hierarchical material containing bundles of triple-helical fibers associated with water and proteoglycan components. Thermal analysis of dehydrated collagen indicates irreversible denaturation at high temperature between 135°C and 200°C, with another reversible event at ∼60-80°C for hydrated samples. We report high temperature Raman spectra for freeze-dried cartilage samples that show an increase in laser-excited fluorescence interpreted as conformational changes associated with denaturation above 140°C. Spectra for separated collagen and proteoglycan fractions extracted from cartilage indicate the changes are associated with collagen. The Raman data also show appearance of new features indicating peptide bond hydrolysis at high temperature implying that molecular H 2 O is retained within the freeze-dried tissue. This is confirmed by thermogravimetric analysis that show 5-7 wt% H 2 O remaining within freeze-dried cartilage that is released progressively upon heating up to 200°C. Spectra obtained after exposure to high temperature and re-hydration following recovery indicate that the capacity of the denatured collagen to re-absorb water is reduced. Our results are important for revealing the presence of bound H 2 O within the collagen component of connective tissue even after freeze-drying and its role in denaturation that is accompanied by or perhaps preceded by breakdown of the primary polypeptide structure. © 2017 Wiley Periodicals, Inc.

Signal processing for a single detector MOEMS based NIR micro spectrometer

NASA Astrophysics Data System (ADS)

Heberer, Andreas; Grüger, Heinrich; Zimmer, Fabian; Schenk, Harald; Kenda, Andreas; Frank, Albert; Scherf, Werner

2005-10-01

The examination of spectra in the NIR range is necessary for applications like process control, element analysis or medical systems. Typically integrated NIR spectrometers are based on optical setups with diffraction grating and detector arrays. The main disadvantage is price and availability of NIR array InGaAs-based detectors. The implementation of a scanning grating chip realized in a MOEMS technology which integrates the diffractive element makes it possible to detect spectra with single detectors time resolved. Either simple InGaAs photodiodes or cooled detectors may be used. The set up is a shrinked Czerny-Turner spectrometer. The light is coupled in by an optical fibre. After focussing the light passes the scanning grating moving at 150-500 Hz in a sinusoidal way. There it is split off in the different wavelength, the monochrome intensity is caught by a second mirror and led to the detector. The detector signal is amplified by a transimpedance stage and converted to digital with 12 bit resolution. The main part of the signal processing is done by a digital signal processor, which is used to unfold the sinusoidal position and calculate the final spectra. The data rate can be up to 3 MHz, then a spectrum is acquired every 2ms by using a 500Hz Mirror. Using the DSP, the spectrometer can operate autarkic without any PC. Then the spectrum is display on a 160 x 80 pixel graphic LCD. A keypad is used to control the functions. For communication a USB port is included, additional interfaces can be realized by a 16-pin expansion port, which is freely programmable, by the system firmware.

Comparative evaluation of the depletion-red cell exchange program with the Spectra Optia and the isovolemic hemodilution-red cell exchange method with the COBE Spectra in sickle cell disease patients.

PubMed

Poullin, Pascale; Sanderson, Frederick; Bernit, Emmanuelle; Brun, Marion; Berdah, Yael; Badens, Catherine

2016-10-01

This study aims to compare in patients with sickle cell disease (SCD), the technical performance and packed red blood cell unit consumption between the automated depletion/Red Blood Cell exchange (RBCx) program (Spectra Optia Apheresis System) with the isovolemic hemodilution (IHD)/RBCx procedure (COBE Spectra Apheresis System) in a routine clinical setting. We retrospectively reviewed the data of 23 patients treated between October 2010 and August 2013 who underwent repeated RBCx on both apheresis systems for preventive indications. Each patient was their own control and had undergone two procedures on each system, totaling 46 sessions per group. On Spectra Optia, we performed the automated depletion/RBCx program. For COBE Spectra, we used a modified IHD/RBCx protocol. All patients had an initial 250 mL depletion offset by a 5% albumin prior to the exchange procedure, for the respective device, with leucodepleted Rh/Kell compatible and cross-matched RBC packs. All procedures were well tolerated except three mild febrile nonhemolytic reactions. Postprocedure hemoglobin S (HbS), fraction of cells remaining (FCR), procedure duration and processed blood and anticoagulant volumes were comparable in the two groups. However, the RBCx volume was significantly higher for the Spectra Optia group (+71 mL, P = 0.01), with no significant difference in the number of RBC units used. Technical performance and packed RBC unit consumption were not compromised when switching from the COBE Spectra IHD/RBCx protocol to the depletion/RBCx protocol on the Spectra Optia. Tolerability was equal for both protocols. J. Clin. Apheresis 31:429-433, 2016. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

The reversible two-state unfolding of a monocot mannose-binding lectin from garlic bulbs reveals the dominant role of the dimeric interface in its stabilization.

PubMed

Bachhawat, K; Kapoor, M; Dam, T K; Surolia, A

2001-06-19

Allium sativum agglutinin (ASAI) is a heterodimeric mannose-specific bulb lectin possessing two polypeptide chains of molecular mass 11.5 and 12.5 kDa. The thermal unfolding of ASAI, characterized by differential scanning calorimetry and circular dichroism, shows it to be highly reversible and can be defined as a two-state process in which the folded dimer is converted directly to the unfolded monomers (A2 if 2U). Its conformational stability has been determined as a function of temperature, GdnCl concentration, and pH using a combination of thermal and isothermal GdnCl-induced unfolding monitored by DSC, far-UV CD, and fluorescence, respectively. Analyses of these data yielded the heat capacity change upon unfolding (DeltaC(p) and also the temperature dependence of the thermodynamic parameters, namely, DeltaG, DeltaH, and DeltaS. The fit of the stability curve to the modified Gibbs-Helmholtz equation provides an estimate of the thermodynamic parameters DeltaH(g), DeltaS(g), and DeltaC(p) as 174.1 kcal x mol(-1), 0.512 kcal x mol(-1) x K(-1), and 3.41 kcal x mol(-1) x K(-1), respectively, at T(g) = 339.4 K. Also, the free energy of unfolding, DeltaG(s), at its temperature of maximum stability (T(s) = 293 K) is 13.13 kcal x mol(-1). Unlike most oligomeric proteins studied so far, the lectin shows excellent agreement between the experimentally determined DeltaC(p) (3.2 +/- 0.28 kcal x mol(-1) x K(-1)) and those evaluated from a calculation of its accessible surface area. This in turn suggests that the protein attains a completely unfolded state irrespective of the method of denaturation. The absence of any folding intermediates suggests the quaternary interactions to be the major contributor to the conformational stability of the protein, which correlates well with its X-ray structure. The small DeltaC(p) for the unfolding of ASAI reflects a relatively small, buried hydrophobic core in the folded dimeric protein.

Trehalose delays the reversible but not the irreversible thermal denaturation of cutinase.

PubMed

Baptista, R P; Cabral, J M; Melo, E P

2000-12-20

The effect of trehalose (0.5 M) on the thermal stability of cutinase in the alkaline pH range was studied. The thermal unfolding induced by increasing temperature was analyzed in the absence and in the presence of trehalose according to a two-state model (which assumes that only the folded and unfolded states of cutinase were present). Trehalose delays the reversible unfolding. The midpoint temperature of the unfolding transition (Tm) increases by 4.0 degrees C and 2. 6 degrees C at pH 9.2 and 10.5, respectively, in the presence of trehalose. At pH 9.2 the thermal unfolding occurs at higher temperatures (Tm is 52.6 degrees C compared to 42.0 degrees C at pH 10.5) and a refolding yield of around 80% was obtained upon cooling. This pH value was chosen to study the irreversible inactivation (long-term stability) of cutinase. Temperatures in the transition range from folded to unfolded state were selected and the rate constants of irreversible inactivation determined. Inactivation followed first-order kinetics and trehalose reduced the observed rate constants of inactivation, pointing to a stabilizing effect on the irreversible inactivation step of thermal denaturation. However, if the contribution of reversible unfolding on the irreversible inactivation of cutinase was taken into account, i.e., considering the fraction of cutinase molecules in the reversible unfolded conformation, the intrinsic rate constants can be calculated. Based on the intrinsic rate constants it was concluded that trehalose does not delay the irreversible inactivation. This conclusion was further supported by comparing the activation energy of the irreversible inactivation in the absence and in the presence of trehalose. The apparent activation energy in the absence and in the presence of trehalose were 67 and 99 Kcal/mol, respectively. The activation energy calculated from intrinsic rate constants was higher in the absence (30 Kcal/mol) than in the presence of trehalose (16 Kcal/mol), showing that kinetics of the irreversible inactivation step increased in the presence of trehalose. In fact, trehalose stabilized only the reversible step of thermal denaturation of cutinase.

Benchmarking of MCNP for calculating dose rates at an interim storage facility for nuclear waste.

PubMed

Heuel-Fabianek, Burkhard; Hille, Ralf

2005-01-01

During the operation of research facilities at Research Centre Jülich, Germany, nuclear waste is stored in drums and other vessels in an interim storage building on-site, which has a concrete shielding at the side walls. Owing to the lack of a well-defined source, measured gamma spectra were unfolded to determine the photon flux on the surface of the containers. The dose rate simulation, including the effects of skyshine, using the Monte Carlo transport code MCNP is compared with the measured dosimetric data at some locations in the vicinity of the interim storage building. The MCNP data for direct radiation confirm the data calculated using a point-kernel method. However, a comparison of the modelled dose rates for direct radiation and skyshine with the measured data demonstrate the need for a more precise definition of the source. Both the measured and the modelled dose rates verified the fact that the legal limits (<1 mSv a(-1)) are met in the area outside the perimeter fence of the storage building to which members of the public have access. Using container surface data (gamma spectra) to define the source may be a useful tool for practical calculations and additionally for benchmarking of computer codes if the discussed critical aspects with respect to the source can be addressed adequately.

Maintenance of the secondary structure of horse cytochrome c during the conversion process of monomers to oligomers by addition of ethanol.

PubMed

Hirota, Shun; Ueda, Mariko; Hayashi, Yugo; Nagao, Satoshi; Kamikubo, Hironari; Kataoka, Mikio

2012-12-01

We have previously shown that polymerization of cytochrome c (cyt c) occurs by successively domain swapping its C-terminal α-helix in the presence of ethanol. However, the factors that govern the conversion process of monomers to domain-swapped oligomers remain unknown. We found that oligomeric cyt c is produced in the presence of ethanol and the oligomers precipitate due to low solubility. The optical absorption spectra revealed that in the presence of 30-40% ethanol, the Met-heme coordination in cyt c is dissociated. However, according to circular dichroism and small-angle X-ray scattering measurements, the α-helical structure of cyt c is maintained in solution with a little perturbation and the radius of gyration increases slightly but without dissociation of the C-terminal α-helix from the rest of the protein by the addition of ethanol. Solid-state nuclear magnetic resonance spectra showed that oligomeric cyt c in the precipitate also retains most of its α-helical structure. In the transmission electron microscopic image of the precipitate obtained by the addition of ethanol to cyt c, spherical particles with diameters of about 3 nm were detected. These results indicate that oligomeric cyt c forms through a state with the Met80 region locally unfolded, while maintaining the secondary structure, possibly an open monomer.

Collision induced unfolding of isolated proteins in the gas phase: past, present, and future.

PubMed

Dixit, Sugyan M; Polasky, Daniel A; Ruotolo, Brandon T

2018-02-01

Rapidly characterizing the three-dimensional structures of proteins and the multimeric machines they form remains one of the great challenges facing modern biological and medical sciences. Ion mobility-mass spectrometry based techniques are playing an expanding role in characterizing these functional complexes, especially in drug discovery and development workflows. Despite this expansion, ion mobility-mass spectrometry faces many challenges, especially in the context of detecting small differences in protein tertiary structure that bear functional consequences. Collision induced unfolding is an ion mobility-mass spectrometry method that enables the rapid differentiation of subtly-different protein isoforms based on their unfolding patterns and stabilities. In this review, we summarize the modern implementation of such gas-phase unfolding experiments and provide an overview of recent developments in both methods and applications. Copyright © 2017 Elsevier Ltd. All rights reserved.

Understanding disordered and unfolded proteins using single-molecule FRET and polymer theory.

PubMed

Hofmann, Hagen

2016-11-17

Understanding protein folding and the functional properties of intrinsically disordered proteins (IDPs) requires detailed knowledge of the forces that act in polypeptide chains. These forces determine the dimensions and dynamics of unfolded and disordered proteins and have been suggested to impact processes such as the coupled binding and folding of IDPs, or the rate of protein folding reactions. Much of the progress in understanding the physical and chemical properties of unfolded and intrinsically disordered polypeptide chains has been made possible by the recent developments in single-molecule fluorescence techniques. However, the interpretation of the experimental results requires concepts from polymer physics in order to be understood. Here, I review some of the theories used to describe the dimensions of unfolded polypeptide chains under varying solvent conditions together with their more recent application to experimental data.

Molecular dynamics study of unfolding of lysozyme in water and its mixtures with dimethyl sulfoxide.

PubMed

Sedov, Igor A; Magsumov, Timur I

2017-09-01

All-atom explicit solvent molecular dynamics was used to study the process of unfolding of hen egg white lysozyme in water and mixtures of water with dimethyl sulfoxide at different compositions. We have determined the kinetic parameters of unfolding at a constant temperature 450K. For each run, the time of disruption of the tertiary structure of lysozyme t u was defined as the moment when a certain structural criterion computed from the trajectory reaches its critical value. A good agreement is observed between the results obtained using several different criteria. The secondary structure according to DSSP calculations is found to be partially unfolded to the moment of disruption of tertiary structure, but some of its elements keep for a long time after that. The values of t u averaged over ten 30ns-long trajectories for each solvent composition are shown to decrease very rapidly with addition of dimethyl sulfoxide, and rather small amounts of dimethyl sulfoxide are found to change the pathway of unfolding. In pure water, despite the loss of tertiary contacts and disruption of secondary structure elements, the protein preserves its compact globular state at least over 130ns of simulation, while even at 5mol percents of dimethyl sulfoxide it loses its compactness within 30ns. The proposed methodology is a generally applicable tool to quantify the rate of protein unfolding in simulation studies. Copyright © 2017 Elsevier Inc. All rights reserved.

Interactions of urea with native and unfolded proteins: a volumetric study.

PubMed

Son, Ikbae; Shek, Yuen Lai; Tikhomirova, Anna; Baltasar, Eduardo Hidalgo; Chalikian, Tigran V

2014-11-26

We describe a statistical thermodynamic approach to analyzing urea-dependent volumetric properties of proteins. We use this approach to analyze our urea-dependent data on the partial molar volume and adiabatic compressibility of lysozyme, apocytochrome c, ribonuclease A, and α-chymotrypsinogen A. The analysis produces the thermodynamic properties of elementary urea-protein association reactions while also yielding estimates of the effective solvent-accessible surface areas of the native and unfolded protein states. Lysozyme and apocytochrome c do not undergo urea-induced transitions. The former remains folded, while the latter is unfolded between 0 and 8 M urea. In contrast, ribonuclease A and α-chymotrypsinogen A exhibit urea-induced unfolding transitions. Thus, our data permit us to characterize urea-protein interactions in both the native and unfolded states. We interpreted the urea-dependent volumetric properties of the proteins in terms of the equilibrium constant, k, and changes in volume, ΔV0, and compressibility, ΔKT0, for a reaction in which urea binds to a protein with a concomitant release of two waters of hydration to the bulk. Comparison of the values of k, ΔV0, and ΔKT0 with the similar data obtained on small molecules mimicking protein groups reveals lack of cooperative effects involved in urea-protein interactions. In general, the volumetric approach, while providing a unique characterization of cosolvent-protein interactions, offers a practical way for evaluating the effective solvent accessible surface area of biologically significant fully or partially unfolded polypeptides.

Role of block copolymer-micelle nanocomposites in dye-bovine serum albumin binding: a combined experimental and molecular docking study.

PubMed

Manna, Anamika; Chakravorti, Sankar

2013-02-02

The role of a nanocomposite (NC), composed of intercalation of the diblock copolymer polyethylene-b-polyethylene glycol (PE-b-PEG) with the anionic surfactant sodium dodecyl sulphate (SDS), on the binding characteristics of bovine serum albumin (BSA) with a dye (1,8-naphthalimide, NAPMD) compared to the interaction between the same players in aqueous solution has been examined comprehensively in this paper. Static quenching due to complex formation in both NC medium and in buffer solution has been inferred on the basis of considerable changes in the absorption spectra of BSA on addition of NAPMD, of which the interaction is found to be stronger in NC medium. Temperature dependent fluorescence data also confirm an effective static quenching and stronger binding of NAPMD with BSA in NC medium. Peptide chain unfolding and denaturing of BSA in NC medium have been confirmed from steady state and time-resolved emission and circular dichroism data. This exposes both the tyrosine and tryptophan moieties as a unique case. Increased energy transfer between NAPMD and the tryptophan residue in the unfolded form of BSA helps in the appearance of tyrosine fluorescence in NC medium by quenching the tryptophan band. Ionization of the hydroxyl group in the aromatic ring of the tyrosine residue by the PEG group present in the NC medium produces a downshift of the tyrosine fluorescence band. The use of site selective markers confirms that NAPMD is near tryptophan in Sudlow's site I in NC medium and in buffer solution it is away from tryptophan in Sudlow's site II. The theoretical docking studies also vindicate the results of binding of NAPMD with BSA in site I or site II in NC and buffer media, as observed from different emission experiments including the site selective markers study.

Structural characterization by NMR of the natively unfolded extracellular domain of beta-dystroglycan: toward the identification of the binding epitope for alpha-dystroglycan.

PubMed

Bozzi, Manuela; Bianchi, Marzia; Sciandra, Francesca; Paci, Maurizio; Giardina, Bruno; Brancaccio, Andrea; Cicero, Daniel O

2003-11-25

Dystroglycan (DG) is an adhesion molecule playing a crucial role for tissue stability during both early embriogenesis and adulthood and is composed by two tightly interacting subunits: alpha-DG, membrane-associated and highly glycosylated, and the transmembrane beta-DG. Recently, by solid-phase binding assays and NMR experiments, we have shown that the C-terminal domain of alpha-DG interacts with a recombinant extracellular fragment of beta-DG (positions 654-750) independently from glycosylation and that the linear binding epitope is located between residues 550 and 565 of alpha-DG. In order to elucidate which moieties of beta-DG are specifically involved in the complex with alpha-DG, the ectodomain has been recombinantly expressed and purified in a labeled ((13)C,(15)N) form and studied by multidimensional NMR. Although it represents a natively unfolded protein domain, we obtained an almost complete backbone assignment. Chemical shift index, (1)H-(15)N heteronuclear single-quantum coherence and nuclear Overhauser effect (HSQC-NOESY) spectra and (3)J(HN,H)(alpha) coupling constant values confirm that this protein is highly disordered, but (1)H-(15)N steady-state NOE experiments indicate that the protein presents two regions of different mobility. The first one, between residues 659 and 722, is characterized by a limited degree of mobility, whereas the C-terminal portion, containing about 30 amino acids, is highly flexible. The binding of beta-DG(654-750) to the C-terminal region of the alpha subunit, alpha-DG(485-620), has been investigated, showing that the region of beta-DG(654-750) between residues 691 and 719 is involved in the interaction.

Molecular dynamics simulation of phosphorylated KID post-translational modification.

PubMed

Chen, Hai-Feng

2009-08-05

Kinase-inducible domain (KID) as transcriptional activator can stimulate target gene expression in signal transduction by associating with KID interacting domain (KIX). NMR spectra suggest that apo-KID is an unstructured protein. After post-translational modification by phosphorylation, KID undergoes a transition from disordered to well folded protein upon binding to KIX. However, the mechanism of folding coupled to binding is poorly understood. To get an insight into the mechanism, we have performed ten trajectories of explicit-solvent molecular dynamics (MD) for both bound and apo phosphorylated KID (pKID). Ten MD simulations are sufficient to capture the average properties in the protein folding and unfolding. Room-temperature MD simulations suggest that pKID becomes more rigid and stable upon the KIX-binding. Kinetic analysis of high-temperature MD simulations shows that bound pKID and apo-pKID unfold via a three-state and a two-state process, respectively. Both kinetics and free energy landscape analyses indicate that bound pKID folds in the order of KIX access, initiation of pKID tertiary folding, folding of helix alpha(B), folding of helix alpha(A), completion of pKID tertiary folding, and finalization of pKID-KIX binding. Our data show that the folding pathways of apo-pKID are different from the bound state: the foldings of helices alpha(A) and alpha(B) are swapped. Here we also show that Asn139, Asp140 and Leu141 with large Phi-values are key residues in the folding of bound pKID. Our results are in good agreement with NMR experimental observations and provide significant insight into the general mechanisms of binding induced protein folding and other conformational adjustment in post-translational modification.

Zinc ion-induced domain organization in metallo-beta-lactamases: a flexible "zinc arm" for rapid metal ion transfer?

PubMed

Selevsek, Nathalie; Rival, Sandrine; Tholey, Andreas; Heinzle, Elmar; Heinz, Uwe; Hemmingsen, Lars; Adolph, Hans W

2009-06-12

The reversible unfolding of metallo-beta-lactamase from Chryseobacterium meningosepticum (BlaB) by guanidinium hydrochloride is best described by a three-state model including folded, intermediate, and unfolded states. The transformation of the folded apoenzyme into the intermediate state requires only very low denaturant concentrations, in contrast to the Zn2-enzyme. Similarly, circular dichroism spectra of both BlaB and metallo-beta-lactamase from Bacillus cereus 569/H/9 (BcII) display distinct differences between metal-free and Zn2-enzymes, indicating that the zinc ions affect the folding of the proteins, giving a larger alpha-helix content. To identify the regions of the protein involved in this zinc ion-induced change, a hydrogen deuterium exchange study with matrix-assisted laser desorption ionization tandem time of flight mass spectrometry on metal-free and Zn1- and Zn2-BcII was carried out. The region spanning the metal binding metallo-beta-lactamases (MBL) superfamily consensus sequence His-X-His-X-Asp motif and the loop connecting the N- and C-terminal domains of the protein undergoes a zinc ion-dependent structural change between intrinsically disordered and ordered states. The inherent flexibility even appears to allow for the formation of metal ion-bridged protein-protein complexes which may account for both electrospray ionization-mass spectroscopy results obtained upon variation of the zinc/protein ratio and stoichiometry-dependent variations of 199mHg-perturbed angular correlation of gamma-rays spectroscopic data. We suggest that this flexible "zinc arm" motif, present in all the MBL subclasses, is disordered in metal-free MBLs and may be involved in metal ion acquisition from zinc-carrying molecules different from MBL in an "activation on demand" regulation of enzyme activity.

Carboxy-terminal glycosyl hydrolase 18 domain of a carbohydrate active protein of Chitinophaga pinensis is a non-processive exochitinase.

PubMed

Ramakrishna, Bellamkonda; Vaikuntapu, PapaRao; Mallakuntla, Mohan Krishna; Bhuvanachandra, Bhoopal; Sivaramakrishna, Dokku; Uikey, Sheetal; Podile, Appa Rao

2018-05-01

The recombinant C-terminal domain of chitinase C of Chitinophaga pinensis (CpChiC-GH18 C ) exhibits the highest activity at pH 6.0 and 35 °C, with a K m of 76.13 (mg -1  ml), a k cat of 10.16 (s -1 ), and a k cat /K m of 0.133 (mg -1  ml s -1 ) on colloidal chitin. Analysis of degradation of (GlcNAc) 3-6 oligomers shows that CpChiC-GH18 C releases (GlcNAc) 2 as the main product, indicating an exo-type cleavage pattern. CpChiC-GH18 C hydrolyzes the chitin polymers yielding GlcNAc, (GlcNAc) 2 , and (GlcNAc) 3 as end products with no sign of processivity. Circular dichroism spectra indicate that the secondary and tertiary structures of CpChiC-GH18 C are unaltered up to 45 °C and the protein denatures without an intermediate state. The urea-induced unfolding is a two-state process and the unfolding of native CpChiC-GH18 C occurs in a single step. Among the metal ions tested, Hg 2+ completely inhibits the enzyme activity. The chemical modulators, p-hydroxymercuribenzoic acid and N-bromosuccinimide considerably decrease the enzyme activity. Sequence analysis and homology modeling suggest that CpChiC-GH18 C lacks a tryptophan residue at the aglycon site. Further, the CpChiC-GH18 C has a shallow and open groove, suggesting that CpChiC-GH18 C is non-processive exo-type chitinase with properties suitable for the bioconversion of chitin waste. Copyright © 2018 Elsevier B.V. All rights reserved.

Stability of Curcuma longa rhizome lectin: Role of N-linked glycosylation.

PubMed

Biswas, Himadri; Chattopadhyaya, Rajagopal

2016-04-01

Curcuma longa rhizome lectin, a mannose-binding protein of non-seed portions of turmeric, is known to have antifungal, antibacterial and α-glucosidase inhibitory activities. We studied the role of complex-type glycans attached to asparagine (Asn) 66 and Asn 110 to elucidate the role of carbohydrates in lectin activity and stability. Apart from the native lectin, the characteristics of a deglycosylated Escherichia coli expressed lectin, high-mannose oligosaccharides at both asparagines and its glycosylation mutants N66Q and N110Q expressed in Pichia pastoris, were compared to understand the relationship between glycosylation and activity. Far UV circular dichroism (CD) spectra, fluorescence emission maximum, hemagglutination assay show no change in secondary or tertiary structures or sugar-binding properties between wild-type and aforementioned recombinant lectins under physiological pH. But reduced agglutination activity and loss of tertiary structure are observed in the acidic pH range for the deglycosylated and the N110Q protein. In thermal and guanidine hydrochloride (GdnCl)-induced unfolding, the wild-type and high-mannose lectins possess higher stability compared with the deglycosylated recombinant lectin and both mutants, as measured by a higher Tm of denaturation or a greater free energy change, respectively. Reversibility experiments after thermal denaturation reveal that deglycosylated proteins tend to aggregate during thermal inactivation but the wild type shows a much greater recovery to the native state upon refolding. These results suggest that N-glycosylation in turmeric lectin is important for the maintenance of its proper folding upon changes in pH, and that the oligosaccharides help in maintaining the active conformation and prevent aggregation in unfolded or partially folded molecules. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

The FTS atomic spectrum tool (FAST) for rapid analysis of line spectra

NASA Astrophysics Data System (ADS)

Ruffoni, M. P.

2013-07-01

The FTS Atomic Spectrum Tool (FAST) is an interactive graphical program designed to simplify the analysis of atomic emission line spectra obtained from Fourier transform spectrometers. Calculated, predicted and/or known experimental line parameters are loaded alongside experimentally observed spectral line profiles for easy comparison between new experimental data and existing results. Many such line profiles, which could span numerous spectra, may be viewed simultaneously to help the user detect problems from line blending or self-absorption. Once the user has determined that their experimental line profile fits are good, a key feature of FAST is the ability to calculate atomic branching fractions, transition probabilities, and oscillator strengths-and their uncertainties-which is not provided by existing analysis packages. Program SummaryProgram title: FAST: The FTS Atomic Spectrum Tool Catalogue identifier: AEOW_v1_0 Program summary URL: http://cpc.cs.qub.ac.uk/summaries/AEOW_v1_0.html Program obtainable from: CPC Program Library, Queen's University, Belfast, N. Ireland Licensing provisions: GNU General Public License version 3 No. of lines in distributed program, including test data, etc.: 293058 No. of bytes in distributed program, including test data, etc.: 13809509 Distribution format: tar.gz Programming language: C++. Computer: Intel x86-based systems. Operating system: Linux/Unix/Windows. RAM: 8 MB minimum. About 50-200 MB for a typical analysis. Classification: 2.2, 2.3, 21.2. Nature of problem: Visualisation of atomic line spectra including the comparison of theoretical line parameters with experimental atomic line profiles. Accurate intensity calibration of experimental spectra, and the determination of observed relative line intensities that are needed for calculating atomic branching fractions and oscillator strengths. Solution method: FAST is centred around a graphical interface, where a user may view sets of experimental line profiles and compare them to calculated data (such as from the Kurucz database [1]), predicted line parameters, and/or previously known experimental results. With additional information on the spectral response of the spectrometer, obtained from a calibrated standard light source, FT spectra may be intensity calibrated. In turn, this permits the user to calculate atomic branching fractions and oscillator strengths, and their respective uncertainties. Running time: Open ended. Defined by the user. References: [1] R.L. Kurucz (2007). URL http://kurucz.harvard.edu/atoms/.

Development of program package for investigation and modeling of carbon nanostructures in diamond like carbon films with the help of Raman scattering and infrared absorption spectra line resolving

NASA Astrophysics Data System (ADS)

Hayrapetyan, David B.; Hovhannisyan, Levon; Mantashyan, Paytsar A.

2013-04-01

The analysis of complex spectra is an actual problem for modern science. The work is devoted to the creation of a software package, which analyzes spectrum in the different formats, possesses by dynamic knowledge database and self-study mechanism, performs automated analysis of the spectra compound based on knowledge database by application of certain algorithms. In the software package as searching systems, hyper-spherical random search algorithms, gradient algorithms and genetic searching algorithms were used. The analysis of Raman and IR spectrum of diamond-like carbon (DLC) samples were performed by elaborated program. After processing the data, the program immediately displays all the calculated parameters of DLC.

Interactive Visualization of Infrared Spectral Data: Synergy of Computation, Visualization, and Experiment for Learning Spectroscopy

NASA Astrophysics Data System (ADS)

Lahti, Paul M.; Motyka, Eric J.; Lancashire, Robert J.

2000-05-01

A straightforward procedure is described to combine computation of molecular vibrational modes using commonly available molecular modeling programs with visualization of the modes using advanced features of the MDL Information Systems Inc. Chime World Wide Web browser plug-in. Minor editing of experimental spectra that are stored in the JCAMP-DX format allows linkage of IR spectral frequency ranges to Chime molecular display windows. The spectra and animation files can be combined by Hypertext Markup Language programming to allow interactive linkage between experimental spectra and computationally generated vibrational displays. Both the spectra and the molecular displays can be interactively manipulated to allow the user maximum control of the objects being viewed. This procedure should be very valuable not only for aiding students through visual linkage of spectra and various vibrational animations, but also by assisting them in learning the advantages and limitations of computational chemistry by comparison to experiment.

Analysis and identification of two similar traditional Chinese medicines by using a three-stage infrared spectroscopy: Ligusticum chuanxiong, Angelica sinensis and their different extracts

NASA Astrophysics Data System (ADS)

Xiang, Li; Wang, Jingjuan; Zhang, Guijun; Rong, Lixin; Wu, Haozhong; Sun, Suqin; Guo, Yizhen; Yang, Yanfang; Lu, Lina; Qu, Lei

2016-11-01

Rhizoma Chuanxiong (CX) and Radix Angelica sinensis (DG) are very important Traditional Chinese Medicine (TCM) and usually used in clinic. They both are from the Umbelliferae family, and have almost similar chemical constituents with each other. It is complicated, time-consuming and laborious to discriminate them by using the chromatographic methods such as high performance liquid chromatography (HPLC) and gas chromatography (GC). Therefore, to find a fast, applicable and effective identification method for two herbs is urged in quality research of TCM. In this paper, by using a three-stage infrared spectroscopy (Fourier transform infrared spectroscopy (FT-IR), the second derivative infrared spectroscopy (SD-IR) and two-dimensional correlation infrared spectroscopy (2D-IR)), we analyzed and discriminated CX, DG and their different extracts (aqueous extract, alcoholic extract and petroleum ether extract). In FT-IR, all the CX and DG samples' spectra seemed similar, but they had their own unique macroscopic fingerprints to identify. Through comparing with the spectra of sucrose and the similarity calculation, we found the content of sucrose in DG raw materials was higher than in CX raw materials. The significant differences in alcoholic extract appeared that in CX alcoholic extract, the peaks at 1743 cm-1 was obviously stronger than the peak at same position in DG alcoholic extract. Besides in petroleum ether extract, we concluded CX contained much more ligustilide than DG by the similarity calculation. With the function of SD-IR, some tiny differences were amplified and overlapped peaks were also unfolded in FT-IR. In the range of 1100-1175 cm-1, there were six peaks in the SD-IR spectra of DG and the intensity, shape and location of those six peaks were similar to that of sucrose, while only two peaks could be observed in that of CX and those two peaks were totally different from sucrose in shape and relative intensity. This result was consistent with that of the FT-IR. Several undetected characteristic fingerprints in FT-IR and SD-IR spectra were further disclosed by 2D-IR spectra. In the range of 1120-1500 cm-1, the FT-IR spectra and the SD-IR spectra of aqueous extract of CX and DG were almost similar and hard to be discriminated, but the 2D-IR spectra were markedly different. These findings indicated that the three-stage infrared spectroscopy can identify not only the main compositions in these two medicinal materials and their different extracts, but also can compare the differences of categories and quantities of chemical constituents between the similar samples. In conclusion, the three-stage infrared spectroscopy could identify the two similar TCM (CX and DG) quickly and effectively.

Simulation of urea-induced protein unfolding: a lesson from bovine β-lactoglobulin.

PubMed

Eberini, Ivano; Emerson, Andrew; Sensi, Cristina; Ragona, Laura; Ricchiuto, Piero; Pedretti, Alessandro; Gianazza, Elisabetta; Tramontano, Anna

2011-09-01

To investigate the molecular mechanisms involved in the very initial stages of protein unfolding, we carried out one long (1 μs) simulation of bovine β-lactoglobulin (BLG) together with three (500 ns) supporting MD runs, in which the unfolding conditions were produced by adding the osmolyte urea to the simulated systems and/or by increasing the thermal energy raising the temperature from 300 to 350 K. BLG was chosen, since it is a well-characterized model protein, for which structural and folding properties have been widely investigated by X-ray and NMR. MD trajectories were analyzed not only in terms of standard progress variables, such as backbone H-bonds, gyration radius width, secondary structure elements, but also through the scrutiny of interactions and dynamical behavior of specific key residues previously pointed out and investigated by NMR and belonging to a well known hydrophobic cluster. MD trajectories simulated in different unfolding conditions suggest that urea destabilizes BLG structure weakening protein::protein hydrophobic interactions and the hydrogen bond network. The early unfolding events, better observed at higher temperature, affect both secondary and tertiary structure of the protein. Copyright © 2011 Elsevier Inc. All rights reserved.

Unfolding of titin immunoglobulin domains by steered molecular dynamics simulation.

PubMed

Lu, H; Isralewitz, B; Krammer, A; Vogel, V; Schulten, K

1998-08-01

Titin, a 1-microm-long protein found in striated muscle myofibrils, possesses unique elastic and extensibility properties in its I-band region, which is largely composed of a PEVK region (70% proline, glutamic acid, valine, and lysine residue) and seven-strand beta-sandwich immunoglobulin-like (Ig) domains. The behavior of titin as a multistage entropic spring has been shown in atomic force microscope and optical tweezer experiments to partially depend on the reversible unfolding of individual Ig domains. We performed steered molecular dynamics simulations to stretch single titin Ig domains in solution with pulling speeds of 0.5 and 1.0 A/ps. Resulting force-extension profiles exhibit a single dominant peak for each Ig domain unfolding, consistent with the experimentally observed sequential, as opposed to concerted, unfolding of Ig domains under external stretching forces. This force peak can be attributed to an initial burst of backbone hydrogen bonds, which takes place between antiparallel beta-strands A and B and between parallel beta-strands A' and G. Additional features of the simulations, including the position of the force peak and relative unfolding resistance of different Ig domains, can be related to experimental observations.

Peripheral Protein Unfolding Drives Membrane Bending.

PubMed

Siaw, Hew Ming Helen; Raghunath, Gokul; Dyer, R Brian

2018-06-20

Dynamic modulation of lipid membrane curvature can be achieved by a number of peripheral protein binding mechanisms such as hy-drophobic insertion of amphipathic helices and membrane scaffolding. Recently, an alternative mechanism was proposed in which crowding of peripherally bound proteins induces membrane curvature through steric pressure generated by lateral collisions. This effect was enhanced using intrinsically disordered proteins that possess high hydrodynamic radii, prompting us to explore whether membrane bending can be triggered by the folding-unfolding transition of surface-bound proteins. We utilized histidine-tagged human serum albumin bound to Ni-NTA-DGS containing liposomes as our model system to test this hypothesis. We found that reduction of the disulfide bonds in the protein resulted in unfolding of HSA, which subsequently led to membrane tubule formation. The frequency of tubule formation was found to be significantly higher when the proteins were unfolded while being localized to a phase-separated domain as opposed to randomly distributed in fluid phase liposomes, indicating that the steric pressure generated from protein unfolding is directly responsible for membrane deformation. Our results are critical for the design of peripheral membrane protein-immobilization strategies and open new avenues for exploring mechanisms of membrane bending driven by conformational changes of peripheral membrane proteins.

The Unfolding MD Simulations of Cyclophilin: Analyzed by Surface Contact Networks and Their Associated Metrics

PubMed Central

Roy, Sourav; Basu, Sankar; Dasgupta, Dipak; Bhattacharyya, Dhananjay; Banerjee, Rahul

2015-01-01

Currently, considerable interest exists with regard to the dissociation of close packed aminoacids within proteins, in the course of unfolding, which could result in either wet or dry moltenglobules. The progressive disjuncture of residues constituting the hydrophobic core ofcyclophilin from L. donovani (LdCyp) has been studied during the thermal unfolding of the molecule, by molecular dynamics simulations. LdCyp has been represented as a surface contactnetwork (SCN) based on the surface complementarity (Sm) of interacting residues within themolecular interior. The application of Sm to side chain packing within proteins make it a very sensitive indicator of subtle perturbations in packing, in the thermal unfolding of the protein. Network based metrics have been defined to track the sequential changes in the disintegration ofthe SCN spanning the hydrophobic core of LdCyp and these metrics prove to be highly sensitive compared to traditional metrics in indicating the increased conformational (and dynamical) flexibility in the network. These metrics have been applied to suggest criteria distinguishing DMG, WMG and transition state ensembles and to identify key residues involved in crucial conformational/topological events during the unfolding process. PMID:26545107

The E. coli thioredoxin folding mechanism: the key role of the C-terminal helix.

PubMed

Vazquez, Diego S; Sánchez, Ignacio E; Garrote, Ana; Sica, Mauricio P; Santos, Javier

2015-02-01

In this work, the unfolding mechanism of oxidized Escherichia coli thioredoxin (EcTRX) was investigated experimentally and computationally. We characterized seven point mutants distributed along the C-terminal α-helix (CTH) and the preceding loop. The mutations destabilized the protein against global unfolding while leaving the native structure unchanged. Global analysis of the unfolding kinetics of all variants revealed a linear unfolding route with a high-energy on-pathway intermediate state flanked by two transition state ensembles TSE1 and TSE2. The experiments show that CTH is mainly unfolded in TSE1 and the intermediate and becomes structured in TSE2. Structure-based molecular dynamics are in agreement with these experiments and provide protein-wide structural information on transient states. In our model, EcTRX folding starts with structure formation in the β-sheet, while the protein helices coalesce later. As a whole, our results indicate that the CTH is a critical module in the folding process, restraining a heterogeneous intermediate ensemble into a biologically active native state and providing the native protein with thermodynamic and kinetic stability. Copyright © 2014 Elsevier B.V. All rights reserved.

Pressure-induced subunit dissociation and unfolding of dimeric beta-lactoglobulin.

PubMed Central

Valente-Mesquita, V L; Botelho, M M; Ferreira, S T

1998-01-01

Effects of hydrostatic pressure on dimeric beta-lactoglobulin A (beta-Lg) were investigated. Application of pressures of up to 3.5 kbar induced a significant red shift ( approximately 11 nm) and a 60% increase in intrinsic fluorescence emission of beta-Lg. These changes were very similar to those induced by guanidine hydrochloride, which caused subunit dissociation and unfolding of beta-Lg. A large hysteresis in the recovery of fluorescence parameters was observed upon decompression of beta-Lg. Pressure-induced dissociation and unfolding were not fully reversible, because of the formation of a nonnative intersubunit disulfide bond that hampered correct refolding of the dimer. Comparison between pressure dissociation/unfolding at 3 degrees C and 23 degrees C revealed a marked destabilization of beta-Lg at low temperature. The stability of beta-Lg toward pressure was significantly enhanced by 1 M NaCl, but not by glycerol (up to 20% v/v). These observations suggest that salt stabilization was not related to a general cosolvent effect, but may reflect charge screening. Interestingly, pressure-induced dissociation/unfolding was completely independent of beta-Lg concentration, in apparent violation of the law of mass action. Possible causes for this anomalous behavior are discussed. PMID:9649408

Residue solvent accessibilities in the unfolded polypeptide chain.

PubMed Central

Zielenkiewicz, P; Saenger, W

1992-01-01

The difference of solvent accessibilities in the native and unfolded states of the protein is used as a measure of the hydrophobic contribution to the free energy of folding. We present a new approximation of amino acids solvent accessibilities in the unfolded state based on the 1-ns molecular dynamics simulation of Ala-X-Ala tripeptides at a temperature of 368 K. The standard accessibility values averaged from the molecular dynamics study are significantly lower from those previously obtained by considering only selected conformations of Ala-X-Ala tripeptides. PMID:1489908

Real-time investigation of protein unfolding at an air–water interface at the 1 s time scale

PubMed Central

Yano, Yohko F.; Arakawa, Etsuo; Voegeli, Wolfgang; Matsushita, Tadashi

2013-01-01

Protein unfolding at an air–water interface has been demonstrated such that the X-ray reflectivity can be measured with an acquisition time of 1 s using a recently developed simultaneous multiple-angle–wavelength-dispersive X-ray reflectometer. This has enabled the electron density profile of the adsorbed protein molecules to be obtained in real time. A globular protein, lysozyme, adsorbed at the air–water interface is found to unfold into a flat shape within 1 s. PMID:24121352

Infusing Training into the Documentation and Culture of Ares I Upper Stage Design and Manufacturing

NASA Technical Reports Server (NTRS)

Scott, David W.

2009-01-01

In roughly two years time, Marshall Space Flight Center's (MSFC) Mission Operations Laboratory (MOL) has incubated a personnel training and certification program for about 1000 learners and multiple phases of the Ares I Upper Stage (US) project. Previous MOL-developed training programs focused on about 100 learners with a focus on operations, and had enough full-time training staff to develop courseware and provide training administration. This paper discusses 1) how creation of a broad, structured training program unfolded as feedback from more narrowly defined tasks, 2) how training philosophy, development methods, and administration are being simplified and tailored so that many Upper Stage organizations can grow their own training yet maintain consistency, accountability, and traceability across the project, and 3) possibilities for interfacing with the production contractor's training system and staff.

Analysis of positron lifetime spectra in polymers

NASA Technical Reports Server (NTRS)

Singh, Jag J.; Mall, Gerald H.; Sprinkle, Danny R.

1988-01-01

A new procedure for analyzing multicomponent positron lifetime spectra in polymers was developed. It requires initial estimates of the lifetimes and the intensities of various components, which are readily obtainable by a standard spectrum stripping process. These initial estimates, after convolution with the timing system resolution function, are then used as the inputs for a nonlinear least squares analysis to compute the estimates that conform to a global error minimization criterion. The convolution integral uses the full experimental resolution function, in contrast to the previous studies where analytical approximations of it were utilized. These concepts were incorporated into a generalized Computer Program for Analyzing Positron Lifetime Spectra (PAPLS) in polymers. Its validity was tested using several artificially generated data sets. These data sets were also analyzed using the widely used POSITRONFIT program. In almost all cases, the PAPLS program gives closer fit to the input values. The new procedure was applied to the analysis of several lifetime spectra measured in metal ion containing Epon-828 samples. The results are described.

Effect of pH, temperature, and salt on the stability of Escherichia coli- and Chinese hamster ovary cell-derived IgG1 Fc.

PubMed

Li, Cynthia H; Narhi, Linda O; Wen, Jie; Dimitrova, Mariana; Wen, Zai-qing; Li, Jenny; Pollastrini, Joseph; Nguyen, Xichdao; Tsuruda, Trace; Jiang, Yijia

2012-12-18

The circulation half-life of a potential therapeutic can be increased by fusing the molecule of interest (an active peptide, the extracellular domain of a receptor, an enzyme, etc.) to the Fc fragment of a monoclonal antibody. For the fusion protein to be a successful therapeutic, it must be stable to process and long-term storage conditions, as well as to physiological conditions. The stability of the Fc used is critical for obtaining a successful therapeutic protein. The effects of pH, temperature, and salt on the stabilities of Escherichia coli- and Chinese hamster ovary cell (CHO)-derived IgG1 Fc high-order structure were probed using a variety of biophysical techniques. Fc molecules derived from both E. coli and CHO were compared. The IgG1 Fc molecules from both sources (glycosylated and aglycosylated) are folded at neutral pH and behave similarly upon heat- and low pH-induced unfolding. The unfolding of both IgG1 Fc molecules occurs via a multistep unfolding process, with the tertiary structure and C(H)2 domain unfolding first, followed by changes in the secondary structure and C(H)3 domain. The acid-induced unfolding of IgG1 Fc molecules is only partially reversible, with the formation of high-molecular weight species. The CHO-derived Fc protein (glycosylated) is more compact (smaller hydrodynamic radius) than the E. coli-derived protein (aglycosylated) at neutral pH. Unfolding is dependent on pH and salt concentration. The glycosylated C(H)2 domain melts at a temperature 4-5 °C higher than that of the aglycosylated domain, and the low-pH-induced unfolding of the glycosylated Fc molecule occurs at a pH ~0.5 pH unit lower than that of the aglycosylated protein. The difference observed between E. coli- and CHO-derived Fc molecules primarily involves the C(H)2 domain, where the glycosylation of the Fc resides.

Conformational stability of pGEX-expressed Schistosoma japonicum glutathione S-transferase: a detoxification enzyme and fusion-protein affinity tag.

PubMed Central

Kaplan, W.; Hüsler, P.; Klump, H.; Erhardt, J.; Sluis-Cremer, N.; Dirr, H.

1997-01-01

A glutathione S-transferase (Sj26GST) from Schistosoma japonicum, which functions in the parasite's Phase II detoxification pathway, is expressed by the Pharmacia pGEX-2T plasmid and is used widely as a fusion-protein affinity tag. It contains all 217 residues of Sj26GST and an additional 9-residue peptide linker with a thrombin cleavage site at its C-terminus. Size-exclusion HPLC (SEC-HPLC) and SDS-PAGE studies indicate that purification of the homodimeric protein under nonreducing conditions results in the reversible formation of significant amounts of 160-kDa and larger aggregates without a loss in catalytic activity. The basis for oxidative aggregation can be ascribed to the high degree of exposure of the four cysteine residues per subunit. The conformational stability of the dimeric protein was studied by urea- and temperature-induced unfolding techniques. Fluorescence-spectroscopy, SEC-HPLC, urea- and temperature-gradient gel electrophoresis, differential scanning microcalorimetry, and enzyme activity were employed to monitor structural and functional changes. The unfolding data indicate the absence of thermodynamically stable intermediates and that the unfolding/refolding transition is a two-state process involving folded native dimer and unfolded monomer. The stability of the protein was found to be dependent on its concentration, with a delta G degree (H2O) = 26.0 +/- 1.7 kcal/mol. The strong relationship observed between the m-value and the size of the protein indicates that the amount of protein surface area exposed to solvent upon unfolding is the major structural determinant for the dependence of the protein's free energy of unfolding on urea concentration. Thermograms obtained by differential scanning microcalorimetry also fitted a two-state unfolding transition model with values of delta Cp = 7,440 J/mol per K, delta H = 950.4 kJ/mol, and delta S = 1,484 J/mol. PMID:9041642

Conformational stability of pGEX-expressed Schistosoma japonicum glutathione S-transferase: a detoxification enzyme and fusion-protein affinity tag.

PubMed

Kaplan, W; Hüsler, P; Klump, H; Erhardt, J; Sluis-Cremer, N; Dirr, H

1997-02-01

A glutathione S-transferase (Sj26GST) from Schistosoma japonicum, which functions in the parasite's Phase II detoxification pathway, is expressed by the Pharmacia pGEX-2T plasmid and is used widely as a fusion-protein affinity tag. It contains all 217 residues of Sj26GST and an additional 9-residue peptide linker with a thrombin cleavage site at its C-terminus. Size-exclusion HPLC (SEC-HPLC) and SDS-PAGE studies indicate that purification of the homodimeric protein under nonreducing conditions results in the reversible formation of significant amounts of 160-kDa and larger aggregates without a loss in catalytic activity. The basis for oxidative aggregation can be ascribed to the high degree of exposure of the four cysteine residues per subunit. The conformational stability of the dimeric protein was studied by urea- and temperature-induced unfolding techniques. Fluorescence-spectroscopy, SEC-HPLC, urea- and temperature-gradient gel electrophoresis, differential scanning microcalorimetry, and enzyme activity were employed to monitor structural and functional changes. The unfolding data indicate the absence of thermodynamically stable intermediates and that the unfolding/refolding transition is a two-state process involving folded native dimer and unfolded monomer. The stability of the protein was found to be dependent on its concentration, with a delta G degree (H2O) = 26.0 +/- 1.7 kcal/mol. The strong relationship observed between the m-value and the size of the protein indicates that the amount of protein surface area exposed to solvent upon unfolding is the major structural determinant for the dependence of the protein's free energy of unfolding on urea concentration. Thermograms obtained by differential scanning microcalorimetry also fitted a two-state unfolding transition model with values of delta Cp = 7,440 J/mol per K, delta H = 950.4 kJ/mol, and delta S = 1,484 J/mol.

High- and low-temperature unfolding of human high-density apolipoprotein A-2.

PubMed Central

Gursky, O.; Atkinson, D.

1996-01-01

Human plasma apolipoprotein A-2 (apoA-2) is the second major protein of the high-density lipoproteins that mediate the transport and metabolism of cholesterol. Using CD spectroscopy and differential scanning calorimetry, we demonstrate that the structure of lipid-free apoA-2 in neutral low-salt solutions is most stable at approximately 25 degrees C and unfolds reversibly both upon heating and cooling from 25 degrees C. High-temperature unfolding of apoA-2, monitored by far-UV CD, extends from 25-85 degrees C with midpoint Th = 56 +/- 2 degrees C and vant Hoff's enthalpy delta H(Th) = 17 +/- 2 kcal/mol that is substantially lower than the expected enthalpy of melting of the alpha-helical structure. This suggests low-cooperativity apoA-2 unfolding. The apparent free energy of apoA-2 stabilization inferred from the CD analysis of the thermal unfolding, delta G(app)(25 degrees) = 0.82 +/- 0.15 kcal/mol, agrees with the value determined from chemical denaturation. Enhanced low-temperature stability of apoA-2 observed upon increase in Na2HPO4 concentration from 0.3 mM to 50 mM or addition of 10% glycerol may be linked to reduced water activity. The close proximity of the heat and cold unfolding transitions, that is consistent with low delta G(app)(25 degrees), indicates that lipid-free apoA-2 has a substantial hydrophobic core but is only marginally stable under near-physiological solvent conditions. This suggests that in vivo apoA-2 transfer is unlikely to proceed via the lipid-free state. Low delta H(Th) and low apparent delta Cp approximately 0.52 kcal/mol.K inferred from the far-UV CD analysis of apoA-2 unfolding, and absence of tertiary packing interactions involving Tyr groups suggested by near-UV CD, are consistent with a molten globular-like state of lipid-free apoA-2. PMID:8880911

Identification of Residual Structure in the Unfolded State of Ribonuclease H1 from the Moderately Thermophilic Chlorobium tepidum: Comparison with Thermophilic and Mesophilic Homologues†

PubMed Central

Ratcliff, Kathleen; Marqusee, Susan

2010-01-01

Ribonucleases H from organisms that grow at different temperatures demonstrate a variable change in heat capacity upon unfolding (ΔC°P) [Ratcliff, K., et al. (2009) Biochemistry 48, 5890–5898]. This ΔC°P has been shown to correlate with a tolerance to higher temperatures and residual structure in the unfolded state of the thermophilic proteins. In the RNase H from Thermus thermophilus, the low ΔC°P has been shown to arise from the same region as the folding core of the protein, and mutagenic studies have shown that loss of a hydrophobic residue in this region can disrupt this residual unfolded state structure and result in a return to a more mesophile-like ΔC°P [Robic, S., et al. (2002) Protein Sci. 11, 381–389; Robic, S., et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 11345–11349]. To understand further how residual structure in the unfolded state is encoded in the sequences of these thermophilic proteins, we subjected the RNase H from Chlorobium tepidum to similar studies. Analysis of new chimeric proteins reveals that like T. thermophilus RNase H, the folding core of C. tepidum RNaseH plays an important role in the unfolded state of this protein. Mutagenesis studies, based on both a computational investigation of the hydrophobic networks in the core region and comparisons with similar studies on T. thermophilus RNase H, identify new residues involved in this residual structure and suggest that the residual structure in the unfolded state of C. tepidum RNase H is more restricted than that of T. thermophilus. We conclude that while the folding core region determines the thermophilic-like behavior of this family of proteins, the residue-specific details vary. PMID:20491485

Stabilization of an α/β-hydrolase by introducing proline residues: salicylic binding protein 2 from tobacco

PubMed Central

Huang, Jun; Jones, Bryan J.; Kazlauskas, Romas J.

2015-01-01

α/β-Hydrolases are important enzymes for biocatalysis, but their stability often limits their application. As a model α/β-hydrolase, we investigated a plant esterase, salicylic acid binding protein 2 (SABP2). SABP2 shows typical stability to urea (unfolding free energy 6.9±1.5 kcal/mol) and to heat inactivation (T1/215 min 49.2±0.5 °C). Denaturation in urea occurs in two steps, but heat inactivation occurs in a single step. The first unfolding step in urea eliminates catalytic activity. Surprisingly, we found that the first unfolding likely corresponds to the unfolding of the larger catalytic domain. Replacing selected amino acid residues with proline stabilized SABP2. Proline restricts the flexibility of the unfolded protein, thereby shifting the equilibrium toward the folded conformation. Seven locations for proline substitution were chosen either by amino acid sequence alignment with a more stable homolog or by targeting flexible regions in SABP2. Introducing proline in the catalytic domain stabilized SABP2 to the first unfolding in urea for three of five cases: L46P (+0.2 M urea), S70P (+0.1) and E215P (+0.9). Introducing proline in the cap domain did not (two of two cases), supporting the assignment that the first unfolding corresponds to the catalytic domain. Proline substitutions in both domains stabilized SABP2 to heat inactivation: L46P (ΔT1/215 min = +6.4 °C), S70P (+5.4), S115P (+1.8), S141P (+4.9), and E215P (+4.2). Combining substitutions did not further increase the stability to urea denaturation, but dramatically increased resistance to heat inactivation: L46P-S70P ΔT1/215 min = +25.7 °C. This straightforward proline substitution approach may also stabilize other α/β-hydrolases. PMID:26110207

Single-molecule studies of the Im7 folding landscape.

PubMed

Pugh, Sara D; Gell, Christopher; Smith, D Alastair; Radford, Sheena E; Brockwell, David J

2010-04-23

Under appropriate conditions, the four-helical Im7 (immunity protein 7) folds from an ensemble of unfolded conformers to a highly compact native state via an on-pathway intermediate. Here, we investigate the unfolded, intermediate, and native states populated during folding using diffusion single-pair fluorescence resonance energy transfer by measuring the efficiency of energy transfer (or proximity or P ratio) between pairs of fluorophores introduced into the side chains of cysteine residues placed in the center of helices 1 and 4, 1 and 3, or 2 and 4. We show that while the native states of each variant give rise to a single narrow distribution with high P values, the distributions of the intermediates trapped at equilibrium (denoted I(eqm)) are fitted by two Gaussian distributions. Modulation of the folding conditions from those that stabilize the intermediate to those that destabilize the intermediate enabled the distribution of lower P value to be assigned to the population of the unfolded ensemble in equilibrium with the intermediate state. The reduced stability of the I(eqm) variants allowed analysis of the effect of denaturant concentration on the compaction and breadth of the unfolded state ensemble to be quantified from 0 to 6 M urea. Significant compaction is observed as the concentration of urea is decreased in both the presence and absence of sodium sulfate, as previously reported for a variety of proteins. In the presence of Na(2)SO(4) in 0 M urea, the P value of the unfolded state ensemble approaches that of the native state. Concurrent with compaction, the ensemble displays increased peak width of P values, possibly reflecting a reduction in the rate of conformational exchange among iso-energetic unfolded, but compact conformations. The results provide new insights into the initial stages of folding of Im7 and suggest that the unfolded state is highly conformationally constrained at the outset of folding. (c) 2010 Elsevier Ltd. All rights reserved.

Single-Molecule Studies of the Im7 Folding Landscape

PubMed Central

Pugh, Sara D.; Gell, Christopher; Smith, D. Alastair; Radford, Sheena E.; Brockwell, David J.

2010-01-01

Under appropriate conditions, the four-helical Im7 (immunity protein 7) folds from an ensemble of unfolded conformers to a highly compact native state via an on-pathway intermediate. Here, we investigate the unfolded, intermediate, and native states populated during folding using diffusion single-pair fluorescence resonance energy transfer by measuring the efficiency of energy transfer (or proximity or P ratio) between pairs of fluorophores introduced into the side chains of cysteine residues placed in the center of helices 1 and 4, 1 and 3, or 2 and 4. We show that while the native states of each variant give rise to a single narrow distribution with high P values, the distributions of the intermediates trapped at equilibrium (denoted Ieqm) are fitted by two Gaussian distributions. Modulation of the folding conditions from those that stabilize the intermediate to those that destabilize the intermediate enabled the distribution of lower P value to be assigned to the population of the unfolded ensemble in equilibrium with the intermediate state. The reduced stability of the Ieqm variants allowed analysis of the effect of denaturant concentration on the compaction and breadth of the unfolded state ensemble to be quantified from 0 to 6 M urea. Significant compaction is observed as the concentration of urea is decreased in both the presence and absence of sodium sulfate, as previously reported for a variety of proteins. In the presence of Na2SO4 in 0 M urea, the P value of the unfolded state ensemble approaches that of the native state. Concurrent with compaction, the ensemble displays increased peak width of P values, possibly reflecting a reduction in the rate of conformational exchange among iso-energetic unfolded, but compact conformations. The results provide new insights into the initial stages of folding of Im7 and suggest that the unfolded state is highly conformationally constrained at the outset of folding. PMID:20211187

Single-Molecule Mechanical (Un)folding of RNA Hairpins: Effects of Single A-U to A∙C Pair Substitutions and Single Proton Binding and Implications for mRNA Structure-Induced -1 Ribosomal Frameshifting.

PubMed

Yang, Lixia; Zhong, Zhensheng; Tong, Cailing; Jia, Huan; Liu, Yiran; Chen, Gang

2018-06-08

A wobble A∙C pair can be protonated at near physiological pH to form a more stable wobble A+∙C pair. Here, we constructed an RNA hairpin (rHP) and three mutants with one A-U base pair substituted with an A∙C mismatch on the top (near the loop, U22C), middle (U25C) and bottom (U29C) positions of the stem, respectively. Our results on single-molecule mechanical (un)folding using optical tweezers reveal the destabilization effect of A-U to A∙C pair substitution, and protonation-dependent enhancement of mechanical stability facilitated through an increased folding rate, or decreased unfolding rate, or both. Our data show that protonation may occur rapidly upon the formation of apparent mechanical folding transition state. Furthermore, we measured the bulk -1 ribosomal frameshifting efficiencies of the hairpins by a cell-free translation assay. For the mRNA hairpins studied, -1 frameshifting efficiency correlates with mechanical unfolding force at equilibrium and folding rate at around 15 pN. U29C has a frameshifting efficiency similar to that of rHP (~2%). Accordingly, the bottom 2-4 base pairs of U29C may not form under a stretching force at pH 7.3, which is consistent with the fact that the bottom base pairs of the hairpins may be disrupted by ribosome at the slippery site. U22C and U25C have a similar frameshifting efficiency (~1%), indicating that both unfolding and folding rates of an mRNA hairpin in a crowded environment may affect frameshifting. Our data indicate that mechanical (un)folding of RNA hairpins may mimic how mRNAs unfold and fold in the presence of translating ribosomes.

Beta-Barrel Scaffold of Fluorescent Proteins: Folding, Stability and Role in Chromophore Formation

PubMed Central

Stepanenko, Olesya V.; Stepanenko, Olga V.; Kuznetsova, Irina M.; Verkhusha, Vladislav V.; Turoverov, Konstantin K.

2013-01-01

This review focuses on the current view of the interaction between the β-barrel scaffold of fluorescent proteins and their unique chromophore located in the internal helix. The chromophore originates from the polypeptide chain and its properties are influenced by the surrounding protein matrix of the β-barrel. On the other hand, it appears that a chromophore tightens the β-barrel scaffold and plays a crucial role in its stability. Furthermore, the presence of a mature chromophore causes hysteresis of protein unfolding and refolding. We survey studies measuring protein unfolding and refolding using traditional methods as well as new approaches, such as mechanical unfolding and reassembly of truncated fluorescent proteins. We also analyze models of fluorescent protein unfolding and refolding obtained through different approaches, and compare the results of protein folding in vitro to co-translational folding of a newly synthesized polypeptide chain. PMID:23351712

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.

Contribution of Long-Range Interactions to the Secondary Structure of an Unfolded Globin

PubMed Central

Fedyukina, Daria V.; Rajagopalan, Senapathy; Sekhar, Ashok; Fulmer, Eric C.; Eun, Ye-Jin; Cavagnero, Silvia

2010-01-01

This work explores the effect of long-range tertiary contacts on the distribution of residual secondary structure in the unfolded state of an α-helical protein. N-terminal fragments of increasing length, in conjunction with multidimensional nuclear magnetic resonance, were employed. A protein representative of the ubiquitous globin fold was chosen as the model system. We found that, while most of the detectable α-helical population in the unfolded ensemble does not depend on the presence of the C-terminal region (corresponding to the native G and H helices), specific N-to-C long-range contacts between the H and A-B-C regions enhance the helical secondary structure content of the N terminus (A-B-C regions). The simple approach introduced here, based on the evaluation of N-terminal polypeptide fragments of increasing length, is of general applicability to identify the influence of long-range interactions in unfolded proteins. PMID:20816043

Dynamical properties of α-amylase in the folded and unfolded state: the role of thermal equilibrium fluctuations for conformational entropy and protein stabilisation

NASA Astrophysics Data System (ADS)

Fitter, J.; Herrmann, R.; Hauß, T.; Lechner, R. E.; Dencher, N. A.

2001-07-01

A comparative analysis of thermal equilibrium fluctuations occurring in a mesophilic and in a thermophilic α-amylase was performed to study the effect of structural fluctuations on thermostability. The thermal fluctuations determining the conformational entropy of both enzymes have been characterised for the folded (at 30°C and 60°C) and for the unfolded state by applying neutron spectroscopy (at 30°C). The folded state shows a higher structural flexibility for the thermophilic protein as compared to the mesophilic homologue. In contrast, the unfolded state of both enzymes is rather similar with respect to the structural fluctuations. On the basis of this result, a mechanism characterised by entropic stabilisation (i.e., smaller Δ S for the unfolding transition of thermophilic α-amylase) can be assumed to be responsible for the higher thermostability of the thermophilic enzyme.

Theoretical and Experimental Investigation of the Translational Diffusion of Proteins in the Vicinity of Temperature-Induced Unfolding Transition.

PubMed

Molchanov, Stanislav; Faizullin, Dzhigangir A; Nesmelova, Irina V

2016-10-06

Translational diffusion is the most fundamental form of transport in chemical and biological systems. The diffusion coefficient is highly sensitive to changes in the size of the diffusing species; hence, it provides important information on the variety of macromolecular processes, such as self-assembly or folding-unfolding. Here, we investigate the behavior of the diffusion coefficient of a macromolecule in the vicinity of heat-induced transition from folded to unfolded state. We derive the equation that describes the diffusion coefficient of the macromolecule in the vicinity of the transition and use it to fit the experimental data from pulsed-field-gradient nuclear magnetic resonance (PFG NMR) experiments acquired for two globular proteins, lysozyme and RNase A, undergoing temperature-induced unfolding. A very good qualitative agreement between the theoretically derived diffusion coefficient and experimental data is observed.

New Optical Constants for Amorphous and Crystalline H2O-ice

NASA Technical Reports Server (NTRS)

Mastrapa, Rachel; Bernstein, Max; Sandford, Scott

2006-01-01

We have used the infrared spectra of laboratory ices to calculate the real and imaginary indices of refraction for amorphous and crystalline H2O-ice. We create H2O-ice samples in vacuum (approx. 10(exp ^-8)Torr). We measure the thickness of the sample by reflecting a He-Ne laser off of the sample and counting interference fringes as it grows and then collect transmission spectra of the samples in the wavelength range 1.25-22 micrometers. Using the ice thickness and transmission spectrum we calculate the imaginary part of the index of refraction. A Kramers-Kronig calculation is then used to calculate the real part of the index of refraction (Berland et al. 1994; Hudgins et al. 1993). These optical constants can be used to create model spectra for comparison to spectra from Solar System objects. We will summarize the differences between the amorphous and crystalline H2O-ice spectra. These include weakening of features and shifting of features to shorter wavelength in amorphous H,O-ice spectra. We will also discuss methods of using band area ratios to quickly estimate the fraction of amorphous to crystalline H2O-ice. We acknowledge financial support from the NASA Origins of the Solar System Program, the NASA Planetary Geology and Geophysics Program, and the NASA Postdoctoral Program.

IDEN2-A program for visual identification of spectral lines and energy levels in optical spectra of atoms and simple molecules

NASA Astrophysics Data System (ADS)

Azarov, V. I.; Kramida, A.; Vokhmentsev, M. Ya.

2018-04-01

The article describes a Java program that can be used in a user-friendly way to visually identify spectral lines observed in complex spectra with theoretically predicted transitions between atomic or molecular energy levels. The program arranges various information about spectral lines and energy levels in such a way that line identification and determination of positions of experimentally observed energy levels become much easier tasks that can be solved fast and efficiently.

Chilling and heat requirements for leaf unfolding in European beech and sessile oak populations at the southern limit of their distribution range.

PubMed

Dantec, Cécile F; Vitasse, Yann; Bonhomme, Marc; Louvet, Jean-Marc; Kremer, Antoine; Delzon, Sylvain

2014-11-01

With global warming, an advance in spring leaf phenology has been reported worldwide. However, it is difficult to forecast phenology for a given species, due to a lack of knowledge about chilling requirements. We quantified chilling and heat requirements for leaf unfolding in two European tree species and investigated their relative contributions to phenological variations between and within populations. We used an extensive database containing information about the leaf phenology of 14 oak and 10 beech populations monitored over elevation gradients since 2005. In parallel, we studied the various bud dormancy phases, in controlled conditions, by regularly sampling low- and high-elevation populations during fall and winter. Oak was 2.3 times more sensitive to temperature for leaf unfolding over the elevation gradient and had a lower chilling requirement for dormancy release than beech. We found that chilling is currently insufficient for the full release of dormancy, for both species, at the lowest elevations in the area studied. Genetic variation in leaf unfolding timing between and within oak populations was probably due to differences in heat requirement rather than differences in chilling requirement. Our results demonstrate the importance of chilling for leaf unfolding in forest trees and indicate that the advance in leaf unfolding phenology with increasing temperature will probably be less pronounced than forecasted. This highlights the urgent need to determine experimentally the interactions between chilling and heat requirements in forest tree species, to improve our understanding and modeling of changes in phenological timing under global warming.

Microsecond simulations of the folding/unfolding thermodynamics of the Trp-cage mini protein

PubMed Central

Day, Ryan; Paschek, Dietmar; Garcia, Angel E.

2012-01-01

We study the unbiased folding/unfolding thermodynamics of the Trp-cage miniprotein using detailed molecular dynamics simulations of an all-atom model of the protein in explicit solvent, using the Amberff99SB force field. Replica-exchange molecular dynamics (REMD) simulations are used to sample the protein ensembles over a broad range of temperatures covering the folded and unfolded states, and at two densities. The obtained ensembles are shown to reach equilibrium in the 1 μs per replica timescale. The total simulation time employed in the calculations exceeds 100 μs. Ensemble averages of the fraction folded, pressure, and energy differences between the folded and unfolded states as a function of temperature are used to model the free energy of the folding transition, ΔG(P,T), over the whole region of temperature and pressures sampled in the simulations. The ΔG(P,T) diagram describes an ellipse over the range of temperatures and pressures sampled, predicting that the system can undergo pressure induced unfolding and cold denaturation at low temperatures and high pressures, and unfolding at low pressures and high temperatures. The calculated free energy function exhibits remarkably good agreement with the experimental folding transition temperature (Tf = 321 K), free energy and specific heat changes. However, changes in enthalpy and entropy are significantly different than the experimental values. We speculate that these differences may be due to the simplicity of the semi-empirical force field used in the simulations and that more elaborate force fields may be required to describe appropriately the thermodynamics of proteins. PMID:20408169

An alternatively spliced heat shock transcription factor, OsHSFA2dI, functions in the heat stress-induced unfolded protein response in rice.

PubMed

Cheng, Q; Zhou, Y; Liu, Z; Zhang, L; Song, G; Guo, Z; Wang, W; Qu, X; Zhu, Y; Yang, D

2015-03-01

As sessile organisms, plants have evolved a wide range of defence pathways to cope with environmental stress such as heat shock. However, the molecular mechanism of these defence pathways remains unclear in rice. In this study, we found that OsHSFA2d, a heat shock transcriptional factor, encodes two main splice variant proteins, OsHSFA2dI and OsHSFA2dII in rice. Under normal conditions, OsHSFA2dII is the dominant but transcriptionally inactive spliced form. However, when the plant suffers heat stress, OsHSFA2d is alternatively spliced into a transcriptionally active form, OsHSFA2dI, which participates in the heat stress response (HSR). Further study found that this alternative splicing was induced by heat shock rather than photoperiod. We found that OsHSFA2dI is localised to the nucleus, whereas OsHSFA2dII is localised to the nucleus and cytoplasm. Moreover, expression of the unfolded protein response (UNFOLDED PROTEIN RESPONSE) sensors, OsIRE1, OsbZIP39/OsbZIP60 and the UNFOLDED PROTEIN RESPONSE marker OsBiP1, was up-regulated. Interestingly, OsbZIP50 was also alternatively spliced under heat stress, indicating that UNFOLDED PROTEIN RESPONSE signalling pathways were activated by heat stress to re-establish cellular protein homeostasis. We further demonstrated that OsHSFA2dI participated in the unfolded protein response by regulating expression of OsBiP1. © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands.

Engineered Human Antibody Constant Domains with Increased Stability*S⃞

PubMed Central

Gong, Rui; Vu, Bang K.; Feng, Yang; Prieto, DaRue A.; Dyba, Marzena A.; Walsh, Joseph D.; Prabakaran, Ponraj; Veenstra, Timothy D.; Tarasov, Sergey G.; Ishima, Rieko; Dimitrov, Dimiter S.

2009-01-01

The immunoglobulin (Ig) constant CH2 domain is critical for antibody effector functions. Isolated CH2 domains are promising as scaffolds for construction of libraries containing diverse binders that could also confer some effector functions. However, previous work has shown that an isolated murine CH2 domain is relatively unstable to thermally induced unfolding. To explore unfolding mechanisms of isolated human CH2 and increase its stability γ1 CH2 was cloned and a panel of cysteine mutants was constructed. Human γ1 CH2 unfolded at a higher temperature (Tm = 54.1 °C, as measured by circular dichroism) than that previously reported for a mouse CH2 (41 °C). One mutant (m01) was remarkably stable (Tm = 73.8 °C). Similar results were obtained by differential scanning calorimetry. This mutant was also significantly more stable than the wild-type CH2 against urea induced unfolding (50% unfolding at urea concentration of 6.8 m versus 4.2 m). The m01 was highly soluble and monomeric. The existence of the second disulfide bond in m01 and its correct position were demonstrated by mass spectrometry and nuclear magnetic resonance spectroscopy, respectively. The loops were on average more flexible than the framework in both CH2 and m01, and the overall secondary structure was not affected by the additional disulfide bond. These data suggest that a human CH2 domain is relatively stable to unfolding at physiological temperature, and that both CH2 and the highly stable mutant m01 are promising new scaffolds for the development of therapeutics against human diseases. PMID:19307178

Force-dependent switch in protein unfolding pathways and transition-state movements

PubMed Central

Zhuravlev, Pavel I.; Hinczewski, Michael; Chakrabarti, Shaon; Marqusee, Susan; Thirumalai, D.

2016-01-01

Although it is known that single-domain proteins fold and unfold by parallel pathways, demonstration of this expectation has been difficult to establish in experiments. Unfolding rate, ku(f), as a function of force f, obtained in single-molecule pulling experiments on src SH3 domain, exhibits upward curvature on a log⁡ku(f) plot. Similar observations were reported for other proteins for the unfolding rate ku([C]). These findings imply unfolding in these single-domain proteins involves a switch in the pathway as f or [C] is increased from a low to a high value. We provide a unified theory demonstrating that if log⁡ku as a function of a perturbation (f or [C]) exhibits upward curvature then the underlying energy landscape must be strongly multidimensional. Using molecular simulations we provide a structural basis for the switch in the pathways and dramatic shifts in the transition-state ensemble (TSE) in src SH3 domain as f is increased. We show that a single-point mutation shifts the upward curvature in log⁡ku(f) to a lower force, thus establishing the malleability of the underlying folding landscape. Our theory, applicable to any perturbation that affects the free energy of the protein linearly, readily explains movement in the TSE in a β-sandwich (I27) protein and single-chain monellin as the denaturant concentration is varied. We predict that in the force range accessible in laser optical tweezer experiments there should be a switch in the unfolding pathways in I27 or its mutants. PMID:26818842

Integrated view of internal friction in unfolded proteins from single-molecule FRET, contact quenching, theory, and simulations

PubMed Central

Soranno, Andrea; Holla, Andrea; Dingfelder, Fabian; Nettels, Daniel; Makarov, Dmitrii E.; Schuler, Benjamin

2017-01-01

Internal friction is an important contribution to protein dynamics at all stages along the folding reaction. Even in unfolded and intrinsically disordered proteins, internal friction has a large influence, as demonstrated with several experimental techniques and in simulations. However, these methods probe different facets of internal friction and have been applied to disparate molecular systems, raising questions regarding the compatibility of the results. To obtain an integrated view, we apply here the combination of two complementary experimental techniques, simulations, and theory to the same system: unfolded protein L. We use single-molecule Förster resonance energy transfer (FRET) to measure the global reconfiguration dynamics of the chain, and photoinduced electron transfer (PET), a contact-based method, to quantify the rate of loop formation between two residues. This combination enables us to probe unfolded-state dynamics on different length scales, corresponding to different parts of the intramolecular distance distribution. Both FRET and PET measurements show that internal friction dominates unfolded-state dynamics at low denaturant concentration, and the results are in remarkable agreement with recent large-scale molecular dynamics simulations using a new water model. The simulations indicate that intrachain interactions and dihedral angle rotation correlate with the presence of internal friction, and theoretical models of polymer dynamics provide a framework for interrelating the contribution of internal friction observed in the two types of experiments and in the simulations. The combined results thus provide a coherent and quantitative picture of internal friction in unfolded proteins that could not be attained from the individual techniques. PMID:28223518

Integrated view of internal friction in unfolded proteins from single-molecule FRET, contact quenching, theory, and simulations.

PubMed

Soranno, Andrea; Holla, Andrea; Dingfelder, Fabian; Nettels, Daniel; Makarov, Dmitrii E; Schuler, Benjamin

2017-03-07

Internal friction is an important contribution to protein dynamics at all stages along the folding reaction. Even in unfolded and intrinsically disordered proteins, internal friction has a large influence, as demonstrated with several experimental techniques and in simulations. However, these methods probe different facets of internal friction and have been applied to disparate molecular systems, raising questions regarding the compatibility of the results. To obtain an integrated view, we apply here the combination of two complementary experimental techniques, simulations, and theory to the same system: unfolded protein L. We use single-molecule Förster resonance energy transfer (FRET) to measure the global reconfiguration dynamics of the chain, and photoinduced electron transfer (PET), a contact-based method, to quantify the rate of loop formation between two residues. This combination enables us to probe unfolded-state dynamics on different length scales, corresponding to different parts of the intramolecular distance distribution. Both FRET and PET measurements show that internal friction dominates unfolded-state dynamics at low denaturant concentration, and the results are in remarkable agreement with recent large-scale molecular dynamics simulations using a new water model. The simulations indicate that intrachain interactions and dihedral angle rotation correlate with the presence of internal friction, and theoretical models of polymer dynamics provide a framework for interrelating the contribution of internal friction observed in the two types of experiments and in the simulations. The combined results thus provide a coherent and quantitative picture of internal friction in unfolded proteins that could not be attained from the individual techniques.

The secondary structure and the thermal unfolding parameters of the S-layer protein from Lactobacillus salivarius.

PubMed

Lighezan, Liliana; Georgieva, Ralitsa; Neagu, Adrian

2016-09-01

Surface layer (S-layer) proteins have been identified in the cell envelope of many organisms, such as bacteria and archaea. They self-assemble, forming monomolecular crystalline arrays. Isolated S-layer proteins are able to recrystallize into regular lattices, which proved useful in biotechnology. Here we investigate the structure and thermal unfolding of the S-layer protein isolated from Lactobacillus salivarius 16 strain of human origin. Using circular dichroism (CD) spectroscopy, and the software CDSSTR from DICHROWEB, CONTINLL from CDPro, as well as CDNN, we assess the fractions of the protein's secondary structural elements at temperatures ranging between 10 and 90 °C, and predict the tertiary class of the protein. To study the thermal unfolding of the protein, we analyze the temperature dependence of the CD signal in the far- and near-UV domains. Fitting the experimental data by two- and three-state models of thermal unfolding, we infer the midpoint temperatures, the temperature dependence of the changes in Gibbs free energy, enthalpy, and entropy of the unfolding transitions in standard conditions, and the temperature dependence of the equilibrium constant. We also estimate the changes in heat capacity at constant pressure in standard conditions. The results indicate that the thermal unfolding of the S-layer protein from L. salivarius is highly cooperative, since changes in the secondary and tertiary structures occur simultaneously. The thermodynamic analysis predicts a "cold" transition, at about -3 °C, of both the secondary and tertiary structures. Our findings may be important for the use of S-layer proteins in biotechnology and in biomedical applications.

Force-dependent switch in protein unfolding pathways and transition-state movements.

PubMed

Zhuravlev, Pavel I; Hinczewski, Michael; Chakrabarti, Shaon; Marqusee, Susan; Thirumalai, D

2016-02-09

Although it is known that single-domain proteins fold and unfold by parallel pathways, demonstration of this expectation has been difficult to establish in experiments. Unfolding rate, [Formula: see text], as a function of force f, obtained in single-molecule pulling experiments on src SH3 domain, exhibits upward curvature on a [Formula: see text] plot. Similar observations were reported for other proteins for the unfolding rate [Formula: see text]. These findings imply unfolding in these single-domain proteins involves a switch in the pathway as f or [Formula: see text] is increased from a low to a high value. We provide a unified theory demonstrating that if [Formula: see text] as a function of a perturbation (f or [Formula: see text]) exhibits upward curvature then the underlying energy landscape must be strongly multidimensional. Using molecular simulations we provide a structural basis for the switch in the pathways and dramatic shifts in the transition-state ensemble (TSE) in src SH3 domain as f is increased. We show that a single-point mutation shifts the upward curvature in [Formula: see text] to a lower force, thus establishing the malleability of the underlying folding landscape. Our theory, applicable to any perturbation that affects the free energy of the protein linearly, readily explains movement in the TSE in a β-sandwich (I27) protein and single-chain monellin as the denaturant concentration is varied. We predict that in the force range accessible in laser optical tweezer experiments there should be a switch in the unfolding pathways in I27 or its mutants.

DXRaySMCS: a user-friendly interface developed for prediction of diagnostic radiology X-ray spectra produced by Monte Carlo (MCNP-4C) simulation.

PubMed

Bahreyni Toossi, M T; Moradi, H; Zare, H

2008-01-01

In this work, the general purpose Monte Carlo N-particle radiation transport computer code (MCNP-4C) was used for the simulation of X-ray spectra in diagnostic radiology. The electron's path in the target was followed until its energy was reduced to 10 keV. A user-friendly interface named 'diagnostic X-ray spectra by Monte Carlo simulation (DXRaySMCS)' was developed to facilitate the application of MCNP-4C code for diagnostic radiology spectrum prediction. The program provides a user-friendly interface for: (i) modifying the MCNP input file, (ii) launching the MCNP program to simulate electron and photon transport and (iii) processing the MCNP output file to yield a summary of the results (relative photon number per energy bin). In this article, the development and characteristics of DXRaySMCS are outlined. As part of the validation process, output spectra for 46 diagnostic radiology system settings produced by DXRaySMCS were compared with the corresponding IPEM78. Generally, there is a good agreement between the two sets of spectra. No statistically significant differences have been observed between IPEM78 reported spectra and the simulated spectra generated in this study.

Kinetic evidence for folding and unfolding intermediates in staphylococcal nuclease.

PubMed

Walkenhorst, W F; Green, S M; Roder, H

1997-05-13

The complex kinetic behavior commonly observed in protein folding studies suggests that a heterogeneous population of molecules exists in solution and that a number of discrete steps are involved in the conversion of unfolded molecules to the fully native form. A central issue in protein folding is whether any of these kinetic events represent conformational steps important for efficient folding rather than side reactions caused by slow steps such as proline isomerization or misfolding of the polypeptide chain. In order to address this question, we used stopped-flow fluorescence techniques to characterize the kinetic mechanism of folding and unfolding for a Pro- variant of SNase in which all six proline residues were replaced by glycines or alanines. Compared to the wild-type protein, which exhibits a series of proline-dependent slow folding phases, the folding kinetics of Pro- SNase were much simpler, which made quantitative kinetic analysis possible. Despite the absence of prolines or other complicating factors, the folding kinetics still contain several phases and exhibit a complex denaturant dependence. The GuHCl dependence of the major observable folding phase and a distinct lag in the appearance of the native state provide clear evidence for an early folding intermediate. The fluorescence of Trp140 in the alpha-helical domain is insensitive to the formation of this early intermediate, which is consistent with a partially folded state with a stable beta-domain and a largely disordered alpha-helical region. A second intermediate is required to model the kinetics of unfolding for the Pro- variant, which shows evidence for a denaturant-induced change in the rate-limiting unfolding step. With the inclusion of these two intermediates, we are able to completely model the major phase(s) in both folding and unfolding across a wide range of denaturant concentrations using a sequential four-state folding mechanism. In order to model the minor slow phase observed for the Pro- mutant, a six-state scheme containing a parallel pathway originating from a distinct unfolded state was required. The properties of this alternate unfolded conformation are consistent with those expected due to the presence of a non-prolyl cis peptide bond. To test the kinetic model, we used simulations based on the six-state scheme and were able to completely reproduce the folding kinetics for Pro- SNase across a range of denaturant concentrations.

Prediction and analysis of structure, stability and unfolding of thermolysin-like proteases

NASA Astrophysics Data System (ADS)

Vriend, Gert; Eijsink, Vincent

1993-08-01

Bacillus neutral proteases (NPs) form a group of well-characterized homologous enzymes, that exhibit large differences in thermostability. The three-dimensional (3D) structures of several of these enzymes have been modelled on the basis of the crystal structures of the NPs of B. thermoproteolyticus (thermolysin) and B. cercus. Several new techniques have been developed to improve the model-building procedures. Also a model-building by mutagenesis' strategy was used, in which mutants were designed just to shed light on parts of the structures that were particularly hard to model. The NP models have been used for the prediction of site-directed mutations aimed at improving the thermostability of the enzymes. Predictions were made using several novel computational techniques, such as position-specific rotamer searching, packing quality analysis and property-profile database searches. Many stabilizing mutations were predicted and produced: improvement of hydrogen bonding, exclusion of buried water molecules, capping helices, improvement of hydrophobic interactions and entropic stabilization have been applied successfully. At elevated temperatures NPs are irreversibly inactivated as a result of autolysis. It has been shown that this denaturation process is independent of the protease activity and concentration and that the inactivation follows first-order kinetics. From this it has been conjectured that local unfolding of (surface) loops, which renders the protein susceptible to autolysis, is the rate-limiting step. Despite the particular nature of the thermal denaturation process, normal rules for protein stability can be applied to NPs. However, rather than stabilizing the whole protein against global unfolding, only a small region has to be protected against local unfolding. In contrast to proteins in general, mutational effects in proteases are not additive and their magnitude is strongly dependent on the location of the mutation. Mutations that alter the stability of the NP by a large amount are located in a relatively weak region (or more precisely, they affect a local unfolding pathway with a relatively low free energy of activation). One weak region, that is supposedly important in the early steps of NP unfolding, has been determined in the NP of B. stearothermophilus. After eliminating this weakest link a drastic increase in thermostability was observed and the search for the second-weakest link, or the second-lowest energy local unfolding pathway is now in progress. Hopefully, this approach can be used to unravel the entire early phase of unfolding.

The National Aeronautics and Space Administration Nondestructive Evaluation Program for Safe and Reliable Operations

NASA Technical Reports Server (NTRS)

Generazio, Ed

2005-01-01

The National Aeronautics and Space Administration (NASA) Nondestructive Evaluation (NDE) Program is presented. As a result of the loss of seven astronauts and the Space Shuttle Columbia on February 1, 2003, NASA has undergone many changes in its organization. NDE is one of the key areas that are recognized by the Columbia Accident Investigation Board (CAIB) that needed to be strengthened by warranting NDE as a discipline with Independent Technical Authority (iTA). The current NASA NDE system and activities are presented including the latest developments in inspection technologies being applied to the Space Transportation System (STS). The unfolding trends and directions in NDE for the future are discussed as they apply to assuring safe and reliable operations.

A computer program for analyzing unresolved Mossbauer hyperfine spectra

NASA Technical Reports Server (NTRS)

Schiess, J. R.; Singh, J. J.

1978-01-01

The program for analyzing unresolved Mossbauer hyperfine spectra was written in FORTRAN 4 language for the Control Data CYBER 170 series digital computer system with network operating system 1.1. With the present dimensions, the program requires approximately 36,000 octal locations of core storage. A typical case involving two innermost coordination shells in which the amplitudes and the peak positions of all three components were estimated in 25 iterations requires 30 seconds on CYBER 173. The program was applied to determine the effects of various near neighbor impurity shells on hyperfine fields in dilute FeAl alloys.

Developmental biology in marine invertebrate symbioses.

PubMed

McFall-Ngai, M J; Ruby, E G

2000-12-01

Associations between marine invertebrates and their cooperative bacterial symbionts offer access to an understanding of the roots of host-microbe interaction; for example, several symbioses like the squid-vibrio light organ association serve as models for investigating how each partner affects the developmental biology of the other. Previous results have identified a program of specific developmental events that unfolds as the association is initiated. In the past year, published studies have focused primarily on describing the mechanisms underlying the signaling processes that occur between the juvenile squid and the luminous bacteria that colonize it.

An Approach for Peptide Identification by De Novo Sequencing of Mixture Spectra.

PubMed

Liu, Yi; Ma, Bin; Zhang, Kaizhong; Lajoie, Gilles

2017-01-01

Mixture spectra occur quite frequently in a typical wet-lab mass spectrometry experiment, which result from the concurrent fragmentation of multiple precursors. The ability to efficiently and confidently identify mixture spectra is essential to alleviate the existent bottleneck of low mass spectra identification rate. However, most of the traditional computational methods are not suitable for interpreting mixture spectra, because they still take the assumption that the acquired spectra come from the fragmentation of a single precursor. In this manuscript, we formulate the mixture spectra de novo sequencing problem mathematically, and propose a dynamic programming algorithm for the problem. Additionally, we use both simulated and real mixture spectra data sets to verify the merits of the proposed algorithm.

Unfolding the values of work - therapists´ experience of addressing the return to work process in occupational rehabilitation based on Acceptance and Commitment Therapy.

PubMed

Klevanger, Nina E; Fimland, Marius S; Johnsen, Roar; Rise, Marit B

2018-04-27

Facilitating return to work can be challenging due to the complexity of work disability. Few studies have examined rehabilitation programs based on Acceptance and Commitment Therapy that intend to support return to work, and none have investigated therapists' experience with providing such programs. The aim of this study was therefore to explore therapists' experience of addressing the return to work process in an inpatient occupational rehabilitation program based on Acceptance and Commitment Therapy. This was a qualitative interview study supported by participant observation. Therapists were interviewed regarding their experiences with addressing return to work in an inpatient occupational rehabilitation program based on Acceptance and Commitment Therapy. In addition, the rehabilitation program was investigated through participant observation. The interviews were analysed according to Interpretative Phenomenological Analysis and informed by an analysis of field notes from the participant observation. Acceptance and Commitment Therapy was experienced as a meaningful approach to facilitate return to work, as it allowed therapists to address all relevant aspects of the individual participant's life that might influence work participation. The therapists' twofold goal was to support participants in building both a meaningful life and sustainable work participation. To do so, they attempted to instil long-term and interrelated processes concerning ownership, causes of sick leave, relation to expectations, the values of work, and the scope of agency. Unfolding values connected to work participation might reconcile the tension between work and family life by integrating work with other areas of life. Providing work participation with personal meaning also seems especially commensurable with a context where economy presents a poor incentive for return to work. Therapists should, however, be attentive to the need to secure the prominence of return to work by relating participants' chosen themes explicitly to their return to work process. Therapists should also be aware of the dilemma that may arise when they attempt to refrain from providing advice while simultaneously encouraging actions they consider appropriate to facilitate sustainable work participation. In addition, having an individual-oriented approach to occupational rehabilitation may obscure the extent to which return to work is a multi-stakeholder process.

How Adequate are One- and Two-Dimensional Free Energy Landscapes for Protein Folding Dynamics?

NASA Astrophysics Data System (ADS)

Maisuradze, Gia G.; Liwo, Adam; Scheraga, Harold A.

2009-06-01

The molecular dynamics trajectories of protein folding or unfolding, generated with the coarse-grained united-residue force field for the B domain of staphylococcal protein A, were analyzed by principal component analysis (PCA). The folding or unfolding process was examined by using free-energy landscapes (FELs) in PC space. By introducing a novel multidimensional FEL, it was shown that the low-dimensional FELs are not always sufficient for the description of folding or unfolding processes. Similarities between the topographies of FELs along low- and high-indexed principal components were observed.

Single molecule FRET investigation of pressure-driven unfolding of cold shock protein A

NASA Astrophysics Data System (ADS)

Schneider, Sven; Paulsen, Hauke; Reiter, Kim Colin; Hinze, Erik; Schiene-Fischer, Cordelia; Hübner, Christian G.

2018-03-01

We demonstrate that fused silica capillaries are suitable for single molecule fluorescence resonance energy transfer (smFRET) measurements at high pressure with an optical quality comparable to the measurement on microscope coverslips. Therefore, we optimized the imaging conditions in a standard square fused silica capillary with an adapted arrangement and evaluated the performance by imaging the focal volume, fluorescence correlation spectroscopy benchmarks, and FRET measurements. We demonstrate single molecule FRET measurements of cold shock protein A unfolding at a pressure up to 2000 bars and show that the unfolded state exhibits an expansion almost independent of pressure.

A fluorescence approach to the unfolding thermodynamics of horseradish peroxidase based on heme degradation by hydrogen peroxide

NASA Astrophysics Data System (ADS)

Ke, Zhigang; Ma, Shanshan; Li, Lamei; Huang, Qing

2016-07-01

Horseradish peroxidase (HRP) is a classical heme-containing protein which has been applied in many fields. The prosthetic group heme in HRP, especially in unfolded state, can react with hydrogen peroxide (H2O2) to produce a fluorescent product with the maximum emission wavelength at 450 nm. Utilizing this emission band as a fluorescence probe, the unfolding process of HRP in urea can be assessed quantitatively, and the calculated thermodynamic parameters are consistent with those determined by circular dichroism (CD) at 222 nm and steady-state tryptophan (Trp) fluorescence methods.

Role of electrostatic interaction on surfactant induced protein unfolding

NASA Astrophysics Data System (ADS)

Sumit, Kumar, Sugam; Aswal, V. K.

2013-02-01

Small Angle Neutron Scattering has been used to examine the effect of electrostatic interaction on surfactant induced protein unfolding. Measurements are carried out from 1 wt% Bovine Serum Albumin (BSA) protein with 1 wt% Sodium Dodecyl Sulphate (SDS) surfactant at pH 7 in presence of varying concentration of NaCl. It is found that both the components (protein and surfactant micelle which are likely charged) exist individually without any interaction in absence of salt, whereas their interaction and protein unfolding is enhanced with the increase in salt concentration. The structure of protein-surfactant interaction is characterized by fractal bead-necklace model.

Breakdown of a 2D Heteroclinic Connection in the Hopf-Zero Singularity (I)

NASA Astrophysics Data System (ADS)

Baldomá, I.; Castejón, O.; Seara, T. M.

2018-04-01

In this paper we study a beyond all orders phenomenon which appears in the analytic unfoldings of the Hopf-zero singularity. It consists in the breakdown of a two-dimensional heteroclinic surface which exists in the truncated normal form of this singularity at any order. The results in this paper are twofold: on the one hand, we give results for generic unfoldings which lead to sharp exponentially small upper bounds of the difference between these manifolds. On the other hand, we provide asymptotic formulas for this difference by means of the Melnikov function for some non-generic unfoldings.

Unfolding and folding internal friction of β-hairpins is smaller than that of α-helices.

PubMed

Schulz, Julius C F; Miettinen, Markus S; Netz, R R

2015-04-02

By the forced unfolding of polyglutamine and polyalanine homopeptides in competing α-helix and β-hairpin secondary structures, we disentangle equilibrium free energetics from nonequilibrium dissipative effects. We find that α-helices are characterized by larger friction or dissipation upon unfolding, regardless of whether they are free energetically preferred over β-hairpins or not. Our analysis, based on MD simulations for atomistic peptide models with explicit water, suggests that this difference is related to the internal friction and mostly caused by the different number of intrapeptide hydrogen bonds in the α-helix and β-hairpin states.

The impact of the unfolded protein response on human disease

PubMed Central

Wang, Shiyu

2012-01-01

A central function of the endoplasmic reticulum (ER) is to coordinate protein biosynthetic and secretory activities in the cell. Alterations in ER homeostasis cause accumulation of misfolded/unfolded proteins in the ER. To maintain ER homeostasis, eukaryotic cells have evolved the unfolded protein response (UPR), an essential adaptive intracellular signaling pathway that responds to metabolic, oxidative stress, and inflammatory response pathways. The UPR has been implicated in a variety of diseases including metabolic disease, neurodegenerative disease, inflammatory disease, and cancer. Signaling components of the UPR are emerging as potential targets for intervention and treatment of human disease. PMID:22733998

The RtcB RNA ligase is an essential component of the metazoan unfolded protein response.

PubMed

Kosmaczewski, Sara Guckian; Edwards, Tyson James; Han, Sung Min; Eckwahl, Matthew J; Meyer, Benjamin Isaiah; Peach, Sally; Hesselberth, Jay R; Wolin, Sandra L; Hammarlund, Marc

2014-12-01

RNA ligation can regulate RNA function by altering RNA sequence, structure and coding potential. For example, the function of XBP1 in mediating the unfolded protein response requires RNA ligation, as does the maturation of some tRNAs. Here, we describe a novel in vivo model in Caenorhabditis elegans for the conserved RNA ligase RtcB and show that RtcB ligates the xbp-1 mRNA during the IRE-1 branch of the unfolded protein response. Without RtcB, protein stress results in the accumulation of unligated xbp-1 mRNA fragments, defects in the unfolded protein response, and decreased lifespan. RtcB also ligates endogenous pre-tRNA halves, and RtcB mutants have defects in growth and lifespan that can be bypassed by expression of pre-spliced tRNAs. In addition, animals that lack RtcB have defects that are independent of tRNA maturation and the unfolded protein response. Thus, RNA ligation by RtcB is required for the function of multiple endogenous target RNAs including both xbp-1 and tRNAs. RtcB is uniquely capable of performing these ligation functions, and RNA ligation by RtcB mediates multiple essential processes in vivo. © 2014 The Authors.

The RtcB RNA ligase is an essential component of the metazoan unfolded protein response

PubMed Central

Kosmaczewski, Sara Guckian; Edwards, Tyson James; Han, Sung Min; Eckwahl, Matthew J; Meyer, Benjamin Isaiah; Peach, Sally; Hesselberth, Jay R; Wolin, Sandra L; Hammarlund, Marc

2014-01-01

RNA ligation can regulate RNA function by altering RNA sequence, structure and coding potential. For example, the function of XBP1 in mediating the unfolded protein response requires RNA ligation, as does the maturation of some tRNAs. Here, we describe a novel in vivo model in Caenorhabditis elegans for the conserved RNA ligase RtcB and show that RtcB ligates the xbp-1 mRNA during the IRE-1 branch of the unfolded protein response. Without RtcB, protein stress results in the accumulation of unligated xbp-1 mRNA fragments, defects in the unfolded protein response, and decreased lifespan. RtcB also ligates endogenous pre-tRNA halves, and RtcB mutants have defects in growth and lifespan that can be bypassed by expression of pre-spliced tRNAs. In addition, animals that lack RtcB have defects that are independent of tRNA maturation and the unfolded protein response. Thus, RNA ligation by RtcB is required for the function of multiple endogenous target RNAs including both xbp-1 and tRNAs. RtcB is uniquely capable of performing these ligation functions, and RNA ligation by RtcB mediates multiple essential processes in vivo. Subject Categories Protein Biosynthesis & Quality Control; RNA Biology PMID:25366321

RPA-mediated unfolding of systematically varying G-quadruplex structures.

PubMed

Ray, Sujay; Qureshi, Mohammad H; Malcolm, Dominic W; Budhathoki, Jagat B; Celik, Uğur; Balci, Hamza

2013-05-21

G-quadruplex (GQ) is a noncanonical nucleic acid structure that is formed by guanine rich sequences. Unless it is destabilized by proteins such as replication protein A (RPA), GQ could interfere with DNA metabolic functions, such as replication or repair. We studied RPA-mediated GQ unfolding using single-molecule FRET on two groups of GQ structures that have different loop lengths and different numbers of G-tetrad layers. We observed a linear increase in the steady-state stability of the GQ against RPA-mediated unfolding with increasing number of layers or decreasing loop length. The stability demonstrated by different GQ structures varied by at least three orders of magnitude. Those with shorter loops (less than three nucleotides long) or a greater number of layers (more than three layers) maintained a significant folded population even at physiological RPA concentration (≈1 μM), raising the possibility of physiological viability of such GQ structures. Finally, we measured the transition time between the start and end of the RPA-mediated GQ unfolding process to be 0.35 ± 0.10 s for all GQ constructs we studied, despite significant differences in their steady-state stabilities. We propose a two-step RPA-mediated GQ unfolding mechanism that is consistent with our observations. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

RPA-Mediated Unfolding of Systematically Varying G-Quadruplex Structures

PubMed Central

Ray, Sujay; Qureshi, Mohammad H.; Malcolm, Dominic W.; Budhathoki, Jagat B.; Çelik, Uğur; Balci, Hamza

2013-01-01

G-quadruplex (GQ) is a noncanonical nucleic acid structure that is formed by guanine rich sequences. Unless it is destabilized by proteins such as replication protein A (RPA), GQ could interfere with DNA metabolic functions, such as replication or repair. We studied RPA-mediated GQ unfolding using single-molecule FRET on two groups of GQ structures that have different loop lengths and different numbers of G-tetrad layers. We observed a linear increase in the steady-state stability of the GQ against RPA-mediated unfolding with increasing number of layers or decreasing loop length. The stability demonstrated by different GQ structures varied by at least three orders of magnitude. Those with shorter loops (less than three nucleotides long) or a greater number of layers (more than three layers) maintained a significant folded population even at physiological RPA concentration (≈1 μM), raising the possibility of physiological viability of such GQ structures. Finally, we measured the transition time between the start and end of the RPA-mediated GQ unfolding process to be 0.35 ± 0.10 s for all GQ constructs we studied, despite significant differences in their steady-state stabilities. We propose a two-step RPA-mediated GQ unfolding mechanism that is consistent with our observations. PMID:23708363

Binding polarity of RPA to telomeric sequences and influence of G-quadruplex stability.

PubMed

Safa, Layal; Delagoutte, Emmanuelle; Petruseva, Irina; Alberti, Patrizia; Lavrik, Olga; Riou, Jean-François; Saintomé, Carole

2014-08-01

Replication protein A (RPA) is a single-stranded DNA binding protein that plays an essential role in telomere maintenance. RPA binds to and unfolds G-quadruplex (G4) structures formed in telomeric DNA, thus facilitating lagging strand DNA replication and telomerase activity. To investigate the effect of G4 stability on the interactions with human RPA (hRPA), we used a combination of biochemical and biophysical approaches. Our data revealed an inverse relationship between G4 stability and ability of hRPA to bind to telomeric DNA; notably small G4 ligands that enhance G4 stability strongly impaired G4 unfolding by hRPA. To gain more insight into the mechanism of binding and unfolding of telomeric G4 structures by RPA, we carried out photo-crosslinking experiments to elucidate the spatial arrangement of the RPA subunits along the DNA strands. Our results showed that RPA1 and RPA2 are arranged from 5' to 3' along the unfolded telomeric G4, as already described for unstructured single-stranded DNA, while no contact is possible with RPA3 on this short oligonucleotide. In addition, these data are compatible with a 5' to 3' directionality in G4 unfolding by hRPA. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Origin and propagation of galactic cosmic rays

NASA Technical Reports Server (NTRS)

Cesarsky, Catherine J.; Ormes, Jonathan F.

1987-01-01

The study of systematic trends in elemental abundances is important for unfolding the nuclear and/or atomic effects that should govern the shaping of source abundances and in constraining the parameters of cosmic ray acceleration models. In principle, much can be learned about the large-scale distributions of cosmic rays in the galaxy from all-sky gamma ray surveys such as COS-B and SAS-2. Because of the uncertainties in the matter distribution which come from the inability to measure the abundance of molecular hydrogen, the results are somewhat controversial. The leaky-box model accounts for a surprising amount of the data on heavy nuclei. However, a growing body of data indicates that the simple picture may have to be abandoned in favor of more complex models which contain additional parameters. Future experiments on the Spacelab and space station will hopefully be made of the spectra of individual nuclei at high energy. Antiprotons must be studied in the background free environment above the atmosphere with much higher reliability and presion to obtain spectral information.

Testing Bonner sphere spectrometers in the JRC-IRMM mono-energetic neutron beams

NASA Astrophysics Data System (ADS)

Bedogni, R.; Domingo, C.; Esposito, A.; Chiti, M.; García-Fusté, M. J.; Lovestam, G.

2010-08-01

Within the framework of the Euratom Transnational Access programme, a specific sub-programme, called NUDAME (neutron data measurements at IRMM), was dedicated to support neutron measurement experiments at the accelerator-based facilities of the JRC-IRMM Geel, Belgium. In this context, the INFN-LNF and UAB groups undertook two separate experiments at the 7 MV Van de Graaff facility, aimed at testing their Bonner sphere spectrometers (BSS) with mono-energetic neutron beams. Both research groups routinely employ the BSS in neutron spectra measurements for radiation protection dosimetry purposes, where accurate knowledge of the BSS response is a mandatory condition for correct dose evaluations. This paper presents the results obtained by both groups, focusing on: (1) the comparison between the value of neutron fluence provided as reference data and that obtained by applying the FRUIT unfolding code to the measured BSS data and (2) the experimental validation of the response matrices of the BSSs, previously derived with Monte Carlo simulations.

The physics of the knee in the cosmic ray spectrum

NASA Astrophysics Data System (ADS)

Kampert, K.-H.; Antoni, T.; Apel, W. D.; Badea, F.; Bekk, K.; Bercuci, A.; Blümer, H.; Bollmann, E.; Bozdog, H.

Recent results from the KASCADE extensive air shower experiment are presented. After briefly reviewing the status of the experiment we report on tests of hadronic interaction models and emphasize the progress being made in understanding the properties and origin of the knee at Eknee ˜= 4 · 1015 eV. Analysing the muonand hadron trigger rates in the KASCADE calorimeter as well as the global properties of high energy hadrons in the shower core leads us to conclude that QGSJET still provides the best overall description of EAS data, being superior to DPMJET II-5 and NEXUS 2, for example. Performing high statistics CORSIKA simulations and applying sophisticated unfolding techniques to the electron and muon shower size distributions, we are able to successfully deconvolute the all-particle energy spectrum into energy spectra of 4 individual primary mass groups (p, He, C, Fe). Each of these preliminary energy distributions exhibits a knee like structure with a change of their knee positions suggesting a constant rigidity of R ˜= 2-3 PV.

Neutron spectrometry in a mixed field of neutrons and protons with a phoswich neutron detector Part I: response functions for photons and neutrons of the phoswich neutron detector

NASA Astrophysics Data System (ADS)

Takada, M.; Taniguchi, S.; Nakamura, T.; Nakao, N.; Uwamino, Y.; Shibata, T.; Fujitaka, K.

2001-06-01

We have developed a phoswich neutron detector consisting of an NE213 liquid scintillator surrounded by an NE115 plastic scintillator to distinguish photon and neutron events in a charged-particle mixed field. To obtain the energy spectra by unfolding, the response functions to neutrons and photons were obtained by the experiment and calculation. The response functions to photons were measured with radionuclide sources, and were calculated with the EGS4-PRESTA code. The response functions to neutrons were measured with a white neutron source produced by the bombardment of 135 MeV protons onto a Be+C target using a TOF method, and were calculated with the SCINFUL code, which we revised in order to calculate neutron response functions up to 135 MeV. Based on these experimental and calculated results, response matrices for photons up to 20 MeV and neutrons up to 132 MeV could finally be obtained.

Latest results and perspectives of the KASCADE-Grande EAS Facility

NASA Astrophysics Data System (ADS)

Haungs, A.; Apel, W. D.; Arteaga-Velázquez, J. C.; Bekk, K.; Bertaina, M.; Blümer, J.; Bozdog, H.; Brancus, I. M.; Buchholz, P.; Cantoni, E.; Chiavassa, A.; Cossavella, F.; Daumiller, K.; de Souza, V.; di Pierro, F.; Doll, P.; Engel, R.; Engler, J.; Finger, M.; Fuhrmann, D.; Ghia, P. L.; Gils, H. J.; Glasstetter, R.; Grupen, C.; Heck, D.; Hörandel, J. R.; Huege, T.; Isar, P. G.; Kampert, K.-H.; Kang, D.; Kickelbick, D.; Klages, H. O.; Link, K.; Łuczak, P.; Ludwig, M.; Mathes, H. J.; Mayer, H. J.; Melissas, M.; Milke, J.; Mitrica, B.; Morello, C.; Navarra, G.; Nehls, S.; Oehlschläger, J.; Ostapchenko, S.; Over, S.; Palmieri, N.; Petcu, M.; Pierog, T.; Rebel, H.; Roth, M.; Schieler, H.; Schröder, F. G.; Sima, O.; Toma, G.; Trinchero, G. C.; Ulrich, H.; Weindl, A.; Wochele, J.; Wommer, M.; Zabierowski, J.

2012-01-01

KASCADE-Grande is a multi-detector experiment at KIT (Karlsruhe Institute of Technology) in Germany for measuring extensive air showers in the primary energy range of 100 TeV to 1 EeV. This paper does not provide a synopsis of all results of the KASCADE-Grande experiment. Rather it is focused on three aspects of current interests illustrating the advantages of a multi-detector facility. Results on the analysis of individual energy spectra of primary mass groups around the knee obtained by unfolding the shower size measurements of KASCADE with the help of the new hadronic interaction model EPOS and the all-particle energy spectrum at higher energies obtained by Grande measurements will be discussed. As KASCADE-Grande serves also as host of the LOPES radio detection experiment where both experiments measure the same showers, special emphasis will be given in comparing the characteristics and feasibility of both techniques in estimating the main parameters of high-energy primary cosmic rays: energy, composition, and arrival direction.

The ENEA criticality accident dosimetry system: a contribution to the 2002 international intercomparison at the SILENE reactor.

PubMed

Gualdrini, G; Bedogni, R; Fantuzzi, E; Mariotti, F

2004-01-01

The present paper summarises the activity carried out at the ENEA Radiation Protection Institute for updating the methodologies employed for the evaluation of the neutron and photon dose to the exposed workers in case of a criticality accident, in the framework of the 'International Intercomparison of Criticality Accident Dosimetry Systems' (Silène reactor, IRSN-CEA-Valduc June 2002). The evaluation of the neutron spectra and the neutron dosimetric quantities relies on activation detectors and on unfolding algorithms. Thermoluminescent detectors are employed for the gamma dose measurement. The work is aimed at accurately characterising the measurement system and, at the same time, testing the algorithms. Useful spectral information were included, based on Monte Carlo simulations, to take into account the potential accident scenarios of practical interest. All along this exercise intercomparison a particular attention was devoted to the 'traceability' of all the experimental and computational parameters and therefore, aimed at an easy treatment by the user.

Pharmaceutical analysis in solids using front face fluorescence spectroscopy and multivariate calibration with matrix correction by piecewise direct standardization

NASA Astrophysics Data System (ADS)

Alves, Julio Cesar L.; Poppi, Ronei J.

2013-02-01

This paper reports the application of piecewise direct standardization (PDS) for matrix correction in front face fluorescence spectroscopy of solids when different excipients are used in a pharmaceutical preparation based on a mixture of acetylsalicylic acid (ASA), paracetamol (acetaminophen) and caffeine. As verified in earlier studies, the use of different excipients and their ratio can cause a displacement, change in fluorescence intensity or band profile. To overcome this important drawback, a standardization strategy was adopted to convert all the excitation-emission fluorescence spectra into those used for model development. An excitation-emission matrix (EEM) for which excitation and emission wavelengths ranging from 265 to 405 nm and 300 to 480 nm, respectively, was used. Excellent results were obtained using unfolded partial least squares (U-PLS), with RMSEP values of 8.2 mg/g, 10.9 mg/g and 2.7 mg/g for ASA, paracetamol and caffeine, respectively, and with relative errors lesser than 5% for the three analytes.

Binding of (-)-epigallocatechin-3-gallate with thermally-induced bovine serum albumin/ι-carrageenan particles.

PubMed

Li, Jinbing; Wang, Xiaoyong

2015-02-01

Novel thermally-induced BSA/ι-carrageenan particles are used as a protective carrier for (-)-epigallocatechin-3-gallate (EGCG). The addition of EGCG to BSA/ι-carrageenan particles can highly quench the intrinsic fluorescence of BSA, which is explained in terms of the binding of EGCG to the hydrophobic pockets of BSA mainly through the hydrophobic force. According to the double logarithm equation, the binding constant is determined as 1.1×10(8)M(-1) for the binding of EGCG with BSA/ι-carrageenan particles. The high binding affinity is ascribed to both the molecular structure of EGCG and the partial unfolding state of BSA in BSA/ι-carrageenan particles. The circular dichroism spectra and calculated α-helix of BSA suggest that the bound EGCG leads to a more random secondary structure of BSA. Furthermore, BSA/ι-carrageenan particles are found to be superior to native BSA and pure BSA particles for improving the stability and radical scavenging activity of EGCG. Copyright © 2014 Elsevier Ltd. All rights reserved.

Sub-Micellar Concentration of Sodium Dodecyl Sulphate Prevents Thermal Denaturation Induced Aggregation of Plant Lectin, Jacalin.

PubMed

Lavanya, V; Anil Kumar, B; Jamal, Shazia; Khan, Md Khurshid Alam; Ahmed, Neesar

2017-02-01

The irreversible thermal unfolding of jacalin, the lectin purified from jackfruit seeds was accompanied by aggregation, where intermolecular interactions among the subunits are favoured over intramolecular interactions. The extent of aggregation increased as a function of temperature, time and protein concentration. The anionic surfactant, sodium dodecyl sulphate (SDS) significantly suppressed the formation of aggregates as observed by turbidity measurements and Rayleigh scattering assay. Moreover, far UV-CD spectra indicate that the protein β sheet transforms into α helical structure, when denatured in the presence of 3 mM SDS. Further, jacalin when heated in the presence of SDS partially retained the hemagglutination activity when jacalin-SDS mixture was diluted to 1:8 factor since 3 mM SDS was found to lyse the red blood cells. Thus, SDS only altered the aggregation behaviour of jacalin by preventing intermolecular hydrogen bonding among the exposed residues but did not completely stabilize the native conformation.

VIBA-Lab 3.0: Computer program for simulation and semi-quantitative analysis of PIXE and RBS spectra and 2D elemental maps

NASA Astrophysics Data System (ADS)

Orlić, Ivica; Mekterović, Darko; Mekterović, Igor; Ivošević, Tatjana

2015-11-01

VIBA-Lab is a computer program originally developed by the author and co-workers at the National University of Singapore (NUS) as an interactive software package for simulation of Particle Induced X-ray Emission and Rutherford Backscattering Spectra. The original program is redeveloped to a VIBA-Lab 3.0 in which the user can perform semi-quantitative analysis by comparing simulated and measured spectra as well as simulate 2D elemental maps for a given 3D sample composition. The latest version has a new and more versatile user interface. It also has the latest data set of fundamental parameters such as Coster-Kronig transition rates, fluorescence yields, mass absorption coefficients and ionization cross sections for K and L lines in a wider energy range than the original program. Our short-term plan is to introduce routine for quantitative analysis for multiple PIXE and XRF excitations. VIBA-Lab is an excellent teaching tool for students and researchers in using PIXE and RBS techniques. At the same time the program helps when planning an experiment and when optimizing experimental parameters such as incident ions, their energy, detector specifications, filters, geometry, etc. By "running" a virtual experiment the user can test various scenarios until the optimal PIXE and BS spectra are obtained and in this way save a lot of expensive machine time.

Role of cavities and hydration in the pressure unfolding of T4 lysozyme

PubMed Central

Nucci, Nathaniel V.; Fuglestad, Brian; Athanasoula, Evangelia A.; Wand, A. Joshua

2014-01-01

It is well known that high hydrostatic pressures can induce the unfolding of proteins. The physical underpinnings of this phenomenon have been investigated extensively but remain controversial. Changes in solvation energetics have been commonly proposed as a driving force for pressure-induced unfolding. Recently, the elimination of void volumes in the native folded state has been argued to be the principal determinant. Here we use the cavity-containing L99A mutant of T4 lysozyme to examine the pressure-induced destabilization of this multidomain protein by using solution NMR spectroscopy. The cavity-containing C-terminal domain completely unfolds at moderate pressures, whereas the N-terminal domain remains largely structured to pressures as high as 2.5 kbar. The sensitivity to pressure is suppressed by the binding of benzene to the hydrophobic cavity. These results contrast to the pseudo-WT protein, which has a residual cavity volume very similar to that of the L99A–benzene complex but shows extensive subglobal reorganizations with pressure. Encapsulation of the L99A mutant in the aqueous nanoscale core of a reverse micelle is used to examine the hydration of the hydrophobic cavity. The confined space effect of encapsulation suppresses the pressure-induced unfolding transition and allows observation of the filling of the cavity with water at elevated pressures. This indicates that hydration of the hydrophobic cavity is more energetically unfavorable than global unfolding. Overall, these observations point to a range of cooperativity and energetics within the T4 lysozyme molecule and illuminate the fact that small changes in physical parameters can significantly alter the pressure sensitivity of proteins. PMID:25201963

Precursory signatures of protein folding/unfolding: From time series correlation analysis to atomistic mechanisms

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

Hsu, P. J.; Lai, S. K., E-mail: sklai@coll.phy.ncu.edu.tw; Molecular Science and Technology Program, Taiwan International Graduate Program, Academia Sinica, Taipei 115, Taiwan

Folded conformations of proteins in thermodynamically stable states have long lifetimes. Before it folds into a stable conformation, or after unfolding from a stable conformation, the protein will generally stray from one random conformation to another leading thus to rapid fluctuations. Brief structural changes therefore occur before folding and unfolding events. These short-lived movements are easily overlooked in studies of folding/unfolding for they represent momentary excursions of the protein to explore conformations in the neighborhood of the stable conformation. The present study looks for precursory signatures of protein folding/unfolding within these rapid fluctuations through a combination of three techniques: (1)more » ultrafast shape recognition, (2) time series segmentation, and (3) time series correlation analysis. The first procedure measures the differences between statistical distance distributions of atoms in different conformations by calculating shape similarity indices from molecular dynamics simulation trajectories. The second procedure is used to discover the times at which the protein makes transitions from one conformation to another. Finally, we employ the third technique to exploit spatial fingerprints of the stable conformations; this procedure is to map out the sequences of changes preceding the actual folding and unfolding events, since strongly correlated atoms in different conformations are different due to bond and steric constraints. The aforementioned high-frequency fluctuations are therefore characterized by distinct correlational and structural changes that are associated with rate-limiting precursors that translate into brief segments. Guided by these technical procedures, we choose a model system, a fragment of the protein transthyretin, for identifying in this system not only the precursory signatures of transitions associated with α helix and β hairpin, but also the important role played by weaker correlations in such protein folding dynamics.« less

Towards data warehousing and mining of protein unfolding simulation data.

PubMed

Berrar, Daniel; Stahl, Frederic; Silva, Candida; Rodrigues, J Rui; Brito, Rui M M; Dubitzky, Werner

2005-10-01

The prediction of protein structure and the precise understanding of protein folding and unfolding processes remains one of the greatest challenges in structural biology and bioinformatics. Computer simulations based on molecular dynamics (MD) are at the forefront of the effort to gain a deeper understanding of these complex processes. Currently, these MD simulations are usually on the order of tens of nanoseconds, generate a large amount of conformational data and are computationally expensive. More and more groups run such simulations and generate a myriad of data, which raises new challenges in managing and analyzing these data. Because the vast range of proteins researchers want to study and simulate, the computational effort needed to generate data, the large data volumes involved, and the different types of analyses scientists need to perform, it is desirable to provide a public repository allowing researchers to pool and share protein unfolding data. To adequately organize, manage, and analyze the data generated by unfolding simulation studies, we designed a data warehouse system that is embedded in a grid environment to facilitate the seamless sharing of available computer resources and thus enable many groups to share complex molecular dynamics simulations on a more regular basis. To gain insight into the conformational fluctuations and stability of the monomeric forms of the amyloidogenic protein transthyretin (TTR), molecular dynamics unfolding simulations of the monomer of human TTR have been conducted. Trajectory data and meta-data of the wild-type (WT) protein and the highly amyloidogenic variant L55P-TTR represent the test case for the data warehouse. Web and grid services, especially pre-defined data mining services that can run on or 'near' the data repository of the data warehouse, are likely to play a pivotal role in the analysis of molecular dynamics unfolding data.

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.

Mechanism of Unfolding of Human Prion Protein.

PubMed

Singh, Reman K; Chamachi, Neharika G; Chakrabarty, Suman; Mukherjee, Arnab

2017-01-26

Misfolding and aggregation of prion proteins are associated with several neurodegenerative diseases. Therefore, understanding the mechanism of the misfolding process is of enormous interest in the scientific community. It has been speculated and widely discussed that the native cellular prion protein (PrP C ) form needs to undergo substantial unfolding to a more stable PrP C* state, which may further oligomerize into the toxic scrapie (PrP Sc ) form. Here, we have studied the mechanism of the unfolding of the human prion protein (huPrP) using a set of extensive well-tempered metadynamics simulations. Through multiple microsecond-long metadynamics simulations, we find several possible unfolding pathways. We show that each pathway leads to an unfolded state of lower free energy than the native state. Thus, our study may point to the signature of a PrP C* form that corresponds to a global minimum on the conformational free-energy landscape. Moreover, we find that these global minima states do not involve an increased β-sheet content, as was assumed to be a signature of PrP Sc formation in previous simulation studies. We have further analyzed the origin of metastability of the PrP C form through free-energy surfaces of the chopped helical segments to show that the helices, particularly H2 and H3 of the prion protein, have the tendency to form either a random coil or a β-structure. Therefore, the secondary structural elements of the prion protein are only weakly stabilized by tertiary contacts and solvation forces so that relatively weak perturbations induced by temperature, pressure, pH, and so forth can lead to substantial unfolding with characteristics of intrinsically disordered proteins.

Mechanics of collective unfolding

NASA Astrophysics Data System (ADS)

Caruel, M.; Allain, J.-M.; Truskinovsky, L.

2015-03-01

Mechanically induced unfolding of passive crosslinkers is a fundamental biological phenomenon encountered across the scales from individual macro-molecules to cytoskeletal actin networks. In this paper we study a conceptual model of athermal load-induced unfolding and use a minimalistic setting allowing one to emphasize the role of long-range interactions while maintaining full analytical transparency. Our model can be viewed as a description of a parallel bundle of N bistable units confined between two shared rigid backbones that are loaded through a series spring. We show that the ground states in this model correspond to synchronized, single phase configurations where all individual units are either folded or unfolded. We then study the fine structure of the wiggly energy landscape along the reaction coordinate linking the two coherent states and describing the optimal mechanism of cooperative unfolding. Quite remarkably, our study shows the fundamental difference in the size and the structure of the folding-unfolding energy barriers in the hard (fixed displacements) and soft (fixed forces) loading devices which persists in the continuum limit. We argue that both, the synchronization and the non-equivalence of the mechanical responses in hard and soft devices, have their origin in the dominance of long-range interactions. We then apply our minimal model to skeletal muscles where the power-stroke in acto-myosin crossbridges can be interpreted as passive folding. A quantitative analysis of the muscle model shows that the relative rigidity of myosin backbone provides the long-range interaction mechanism allowing the system to effectively synchronize the power-stroke in individual crossbridges even in the presence of thermal fluctuations. In view of the prototypical nature of the proposed model, our general conclusions pertain to a variety of other biological systems where elastic interactions are mediated by effective backbones.

Precursory signatures of protein folding/unfolding: From time series correlation analysis to atomistic mechanisms

NASA Astrophysics Data System (ADS)

Hsu, P. J.; Cheong, S. A.; Lai, S. K.

2014-05-01

Folded conformations of proteins in thermodynamically stable states have long lifetimes. Before it folds into a stable conformation, or after unfolding from a stable conformation, the protein will generally stray from one random conformation to another leading thus to rapid fluctuations. Brief structural changes therefore occur before folding and unfolding events. These short-lived movements are easily overlooked in studies of folding/unfolding for they represent momentary excursions of the protein to explore conformations in the neighborhood of the stable conformation. The present study looks for precursory signatures of protein folding/unfolding within these rapid fluctuations through a combination of three techniques: (1) ultrafast shape recognition, (2) time series segmentation, and (3) time series correlation analysis. The first procedure measures the differences between statistical distance distributions of atoms in different conformations by calculating shape similarity indices from molecular dynamics simulation trajectories. The second procedure is used to discover the times at which the protein makes transitions from one conformation to another. Finally, we employ the third technique to exploit spatial fingerprints of the stable conformations; this procedure is to map out the sequences of changes preceding the actual folding and unfolding events, since strongly correlated atoms in different conformations are different due to bond and steric constraints. The aforementioned high-frequency fluctuations are therefore characterized by distinct correlational and structural changes that are associated with rate-limiting precursors that translate into brief segments. Guided by these technical procedures, we choose a model system, a fragment of the protein transthyretin, for identifying in this system not only the precursory signatures of transitions associated with α helix and β hairpin, but also the important role played by weaker correlations in such protein folding dynamics.

Unfolding of a model protein on ion exchange and mixed mode chromatography surfaces.

PubMed

Gospodarek, Adrian M; Hiser, Diana E; O'Connell, John P; Fernandez, Erik J

2014-08-15

Recent studies with proteins indicate that conformational changes and aggregation can occur during ion exchange chromatography (IEC). Such behavior is not usually expected, but could lead to decreased yield and product degradation from both IEC and multi mode chromatography (MMC) that has ligands of both hydrophobic and charged functionalities. In this study, we used hydrogen exchange mass spectrometry to investigate unfolding of the model protein BSA on IEC and MMC surfaces under different solution conditions at 25°C. Increased solvent exposure, indicating greater unfolding relative to that in solution, was found for protein adsorbed on cationic IEC and MMC surfaces in the pH range of 3.0 to 4.5, where BSA has decreased stability in solution. There was no effect of anionic surfaces at pH values in the range from 6.0 to 9.0. Differences of solvent exposure of whole molecules when adsorbed and in solution suggest that adsorbed BSA unfolds at lower pH values and may show aggregation, depending upon pH and the surface type. Measurements on digested peptides showed that classifications of stability can be made for various regions; these are generally retained as pH is changed. When salt was added to MMC systems, where electrostatic interactions would be minimized, less solvent exposure was seen, implying that it is the cationic moieties, rather than the hydrophobic ligands, which cause greater surface unfolding at low salt concentrations. These results suggest that proteins of lower stability may exhibit unfolding and aggregation during IEC and MMC separations, as they can with hydrophobic interaction chromatography. Copyright © 2014 Elsevier B.V. All rights reserved.

Dynamic properties of bacterial pili measured by optical tweezers

NASA Astrophysics Data System (ADS)

Fallman, Erik G.; Andersson, Magnus J.; Schedin, Staffan S.; Jass, Jana; Uhlin, Bernt Eric; Axner, Ove

2004-10-01

The ability of uropathogenic Escherichia coli (UPEC) to cause urinary tract infections is dependent on their ability to colonize the uroepithelium. Infecting bacteria ascend the urethra to the bladder and then kidneys by attaching to the uroepithelial cells via the differential expression of adhesins. P pili are associated with pyelonephritis, the more severe infection of the kidneys. In order to find means to treat pyelonephritis, it is therefore of interest to investigate the properties P pili. The mechanical behavior of individual P pili of uropathogenic Escherichia coli has recently been investigated using optical tweezers. P pili, whose main part constitutes the PapA rod, composed of ~1000 PapA subunits in a helical arrangement, are distributed over the bacterial surface and mediate adhesion to host cells. We have earlier studied P pili regarding its stretching/elongation properties where we have found and characterized three different elongation regions, of which one constitute an unfolding of the quaternary (helical) structure of the PapA rod. It was shown that this unfolding takes place at an elongation independent force of 27 +/- 2 pN. We have also recently performed studies on its folding properties and shown that the unfolding/folding of the PapA rod is completely reversible. Here we present a study of the dynamical properties of the PapA rod. We show, among other things, that the unfolding force increases and that the folding force decreases with the speed of unfolding and folding respectively. Moreover, the PapA rod can be folded-unfolded a significant number of times without loosing its characteristics, a phenomenon that is believed to be important for the bacterium to keep close contact to the host tissue and consequently helps the bacterium to colonize the host tissue.

Determining the folding and unfolding rate constants of nucleic acids by biosensor. Application to telomere G-quadruplex.

PubMed

Zhao, Yong; Kan, Zhong-yuan; Zeng, Zhi-xiong; Hao, Yu-hua; Chen, Hua; Tan, Zheng

2004-10-20

Nucleic acid molecules may fold into secondary structures, and the formation of such structures is involved in many biological processes and technical applications. The folding and unfolding rate constants define the kinetics of conformation interconversion and the stability of these structures and is important in realizing their functions. We developed a method to determine these kinetic parameters using an optical biosensor based on surface plasmon resonance. The folding and unfolding of a nucleic acid is coupled with a hybridization reaction by immobilization of the target nucleic acid on a sensor chip surface and injection of a complementary probe nucleic acid over the sensor chip surface. By monitoring the time course of duplex formation, both the folding and unfolding rate constants for the target nucleic acid and the association and dissociation rate constants for the target-probe duplex can all be derived from the same measurement. We applied this method to determine the folding and unfolding rate constants of the G-quadruplex of human telomere sequence (TTAGGG)(4) and its association and dissociation rate constants with the complementary strand (CCCTAA)(4). The results show that both the folding and unfolding occur on the time scale of minutes at physiological concentration of K(+). We speculate that this property might be important for telomere elongation. A complete set of the kinetic parameters for both of the structures allows us to study the competition between the formation of the quadruplex and the duplex. Calculations indicate that the formation of both the quadruplex and the duplex is strand concentration-dependent, and the quadruplex can be efficiently formed at low strand concentration. This property may provide the basis for the formation of the quadruplex in vivo in the presence of a complementary strand.

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

Elucidating quantitative stability/flexibility relationships within thioredoxin and its fragments using a distance constraint model.

PubMed

Jacobs, Donald J; Livesay, Dennis R; Hules, Jeremy; Tasayco, Maria Luisa

2006-05-05

Numerous quantitative stability/flexibility relationships, within Escherichia coli thioredoxin (Trx) and its fragments are determined using a minimal distance constraint model (DCM). A one-dimensional free energy landscape as a function of global flexibility reveals Trx to fold in a low-barrier two-state process, with a voluminous transition state. Near the folding transition temperature, the native free energy basin is markedly skewed to allow partial unfolded forms. Under native conditions the skewed shape is lost, and the protein forms a compact structure with some flexibility. Predictions on ten Trx fragments are generally consistent with experimental observations that they are disordered, and that complementary fragments reconstitute. A hierarchical unfolding pathway is uncovered using an exhaustive computational procedure of breaking interfacial cross-linking hydrogen bonds that span over a series of fragment dissociations. The unfolding pathway leads to a stable core structure (residues 22-90), predicted to act as a kinetic trap. Direct connection between degree of rigidity within molecular structure and non-additivity of free energy is demonstrated using a thermodynamic cycle involving fragments and their hierarchical unfolding pathway. Additionally, the model provides insight about molecular cooperativity within Trx in its native state, and about intermediate states populating the folding/unfolding pathways. Native state cooperativity correlation plots highlight several flexibly correlated regions, giving insight into the catalytic mechanism that facilitates access to the active site disulfide bond. Residual native cooperativity correlations are present in the core substructure, suggesting that Trx can function when it is partly unfolded. This natively disordered kinetic trap, interpreted as a molten globule, has a wide temperature range of metastability, and it is identified as the "slow intermediate state" observed in kinetic experiments. These computational results are found to be in overall agreement with a large array of experimental data.

Correlating folding and signaling in a photoreceptor by single molecule measurements and energy landscape calculations

NASA Astrophysics Data System (ADS)

Hoff, Wouter

2007-03-01

Receptor activation is a fundamental process in biological signaling. We study the structural changes during activation of photoactive yellow protein (PYP). This is triggered by photoisomerization of the p-coumaric acid (pCA) chromophore of PYP, which converts the initial pG state into the activated pB state. Mechanical unfolding of Cys-linked PYP multimers probed by atomic force microscopy (AFM) in the presence and absence of illumination reveals that the core of the protein is extended by 3 nm and destabilized by 30 percent in pB. These results establish a generally applicable single molecule approach for mapping functional conformational changes to selected regions of a protein and indicate that stimulus-induced partial protein unfolding can be employed as a signaling mechanism. Comparative measurements, Jarzynski-Hummer-Szabo analysis of the data, and steered MD simulations of two double-Cys PYP mutants reveal strong anisotropy in the unfolding mechanism along the two axes defined by the Cys residues. Unfolding along one axis exhibits a transition-state-like feature where six hydrogen bonds break simultaneously. The other axis displays an unpeaked force profile reflecting a non-cooperative transition, challenging the notion that cooperative unfolding is a universal feature in protein stability. MD simulations with a coarse-grained protein model show that the folding of pG is two-state, consistent with experimental observations. In contrast, the folding free energy surface of a coarse-grained model of pB involves an on-pathway partially unfolded intermediate that closely matches experimental data. The results reveal that interactions between the pCA and its binding pocket can switch the energy landscape for PYP from two- to three-state folding, and show how this can be exploited to trigger large functionally important protein conformational changes.

Protein structural dynamics at the gas/water interface examined by hydrogen exchange mass spectrometry.

PubMed

Xiao, Yiming; Konermann, Lars

2015-08-01

Gas/water interfaces (such as air bubbles or foam) are detrimental to the stability of proteins, often causing aggregation. This represents a potential problem for industrial processes, for example, the production and handling of protein drugs. Proteins possess surfactant-like properties, resulting in a high affinity for gas/water interfaces. The tendency of previously buried nonpolar residues to maximize contact with the gas phase can cause significant structural distortion. Most earlier studies in this area employed spectroscopic tools that could only provide limited information. Here we use hydrogen/deuterium exchange (HDX) mass spectrometry (MS) for probing the conformational dynamics of the model protein myoglobin (Mb) in the presence of N(2) bubbles. HDX/MS relies on the principle that unfolded and/or highly dynamic regions undergo faster deuteration than tightly folded segments. In bubble-free solution Mb displays EX2 behavior, reflecting the occurrence of short-lived excursions to partially unfolded conformers. A dramatically different behavior is seen in the presence of N(2) bubbles; EX2 dynamics still take place, but in addition the protein shows EX1 behavior. The latter results from interconversion of the native state with conformers that are globally unfolded and long-lived. These unfolded species likely correspond to Mb that is adsorbed to the surface of gas bubbles. N(2) sparging also induces aggregation. To explain the observed behavior we propose a simple model, that is, "semi-unfolded" ↔ "native" ↔ "globally unfolded" → "aggregated". This model quantitatively reproduces the experimentally observed kinetics. To the best of our knowledge, the current study marks the first exploration of surface denaturation phenomena by HDX/MS. © 2015 The Protein Society.

Growing a Training System and Culture for the Ares I Upper Stage Project

NASA Technical Reports Server (NTRS)

Scott, David W.

2009-01-01

In roughly two years time, Marshall Space Flight Center s (MSFC) Mission Operations Laboratory (MOL) has incubated a personnel training and certification program for about 1000 learners and multiple phases of the Ares I Upper Stage (US) project. Previous MOL-developed training programs focused on about 100 learners with a focus on operations, and had enough full-time training staff to develop courseware and provide training administration. This paper discusses 1) the basics of MOL's training philosophy, 2) how creation of a broad, structured training program unfolded as feedback from more narrowly defined tasks, 3) how training philosophy, development methods, and administration are being simplified and tailored so that many Upper Stage organizations can "grow their own" training yet maintain consistency, accountability, and traceability across the project, 4) interfacing with the production contractor's training system and staff, and 5) reaping training value from existing materials and events.

Number 2 heating oil/propane program. Final report, 1991/92

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

McBrien, J.

1992-06-01

During the 1991--92 heating season, the Massachusetts Division of Energy Resources (DOER) participated in a joint data collection program between several state energy offices and the federal Department of Energy`s (DOE) Energy Information Administration (EIA). The purpose of the program was to collect and monitor retail and wholesale heating oil and propane prices and inventories from October, 1991 through March, 1992. This final report begins with an overview of the unique events which had an impact on the reporting period. Next, the report summarizes the results from the residential heating oil and propane price surveys conducted by DOER over themore » 1991--1992 heating season. The report also incorporates the wholesale heating oil and propane prices and inventories collected by the EIA and distributed to the states. Finally, the report outlines DOER`s use of the data and responses to the events which unfolded during the 1991--1992 heating season.« less

The Grand Banks ERS-1 SAR wave spectra validation experiment

NASA Technical Reports Server (NTRS)

Vachon, P. W.; Dobson, F. W.; Smith, S. D.; Anderson, R. J.; Buckley, J. R.; Allingham, M.; Vandemark, D.; Walsh, E. J.; Khandekar, M.; Lalbeharry, R.

1993-01-01

As part of the ERS-1 validation program, the ERS-1 Synthetic Aperture Radar (SAR) wave spectra validation experiment was carried out over the Grand Banks of Newfoundland (Canada) in Nov. 1991. The principal objective of the experiment was to obtain complete sets of wind and wave data from a variety of calibrated instruments to validate SAR measurements of ocean wave spectra. The field program activities are described and the rather complex wind and wave conditions which were observed are summarized. Spectral comparisons with ERS-1 SAR image spectra are provided. The ERS-1 SAR is shown to have measured swell and range traveling wind seas, but did not measure azimuth traveling wind seas at any time during the experiment. Results of velocity bunching forward mapping and new measurements of the relationship between wind stress and sea state are also shown.

Mapping the energy landscape for second-stage folding of a single membrane protein

PubMed Central

Min, Duyoung; Jefferson, Robert E; Bowie, James U; Yoon, Tae-Young

2016-01-01

Membrane proteins are designed to fold and function in a lipid membrane, yet folding experiments within a native membrane environment are challenging to design. Here we show that single-molecule forced unfolding experiments can be adapted to study helical membrane protein folding under native-like bicelle conditions. Applying force using magnetic tweezers, we find that a transmembrane helix protein, Escherichia coli rhomboid protease GlpG, unfolds in a highly cooperative manner, largely unraveling as one physical unit in response to mechanical tension above 25 pN. Considerable hysteresis is observed, with refolding occurring only at forces below 5 pN. Characterizing the energy landscape reveals only modest thermodynamic stability (ΔG = 6.5 kBT) but a large unfolding barrier (21.3 kBT) that can maintain the protein in a folded state for long periods of time (t1/2 ~3.5 h). The observed energy landscape may have evolved to limit the existence of troublesome partially unfolded states and impart rigidity to the structure. PMID:26479439

An unfolded protein-induced conformational switch activates mammalian IRE1

PubMed Central

Acosta-Alvear, Diego; Nguyen, Hieu T; Lee, Crystal P; Chu, Feixia

2017-01-01

The unfolded protein response (UPR) adjusts the cell’s protein folding capacity in the endoplasmic reticulum (ER) according to need. IRE1 is the most conserved UPR sensor in eukaryotic cells. It has remained controversial, however, whether mammalian and yeast IRE1 use a common mechanism for ER stress sensing. Here, we show that similar to yeast, human IRE1α’s ER-lumenal domain (hIRE1α LD) binds peptides with a characteristic amino acid bias. Peptides and unfolded proteins bind to hIRE1α LD’s MHC-like groove and induce allosteric changes that lead to its oligomerization. Mutation of a hydrophobic patch at the oligomerization interface decoupled peptide binding to hIRE1α LD from its oligomerization, yet retained peptide-induced allosteric coupling within the domain. Importantly, impairing oligomerization of hIRE1α LD abolished IRE1’s activity in living cells. Our results provide evidence for a unifying mechanism of IRE1 activation that relies on unfolded protein binding-induced oligomerization. PMID:28971800

Unfolding four-helix bundles

NASA Astrophysics Data System (ADS)

Gray, Harry B.; Winkler, Jay R.; Kozak, John J.

2011-03-01

A geometrical model has been developed to describe the early stages of unfolding of cytochromes c‧ and c-b562 . Calculations are based on a step-wise extension of the polypeptide chain subject to the constraint that the spatial relationship among the residues of each triplet is fixed by the native-state crystallographic data. The response of each protein to these structural perturbations allows the evolution of each of the four helices in these two proteins to be differentiated. It is found that the two external helices in c‧ unfold before its two internal helices, whereas exactly the opposite behaviour is demonstrated by c-b562 . Each of these cytochromes has an extended, internal, non-helical ('turning') region that initially lags behind the most labile helix but then, at a certain stage (identified for each cytochrome), unravels before any of the four helices present in the native structure. It is believed that these predictions will be useful in guiding future experimental studies on the unfolding of these two cytochromes.

Molecular dynamics simulations and CD spectroscopy reveal hydration-induced unfolding of the intrinsically disordered LEA proteins COR15A and COR15B from Arabidopsis thaliana.

PubMed

Navarro-Retamal, Carlos; Bremer, Anne; Alzate-Morales, Jans; Caballero, Julio; Hincha, Dirk K; González, Wendy; Thalhammer, Anja

2016-10-07

The LEA (late embryogenesis abundant) proteins COR15A and COR15B from Arabidopsis thaliana are intrinsically disordered under fully hydrated conditions, but obtain α-helical structure during dehydration, which is reversible upon rehydration. To understand this unusual structural transition, both proteins were investigated by circular dichroism (CD) and molecular dynamics (MD) approaches. MD simulations showed unfolding of the proteins in water, in agreement with CD data obtained with both HIS-tagged and untagged recombinant proteins. Mainly intramolecular hydrogen bonds (H-bonds) formed by the protein backbone were replaced by H-bonds with water molecules. As COR15 proteins function in vivo as protectants in leaves partially dehydrated by freezing, unfolding was further assessed under crowded conditions. Glycerol reduced (40%) or prevented (100%) unfolding during MD simulations, in agreement with CD spectroscopy results. H-bonding analysis indicated that preferential exclusion of glycerol from the protein backbone increased stability of the folded state.

Pharmacological chaperone reshapes the energy landscape for folding and aggregation of the prion protein

NASA Astrophysics Data System (ADS)

Gupta, Amar Nath; Neupane, Krishna; Rezajooei, Negar; Cortez, Leonardo M.; Sim, Valerie L.; Woodside, Michael T.

2016-06-01

The development of small-molecule pharmacological chaperones as therapeutics for protein misfolding diseases has proven challenging, partly because their mechanism of action remains unclear. Here we study Fe-TMPyP, a tetrapyrrole that binds to the prion protein PrP and inhibits misfolding, examining its effects on PrP folding at the single-molecule level with force spectroscopy. Single PrP molecules are unfolded with and without Fe-TMPyP present using optical tweezers. Ligand binding to the native structure increases the unfolding force significantly and alters the transition state for unfolding, making it more brittle and raising the barrier height. Fe-TMPyP also binds the unfolded state, delaying native refolding. Furthermore, Fe-TMPyP binding blocks the formation of a stable misfolded dimer by interfering with intermolecular interactions, acting in a similar manner to some molecular chaperones. The ligand thus promotes native folding by stabilizing the native state while also suppressing interactions driving aggregation.

Web-based unfolding cases: a strategy to enhance and evaluate clinical reasoning skills.

PubMed

Johnson, Gail; Flagler, Susan

2013-10-01

Clinical reasoning involves the use of both analytical and nonanalytical intuitive cognitive processes. Fostering student development of clinical reasoning skills and evaluating student performance in this cognitive arena can challenge educators. The use of Web-based unfolding cases is proposed as a strategy to address these challenges. Unfolding cases mimic real-life clinical situations by presenting only partial clinical information in sequential segments. Students receive immediate feedback after submitting a response to a given segment. The student's comparison of the desired and submitted responses provides information to enhance the development of clinical reasoning skills. Each student's set of case responses are saved for the instructor in an individual-student electronic file, providing a record of the student's knowledge and thinking processes for faculty evaluation. For the example case given, the approaches used to evaluate individual components of clinical reasoning are provided. Possible future uses of Web-based unfolding cases are described. Copyright 2013, SLACK Incorporated.

Initiation of Phage Infection by Partial Unfolding and Prolyl Isomerization*♦

PubMed Central

Hoffmann-Thoms, Stephanie; Weininger, Ulrich; Eckert, Barbara; Jakob, Roman P.; Koch, Johanna R.; Balbach, Jochen; Schmid, Franz X.

2013-01-01

Infection of Escherichia coli by the filamentous phage fd starts with the binding of the N2 domain of the phage gene-3-protein to an F pilus. This interaction triggers partial unfolding of the gene-3-protein, cis → trans isomerization at Pro-213, and domain disassembly, thereby exposing its binding site for the ultimate receptor TolA. The trans-proline sets a molecular timer to maintain the binding-active state long enough for the phage to interact with TolA. We elucidated the changes in structure and local stability that lead to partial unfolding and thus to the activation of the gene-3-protein for phage infection. Protein folding and TolA binding experiments were combined with real-time NMR spectroscopy, amide hydrogen exchange measurements, and phage infectivity assays. In combination, the results provide a molecular picture of how a local unfolding reaction couples with prolyl isomerization not only to generate the activated state of a protein but also to maintain it for an extended time. PMID:23486474

The small angle x-ray scattering of globular proteins in solution during heat denaturation

NASA Astrophysics Data System (ADS)

Banuelos, Jose; Urquidi, Jacob

2008-10-01

The ability of proteins to change their conformation in response to changes in their environment has consequences in biological processes like metabolism, chemical regulation in cells, and is believed to play a role in the onset of several neurodegenerative diseases. Factors such as a change in temperature, pressure, and the introduction of ions into the aqueous environment of a protein can give rise to the folding/unfolding of a protein. As a protein unfolds, the ratio of nonpolar to polar groups exposed to water changes, affecting a protein's thermodynamic properties. Using small angle x-ray scattering (SAXS), we are currently studying the intermediate protein conformations that arise during the folding/unfolding process as a function of temperature for five globular proteins. Trends in the observed intermediate structures of these globular proteins, along with correlations with data on protein thermodynamics may help elucidate shared characteristics between all proteins in the folding/unfolding process. Experimental design considerations will be discussed and preliminary results for some of these systems will be presented.

Unfolding of a ClC chloride transporter retains memory of its evolutionary history.

PubMed

Min, Duyoung; Jefferson, Robert E; Qi, Yifei; Wang, Jing Yang; Arbing, Mark A; Im, Wonpil; Bowie, James U

2018-05-01

ClC chloride channels and transporters are important for chloride homeostasis in species from bacteria to human. Mutations in ClC proteins cause genetically inherited diseases, some of which are likely to involve folding defects. The ClC proteins present a challenging and unusual biological folding problem because they are large membrane proteins possessing a complex architecture, with many reentrant helices that go only partway through membrane and loop back out. Here we were able to examine the unfolding of the Escherichia coli ClC transporter, ClC-ec1, using single-molecule forced unfolding methods. We found that the protein could be separated into two stable halves that unfolded independently. The independence of the two domains is consistent with an evolutionary model in which the two halves arose from independently folding subunits that later fused together. Maintaining smaller folding domains of lesser complexity within large membrane proteins may be an advantageous strategy to avoid misfolding traps.

Contribution of long-range interactions to the secondary structure of an unfolded globin.

PubMed

Fedyukina, Daria V; Rajagopalan, Senapathy; Sekhar, Ashok; Fulmer, Eric C; Eun, Ye-Jin; Cavagnero, Silvia

2010-09-08

This work explores the effect of long-range tertiary contacts on the distribution of residual secondary structure in the unfolded state of an alpha-helical protein. N-terminal fragments of increasing length, in conjunction with multidimensional nuclear magnetic resonance, were employed. A protein representative of the ubiquitous globin fold was chosen as the model system. We found that, while most of the detectable alpha-helical population in the unfolded ensemble does not depend on the presence of the C-terminal region (corresponding to the native G and H helices), specific N-to-C long-range contacts between the H and A-B-C regions enhance the helical secondary structure content of the N terminus (A-B-C regions). The simple approach introduced here, based on the evaluation of N-terminal polypeptide fragments of increasing length, is of general applicability to identify the influence of long-range interactions in unfolded proteins. Copyright 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

ATP-induced noncooperative thermal unfolding of hen lysozyme

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

Liu, Honglin; Yin, Peidong; He, Shengnan

To understand the role of ATP underlying the enhanced amyloidosis of hen egg white lysozyme (HEWL), the synchrotron radiation circular dichroism, combined with tryptophan fluorescence, dynamic light-scattering, and differential scanning calorimetry, is used to examine the alterations of the conformation and thermal unfolding pathway of the HEWL in the presence of ATP, Mg{sup 2+}-ATP, ADP, AMP, etc. It is revealed that the binding of ATP to HEWL through strong electrostatic interaction changes the secondary structures of HEWL and makes the exposed residue W62 move into hydrophobic environments. This alteration of W62 decreases the {beta}-domain stability of HEWL, induces a noncooperativemore » unfolding of the secondary structures, and produces a partially unfolded intermediate. This intermediate containing relatively rich {alpha}-helix and less {beta}-sheet structures has a great tendency to aggregate. The results imply that the ease of aggregating of HEWL is related to the extent of denaturation of the amyloidogenic region, rather than the electrostatic neutralizing effect or monomeric {beta}-sheet enriched intermediate.« less

Responses of spring phenology to climate warming reduced over the past decades

NASA Astrophysics Data System (ADS)

Fu, Yongshuo. H.; Zhao, hongfang; piao, Shilong; Peaucelle, Marc; Peng, Shushi; Zhou, Guiyun; Ciais, Philippe; Huang, Mengtian; Menzel, Annette; Penuelas, Josep; Song, Yang; Vitasse, Yann; Zeng, Zhenzhong; Janssens, Ivan. A.

2016-04-01

The phenology of spring leaf unfolding is one of the key indicators of the climate change on ecosystems, and influences regional and hemispheric-scale carbon balances and plant-animal interactions. Changes in the phenology of spring leaf unfolding can also exert biophysical feedbacks on climate by modifying the surface albedo and energy budget. Recent studies have reported significant advances in spring phenology as a result of warming in most northern hemisphere regions. Climate warming is projected to further increase, but the future evolution of the phenology of spring leaf unfolding remains uncertain - in view of the imperfect understanding of how the underlying mechanisms respond to environmental stimuli. In addition, the relative contributions of each environmental stimulus, which together define the apparent temperature sensitivity of the phenology of spring leaf unfolding (advances in days per degree Celsius warming, ST), may also change over time. An improved characterization of the variation in phenological responses to spring temperature is thus valuable, provided that it addresses temporal and spatial scales relevant for regional projections. Using long-term in situ observations of leaf unfolding for seven dominant European tree species at 1,245 sites, we show here that the apparent response of leaf unfolding to climate warming (ST, expressed in days advance per ° C) has significantly decreased from 1980 to 2013 in all monitored tree species. Averaged across all species and sites, ST decreased by 40% from 4.0 ± 1.8 days ° C-1 during 1980-1994 to 2.3 ± 1.6 days ° C-1 during 1999-2013. The declining ST was also simulated by chilling-based phenology models, albeit with a weaker decline (24%-30%) than observed in situ. The reduction in ST is likely to be partly attributable to reduced chilling. Nonetheless, other mechanisms may also play a role, such as 'photoperiod limitation' mechanisms that may become ultimately limiting when leaf unfolding dates occur too early in the season. Our results provide empirical evidence for a declining ST, but also suggest that the predicted strong winter warming in the future may further reduce ST and therefore result in a slowdown in the advance of tree spring phenology.