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Journal articles on the topic 'Ensemble FRET'

Author: Grafiati

Published: 6 September 2023

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1

Dagher, Milad, Michael Kleinman, Andy Ng, and David Juncker. "Ensemble multicolour FRET model enables barcoding at extreme FRET levels." Nature Nanotechnology 13, no. 10 (July 30, 2018): 925–32. http://dx.doi.org/10.1038/s41565-018-0205-0.

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2

Warrier, Anita R., Chithra Parameswaran, Jayachandra Bingi, and C. Vijayan. "FRET controlled photoluminescence inβ-In2S3microflower—Au nanoparticle ensemble." Materials Research Express 3, no. 6 (June 16, 2016): 065016. http://dx.doi.org/10.1088/2053-1591/3/6/065016.

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3

LeBlanc, Sharonda, Prakash Kulkarni, and Keith Weninger. "Single Molecule FRET: A Powerful Tool to Study Intrinsically Disordered Proteins." Biomolecules 8, no. 4 (November 8, 2018): 140. http://dx.doi.org/10.3390/biom8040140.

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Intrinsically disordered proteins (IDPs) are often modeled using ideas from polymer physics that suggest they smoothly explore all corners of configuration space. Experimental verification of this random, dynamic behavior is difficult as random fluctuations of IDPs cannot be synchronized across an ensemble. Single molecule fluorescence (or Förster) resonance energy transfer (smFRET) is one of the few approaches that are sensitive to transient populations of sub-states within molecular ensembles. In some implementations, smFRET has sufficient time resolution to resolve transitions in IDP behaviors. Here we present experimental issues to consider when applying smFRET to study IDP configuration. We illustrate the power of applying smFRET to IDPs by discussing two cases in the literature of protein systems for which smFRET has successfully reported phosphorylation-induced modification (but not elimination) of the disordered properties that have been connected to impacts on the related biological function. The examples we discuss, PAGE4 and a disordered segment of the GluN2B subunit of the NMDA receptor, illustrate the great potential of smFRET to inform how IDP function can be regulated by controlling the detailed ensemble of disordered states within biological networks.

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4

Lai, Wan-Jung C., and Dmitri N. Ermolenko. "Ensemble and single-molecule FRET studies of protein synthesis." Methods 137 (March 2018): 37–48. http://dx.doi.org/10.1016/j.ymeth.2017.12.007.

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5

Vandenberk, Niels. "Comparison of Organic Blue/Red Dye FRET Pairs via Ensemble and Single-Molecule FRET Spectroscopy." Biophysical Journal 114, no. 3 (February 2018): 167a—168a. http://dx.doi.org/10.1016/j.bpj.2017.11.938.

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6

Tang, Chun. "Decomposing NMR Ensemble with the Assistance of Single Molecule FRET." Biophysical Journal 116, no. 3 (February 2019): 470a. http://dx.doi.org/10.1016/j.bpj.2018.11.2541.

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7

Aznauryan, Mikayel, Leonildo Delgado, Andrea Soranno, Daniel Nettels, Jie-rong Huang, Alexander M. Labhardt, Stephan Grzesiek, and Benjamin Schuler. "Comprehensive structural and dynamical view of an unfolded protein from the combination of single-molecule FRET, NMR, and SAXS." Proceedings of the National Academy of Sciences 113, no. 37 (August 26, 2016): E5389—E5398. http://dx.doi.org/10.1073/pnas.1607193113.

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The properties of unfolded proteins are essential both for the mechanisms of protein folding and for the function of the large group of intrinsically disordered proteins. However, the detailed structural and dynamical characterization of these highly dynamic and conformationally heterogeneous ensembles has remained challenging. Here we combine and compare three of the leading techniques for the investigation of unfolded proteins, NMR spectroscopy (NMR), small-angle X-ray scattering (SAXS), and single-molecule Förster resonance energy transfer (FRET), with the goal of quantitatively testing their consistency and complementarity and for obtaining a comprehensive view of the unfolded-state ensemble. Using unfolded ubiquitin as a test case, we find that its average dimensions derived from FRET and from structural ensembles calculated using the program X-PLOR-NIH based on NMR and SAXS restraints agree remarkably well; even the shapes of the underlying intramolecular distance distributions are in good agreement, attesting to the reliability of the approaches. The NMR-based results provide a highly sensitive way of quantifying residual structure in the unfolded state. FRET-based nanosecond fluorescence correlation spectroscopy allows long-range distances and chain dynamics to be probed in a time range inaccessible by NMR. The combined techniques thus provide a way of optimally using the complementarity of the available methods for a quantitative structural and dynamical description of unfolded proteins both at the global and the local level.

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8

Pushina, Mariia, Sepideh Farshbaf, Elena G. Shcherbakova, and Pavel Anzenbacher. "A dual chromophore sensor for the detection of amines, diols, hydroxy acids, and amino alcohols." Chemical Communications 55, no. 31 (2019): 4495–98. http://dx.doi.org/10.1039/c9cc01051c.

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The determination of enantiomeric excess (ee) in various groups of chiral compounds, namely amines, amino alcohols, diols, and hydroxy acids is performed using a dual chromophore FRET/PET based sensor ensemble.

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9

Schaaf, Tory M., Kurt C. Peterson, Benjamin D. Grant, David D. Thomas, and Gregory D. Gillispie. "Spectral Unmixing Plate Reader: High-Throughput, High-Precision FRET Assays in Living Cells." SLAS DISCOVERY: Advancing the Science of Drug Discovery 22, no. 3 (November 23, 2016): 250–61. http://dx.doi.org/10.1177/1087057116679637.

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We have developed a microplate reader that records a complete high-quality fluorescence emission spectrum on a well-by-well basis under true high-throughput screening (HTS) conditions. The read time for an entire 384-well plate is less than 3 min. This instrument is particularly well suited for assays based on fluorescence resonance energy transfer (FRET). Intramolecular protein biosensors with genetically encoded green fluorescent protein (GFP) donor and red fluorescent protein (RFP) acceptor tags at positions sensitive to structural changes were stably expressed and studied in living HEK cells. Accurate quantitation of FRET was achieved by decomposing each observed spectrum into a linear combination of four component (basis) spectra (GFP emission, RFP emission, water Raman, and cell autofluorescence). Excitation and detection are both conducted from the top, allowing for thermoelectric control of the sample temperature from below. This spectral unmixing plate reader (SUPR) delivers an unprecedented combination of speed, precision, and accuracy for studying ensemble-averaged FRET in living cells. It complements our previously reported fluorescence lifetime plate reader, which offers the feature of resolving multiple FRET populations within the ensemble. The combination of these two direct waveform-recording technologies greatly enhances the precision and information content for HTS in drug discovery.

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10

Bhattacharya, Supriyo, and Xingcheng Lin. "Recent Advances in Computational Protocols Addressing Intrinsically Disordered Proteins." Biomolecules 9, no. 4 (April 11, 2019): 146. http://dx.doi.org/10.3390/biom9040146.

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Intrinsically disordered proteins (IDP) are abundant in the human genome and have recently emerged as major therapeutic targets for various diseases. Unlike traditional proteins that adopt a definitive structure, IDPs in free solution are disordered and exist as an ensemble of conformations. This enables the IDPs to signal through multiple signaling pathways and serve as scaffolds for multi-protein complexes. The challenge in studying IDPs experimentally stems from their disordered nature. Nuclear magnetic resonance (NMR), circular dichroism, small angle X-ray scattering, and single molecule Förster resonance energy transfer (FRET) can give the local structural information and overall dimension of IDPs, but seldom provide a unified picture of the whole protein. To understand the conformational dynamics of IDPs and how their structural ensembles recognize multiple binding partners and small molecule inhibitors, knowledge-based and physics-based sampling techniques are utilized in-silico, guided by experimental structural data. However, efficient sampling of the IDP conformational ensemble requires traversing the numerous degrees of freedom in the IDP energy landscape, as well as force-fields that accurately model the protein and solvent interactions. In this review, we have provided an overview of the current state of computational methods for studying IDP structure and dynamics and discussed the major challenges faced in this field.

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11

Pan, Chengjun, Kazunori Sugiyasu, and Masayuki Takeuchi. "Blending conjugated polymers without phase separation for fluorescent colour tuning of polymeric materials through FRET." Chem. Commun. 50, no. 80 (2014): 11814–17. http://dx.doi.org/10.1039/c4cc03594a.

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12

Baltierra-Jasso, Laura E., Michael J. Morten, and Steven W. Magennis. "Sub-Ensemble Monitoring of DNA Strand Displacement Using Multiparameter Single-Molecule FRET." ChemPhysChem 19, no. 5 (January 26, 2018): 551–55. http://dx.doi.org/10.1002/cphc.201800012.

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13

Steffen, Fabio D., Roland K. O. Sigel, and Richard Börner. "An atomistic view on carbocyanine photophysics in the realm of RNA." Physical Chemistry Chemical Physics 18, no. 42 (2016): 29045–55. http://dx.doi.org/10.1039/c6cp04277e.

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The photophysics of cyanine-labeled nucleic acids (NA) are transferred from the ensemble to the molecular level by means of all-atom MD with explicit dye probes. RNA-induced fluorescence enhancement (RIFE) is introduced as a sensor for dye–NA interactions to bridge the distance regimes of PET and FRET.

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14

Diao, Jiajie, Yuji Ishitsuka, and Woo-Ri Bae. "Single-molecule FRET study of SNARE-mediated membrane fusion." Bioscience Reports 31, no. 6 (September 15, 2011): 457–63. http://dx.doi.org/10.1042/bsr20110011.

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Membrane fusion is one of the most important cellular processes by which two initially distinct lipid bilayers merge their hydrophobic cores, resulting in one interconnected structure. Proteins, called SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor), play a central role in the fusion process that is also regulated by several accessory proteins. In order to study the SNARE-mediated membrane fusion, the in vitro protein reconstitution assay involving ensemble FRET (fluorescence resonance energy transfer) has been used over a decade. In this mini-review, we describe several single-molecule-based FRET approaches that have been applied to this field to overcome the shortage of the bulk assay in terms of protein and fusion dynamics.

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15

Klement, Reinhard, Timo Graen, Asaf Grupi, Elisha Haas, and Helmut Grubmüller. "Molecular Dynamics Simulations of Alpha-Synuclein Ensemble FRET Measurements from Different Force Fields." Biophysical Journal 110, no. 3 (February 2016): 551a. http://dx.doi.org/10.1016/j.bpj.2015.11.2949.

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16

Ferri, C. G. L., R. H. Inman, B. Rich, A. Gopinathan, M. Khine, and S. Ghosh. "Plasmon-induced enhancement of intra-ensemble FRET in quantum dots on wrinkled thin films." Optical Materials Express 3, no. 3 (February 6, 2013): 383. http://dx.doi.org/10.1364/ome.3.000383.

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17

Renz, Malte, Brian Daniels, Gyorgy Vamosi, Irwin Arias, and Jennifer Lippincott-Schwartz. "Plasticity of the Asialoglycoprotein Receptor Deciphered by Ensemble FRET and Single-Molecule Counting PALM." Biophysical Journal 102, no. 3 (January 2012): 430a. http://dx.doi.org/10.1016/j.bpj.2011.11.2354.

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18

Schütz, Robert, Shashwat Malhotra, Inara Thomas, Christian Strothkämper, Andreas Bartelt, Klaus Schwarzburg, Thomas Hannappel, Carlo Fasting, and Rainer Eichberger. "Dynamics of a Covalently Conjoined FRET Dye Ensemble for Electron Injection into ZnO Nanorods." Journal of Physical Chemistry C 118, no. 18 (April 25, 2014): 9336–45. http://dx.doi.org/10.1021/jp501062e.

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19

Chung, Hoi Sung, Fanjie Meng, Jae-Yeol Kim, Kevin McHale, Irina V. Gopich, and John M. Louis. "Oligomerization of the tetramerization domain of p53 probed by two- and three-color single-molecule FRET." Proceedings of the National Academy of Sciences 114, no. 33 (July 31, 2017): E6812—E6821. http://dx.doi.org/10.1073/pnas.1700357114.

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We describe a method that combines two- and three-color single-molecule FRET spectroscopy with 2D FRET efficiency–lifetime analysis to probe the oligomerization process of intrinsically disordered proteins. This method is applied to the oligomerization of the tetramerization domain (TD) of the tumor suppressor protein p53. TD exists as a monomer at subnanomolar concentrations and forms a dimer and a tetramer at higher concentrations. Because the dissociation constants of the dimer and tetramer are very close, as we determine in this paper, it is not possible to characterize different oligomeric species by ensemble methods, especially the dimer that cannot be readily separated. However, by using single-molecule FRET spectroscopy that includes measurements of fluorescence lifetime and two- and three-color FRET efficiencies with corrections for submillisecond acceptor blinking, we show that it is possible to obtain structural information for individual oligomers at equilibrium and to determine the dimerization kinetics. From these analyses, we show that the monomer is intrinsically disordered and that the dimer conformation is very similar to that of the tetramer but the C terminus of the dimer is more flexible.

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20

Chan, Kevin, Clive Yik-Sham Chung, and Vivian Wing-Wah Yam. "Parallel folding topology-selective label-free detection and monitoring of conformational and topological changes of different G-quadruplex DNAs by emission spectral changes via FRET of mPPE-Ala–Pt(ii) complex ensemble." Chemical Science 7, no. 4 (2016): 2842–55. http://dx.doi.org/10.1039/c5sc04563k.

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21

Baldwin, Tanya A., Gi-Ho Kim, Tyrel R. Deutscher, Maria E. Moutsoglou, and John M. Robinson. "A Comparison of Donor-Acceptor Distance Distributions Derived from Ensemble and Single-Pair FRET Measurements." Biophysical Journal 102, no. 3 (January 2012): 403a. http://dx.doi.org/10.1016/j.bpj.2011.11.2201.

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22

Renz, M., B. R. Daniels, G. Vamosi, I. M. Arias, and J. Lippincott-Schwartz. "Plasticity of the asialoglycoprotein receptor deciphered by ensemble FRET imaging and single-molecule counting PALM imaging." Proceedings of the National Academy of Sciences 109, no. 44 (October 4, 2012): E2989—E2997. http://dx.doi.org/10.1073/pnas.1211753109.

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23

Grossman-Haham, Iris, Gabriel Rosenblum, Trishool Namani, and Hagen Hofmann. "Slow domain reconfiguration causes power-law kinetics in a two-state enzyme." Proceedings of the National Academy of Sciences 115, no. 3 (January 3, 2018): 513–18. http://dx.doi.org/10.1073/pnas.1714401115.

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Protein dynamics are typically captured well by rate equations that predict exponential decays for two-state reactions. Here, we describe a remarkable exception. The electron-transfer enzyme quiescin sulfhydryl oxidase (QSOX), a natural fusion of two functionally distinct domains, switches between open- and closed-domain arrangements with apparent power-law kinetics. Using single-molecule FRET experiments on time scales from nanoseconds to milliseconds, we show that the unusual open-close kinetics results from slow sampling of an ensemble of disordered domain orientations. While substrate accelerates the kinetics, thus suggesting a substrate-induced switch to an alternative free energy landscape of the enzyme, the power-law behavior is also preserved upon electron load. Our results show that the slow sampling of open conformers is caused by a variety of interdomain interactions that imply a rugged free energy landscape, thus providing a generic mechanism for dynamic disorder in multidomain enzymes.

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24

Osa-Andrews, Bremansu, Kee Tan, Angelina Sampson, and Surtaj Iram. "Development of Novel Intramolecular FRET-Based ABC Transporter Biosensors to Identify New Substrates and Modulators." Pharmaceutics 10, no. 4 (October 13, 2018): 186. http://dx.doi.org/10.3390/pharmaceutics10040186.

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Multidrug resistance protein 1 (MRP1) can efflux a wide variety of molecules including toxic chemicals, drugs, and their derivatives out of cells. Substrates of MRP1 include anti-cancer agents, antibiotics, anti-virals, anti-human immunodeficiency virus (HIV), and many other drugs. To identify novel substrates and modulators of MRP1 by exploiting intramolecular fluorescence resonance energy transfer (FRET), we genetically engineered six different two-color MRP1 proteins by changing green fluorescent protein (GFP) insertion sites, while keeping the red fluorescent protein (RFP) at the C-terminal of MRP1. Four of six recombinant proteins showed normal expression, localization, and transport activity. We quantified intramolecular FRET using ensemble fluorescence spectroscopy in response to binding of known substrate or ATP alone, substrate/ATP, and trapping of the transporter in closed conformation by vanadate. Recombinant MRP1 proteins GR-881, GR-888, and GR-905 exhibited reproducible and higher FRET changes under all tested conditions and are very promising for use as MRP1 biosensors. Furthermore, we used GR-881 to screen 40 novel anti-cancer drugs and identified 10 hits that potentially directly interact with MRP1 and could be substrates or modulators. Profiling of drug libraries for interaction with MRP1 can provide very useful information to improve the efficacy and reduce the toxicity of various therapies.

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25

Baltierra-Jasso, Laura E., Michael J. Morten, and Steven W. Magennis. "Cover Feature: Sub-Ensemble Monitoring of DNA Strand Displacement Using Multiparameter Single-Molecule FRET (ChemPhysChem 5/2018)." ChemPhysChem 19, no. 5 (February 28, 2018): 548. http://dx.doi.org/10.1002/cphc.201800146.

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26

KOBITSKI, ANDREI YU, ALEXANDER NIERTH, MARTIN HENGESBACH, ANDRES JÄSCHKE, MARK HELM, and G. ULRICH NIENHAUS. "EXPLORING THE FOLDING FREE ENERGY LANDSCAPE OF SMALL RNA MOLECULES BY SINGLE-PAIR FÖRSTER RESONANCE ENERGY TRANSFER." Biophysical Reviews and Letters 03, no. 04 (October 2008): 439–57. http://dx.doi.org/10.1142/s1793048008000873.

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Proteins and RNA are biological macromolecules built from linear polymers. The process by which they fold into compact, well-defined, three-dimensional architectures to perform their functional tasks is still not well understood. It can be visualized by Brownian motion of an ensemble of molecules through a rugged energy landscape in search of an energy minimum corresponding to the native state. To explore the conformational energy landscape of small RNAs, single pair Förster resonance energy transfer (spFRET) experiments on solutions as well as on surface-immobilized samples have provided new insights. In this review, we focus on our recent work on two FRET-labeled small RNAs, the Diels-Alderase (DAse) ribozyme and the human mitochondrial tRNA Lys . For both RNAs, three different conformational states can be distinguished, and the associated mean FRET efficiencies provide clues about their structural properties. The systematic variation of their free energies with the concentration of Mg 2+ counterions was analyzed quantitatively by using a thermodynamic model that separates conformational changes from Mg 2+ binding. Furthermore, time-resolved spFRET studies on immobilized DAse reveal slow interconversions between intermediate and folded states on the time scale of ~ 100 ms. The quantitative data obtained from spFRET experiments may likely assist in the further development of theories and models addressing the folding dynamics and (counterion-dependent) energetics of RNA molecules.

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27

Rahamim, Gil, Marina Chemerovski-Glikman, Shai Rahimipour, Dan Amir, and Elisha Haas. "Resolution of Two Sub-Populations of Conformers and Their Individual Dynamics by Time Resolved Ensemble Level FRET Measurements." PLOS ONE 10, no. 12 (December 23, 2015): e0143732. http://dx.doi.org/10.1371/journal.pone.0143732.

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28

Fegan, Adrian, Pravin S. Shirude, Liming Ying, and Shankar Balasubramanian. "Ensemble and single molecule FRET analysis of the structure and unfolding kinetics of the c-kit promoter quadruplexes." Chem. Commun. 46, no. 6 (2010): 946–48. http://dx.doi.org/10.1039/b920680a.

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29

Espinoza-Sanchez, Sofia, Lauren Ann Metskas, Steven Z. Chou, Elizabeth Rhoades, and Thomas D. Pollard. "Conformational changes in Arp2/3 complex induced by ATP, WASp-VCA, and actin filaments." Proceedings of the National Academy of Sciences 115, no. 37 (August 27, 2018): E8642—E8651. http://dx.doi.org/10.1073/pnas.1717594115.

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We used fluorescence spectroscopy and EM to determine how binding of ATP, nucleation-promoting factors, actin monomers, and actin filaments changes the conformation of Arp2/3 complex during the process that nucleates an actin filament branch. We mutated subunits of Schizosaccharomyces pombe Arp2/3 complex for labeling with fluorescent dyes at either the C termini of Arp2 and Arp3 or ArpC1 and ArpC3. We measured Förster resonance energy transfer (FRET) efficiency (ETeff) between the dyes in the presence of the various ligands. We also computed class averages from electron micrographs of negatively stained specimens. ATP binding made small conformational changes of the nucleotide-binding cleft of the Arp2 subunit. WASp-VCA, WASp-CA, and WASp-actin-VCA changed the ETeff between the dyes on the Arp2 and Arp3 subunits much more than between dyes on ArpC1 and ArpC3. Ensemble FRET detected an additional structural change that brought ArpC1 and ArpC3 closer together when Arp2/3 complex bound actin filaments. VCA binding to Arp2/3 complex causes a conformational change that favors binding to the side of an actin filament, which allows further changes required to nucleate a daughter filament.

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30

Wang, Xin, Jingyuan Nie, Yi Li, Hai Pan, Peng Zheng, Meng Qin, Yi Cao, and Wei Wang. "A versatile platform for single-molecule enzymology of restriction endonuclease." Journal of Innovative Optical Health Sciences 12, no. 01 (January 2019): 1841002. http://dx.doi.org/10.1142/s179354581841002x.

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Enzymes are the major players for many biological processes. Fundamental studies of the enzymatic activity at the single-molecule level provides important information that is otherwise inaccessible at the ensemble level. Yet, these single-molecule experiments are technically difficult and generally require complicated experimental design. Here, we develop a Holliday junction (HJ)-based platform to study the activity of restriction endonucleases at the single-molecule level using single-molecule FRET (sm-FRET). We show that the intrinsic dynamics of HJ can be used as the reporter for both the enzyme-binding and the substrate-release events. Thanks to the multiple-arms structure of HJ, the fluorophore-labeled arms can be different from the surface anchoring arm and the substrate arm. Therefore, it is possible to independently change the substrate arm to study different enzymes with similar functions. Such a design is extremely useful for the systematic study of enzymes from the same family or enzymes bearing different pathologic mutations. Moreover, this method can be easily extended to study other types of DNA-binding enzymes without too much modification of the design. We anticipate it can find broad applications in single-molecule enzymology.

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31

Goswami, Shyamaprosad, Sima Paul, and Abhishek Manna. "FRET based selective and ratiometric ‘naked-eye’ detection of CN− in aqueous solution on fluorescein–Zn–naphthalene ensemble platform." Tetrahedron Letters 55, no. 29 (July 2014): 3946–49. http://dx.doi.org/10.1016/j.tetlet.2014.05.018.

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32

Husada, Florence, Giorgos Gouridis, Ruslan Vietrov, Gea K. Schuurman-Wolters, Evelyn Ploetz, Marijn de Boer, Bert Poolman, and Thorben Cordes. "Watching conformational dynamics of ABC transporters with single-molecule tools." Biochemical Society Transactions 43, no. 5 (October 1, 2015): 1041–47. http://dx.doi.org/10.1042/bst20150140.

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ATP-binding cassette (ABC) transporters play crucial roles in cellular processes, such as nutrient uptake, drug resistance, cell-volume regulation and others. Despite their importance, all proposed molecular models for transport are based on indirect evidence, i.e. functional interpretation of static crystal structures and ensemble measurements of function and structure. Thus, classical biophysical and biochemical techniques do not readily visualize dynamic structural changes. We recently started to use single-molecule fluorescence techniques to study conformational states and changes of ABC transporters in vitro, in order to observe directly how the different steps during transport are coordinated. This review summarizes our scientific strategy and some of the key experimental advances that allowed the substrate-binding mechanism of prokaryotic ABC importers and the transport cycle to be explored. The conformational states and transitions of ABC-associated substrate-binding domains (SBDs) were visualized with single-molecule FRET, permitting a direct correlation of structural and kinetic information of SBDs. We also delineated the different steps of the transport cycle. Since information in such assays are restricted by proper labelling of proteins with fluorescent dyes, we present a simple approach to increase the amount of protein with FRET information based on non-specific interactions between a dye and the size-exclusion chromatography (SEC) column material used for final purification.

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33

Wälti, Marielle A., Joseph Steiner, Fanjie Meng, Hoi Sung Chung, John M. Louis, Rodolfo Ghirlando, Vitali Tugarinov, Avindra Nath, and G. Marius Clore. "Probing the mechanism of inhibition of amyloid-β(1–42)–induced neurotoxicity by the chaperonin GroEL." Proceedings of the National Academy of Sciences 115, no. 51 (December 3, 2018): E11924—E11932. http://dx.doi.org/10.1073/pnas.1817477115.

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The human chaperonin Hsp60 is thought to play a role in the progression of Alzheimer’s disease by mitigating against intracellular β-amyloid stress. Here, we show that the bacterial homolog GroEL (51% sequence identity) reduces the neurotoxic effects of amyloid-β(1–42) (Aβ42) on human neural stem cell-derived neuronal cultures. To understand the mechanism of GroEL-mediated abrogation of neurotoxicity, we studied the interaction of Aβ42 with GroEL using a variety of biophysical techniques. Aβ42 binds to GroEL as a monomer with a lifetime of ∼1 ms, as determined from global analysis of multiple relaxation-based NMR experiments. Dynamic light scattering demonstrates that GroEL dissolves small amounts of high–molecular-weight polydisperse aggregates present in fresh soluble Aβ42 preparations. The residue-specific transverse relaxation rate profile for GroEL-bound Aβ42 reveals the presence of three anchor-binding regions (residues 16–21, 31–34, and 40–41) located within the hydrophobic GroEL-consensus binding sequences. Single-molecule FRET analysis of Aβ42 binding to GroEL results in no significant change in the FRET efficiency of a doubly labeled Aβ42 construct, indicating that Aβ42 samples a random coil ensemble when bound to GroEL. Finally, GroEL substantially slows down the disappearance of NMR visible Aβ42 species and the appearance of Aβ42 protofibrils and fibrils as monitored by electron and atomic force microscopies. The latter observations correlate with the effect of GroEL on the time course of Aβ42-induced neurotoxicity. These data provide a physical basis for understanding how Hsp60 may serve to slow down the progression of Alzheimer’s disease.

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34

Dietrich, Anja, Volker Buschmann, Christian Müller, and Markus Sauer. "Fluorescence resonance energy transfer (FRET) and competing processes in donor–acceptor substituted DNA strands: a comparative study of ensemble and single-molecule data." Reviews in Molecular Biotechnology 82, no. 3 (January 2002): 211–31. http://dx.doi.org/10.1016/s1389-0352(01)00039-3.

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35

Haas, Elisha. "The Study of Protein Folding and Dynamics by Determination of Intramolecular Distance Distributions and Their Fluctuations Using Ensemble and Single-Molecule FRET Measurements." ChemPhysChem 6, no. 5 (May 13, 2005): 858–70. http://dx.doi.org/10.1002/cphc.200400617.

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36

Peran, Ivan, Alex S. Holehouse, Isaac S. Carrico, Rohit V. Pappu, Osman Bilsel, and Daniel P. Raleigh. "Unfolded states under folding conditions accommodate sequence-specific conformational preferences with random coil-like dimensions." Proceedings of the National Academy of Sciences 116, no. 25 (June 5, 2019): 12301–10. http://dx.doi.org/10.1073/pnas.1818206116.

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Proteins are marginally stable molecules that fluctuate between folded and unfolded states. Here, we provide a high-resolution description of unfolded states under refolding conditions for the N-terminal domain of the L9 protein (NTL9). We use a combination of time-resolved Förster resonance energy transfer (FRET) based on multiple pairs of minimally perturbing labels, time-resolved small-angle X-ray scattering (SAXS), all-atom simulations, and polymer theory. Upon dilution from high denaturant, the unfolded state undergoes rapid contraction. Although this contraction occurs before the folding transition, the unfolded state remains considerably more expanded than the folded state and accommodates a range of local and nonlocal contacts, including secondary structures and native and nonnative interactions. Paradoxically, despite discernible sequence-specific conformational preferences, the ensemble-averaged properties of unfolded states are consistent with those of canonical random coils, namely polymers in indifferent (theta) solvents. These findings are concordant with theoretical predictions based on coarse-grained models and inferences drawn from single-molecule experiments regarding the sequence-specific scaling behavior of unfolded proteins under folding conditions.

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37

Grecco, H. E., K. A. Lidke, R. Heintzmann, D. S. Lidke, C. Spagnuolo, O. E. Martinez, E. A. Jares-Erijman, and T. M. Jovin. "Ensemble and single particle photophysical properties (two-photon excitation, anisotropy, FRET, lifetime, spectral conversion) of commercial quantum dots in solution and in live cells." Microscopy Research and Technique 65, no. 4-5 (November 2004): 169–79. http://dx.doi.org/10.1002/jemt.20129.

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38

JØRGENSEN, SUNE K., and NIKOS S. HATZAKIS. "INSIGHTS IN ENZYME FUNCTIONAL DYNAMICS AND ACTIVITY REGULATION BY SINGLE MOLECULE STUDIES." Biophysical Reviews and Letters 08, no. 03n04 (December 2013): 137–60. http://dx.doi.org/10.1142/s1793048013300028.

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The advent of advanced single molecule measurements heralded the arrival of a wealth of dynamic information revolutionizing our understanding of protein dynamics and behavior in ways not deducible by conventional bulk assays. They offered the direct observation and quantification of the abundance and life time of multiple states and transient intermediates in the energy landscape that are typically averaged out in non-synchronized ensemble measurements, thus providing unprecedented insights into complex biological processes. Here we survey the current state of the art in single-molecule fluorescence microscopy methodology for studying the mechanism of enzymatic activity and the insights on protein functional dynamics. We will initially discuss the strategies employed to date, their limitations and possible ways to overcome them, and finally how single enzyme kinetics can advance our understanding on mechanisms underlying function and regulation of proteins. [Formula: see text]Special Issue Comment: This review focuses on functional dynamics of individual enzymes and is related to the review on ion channels by Lu,44 the reviews on mathematical treatment of Flomenbom45 and Sach et al.,46 and review on FRET by Ruedas-Rama et al.41

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39

Davydova, Nadezhda Y., Bikash Dangi, Marc A. Maldonado, Nikita E. Vavilov, Victor G. Zgoda, and Dmitri R. Davydov. "Toward a systems approach to cytochrome P450 ensemble: interactions of CYP2E1 with other P450 species and their impact on CYP1A2." Biochemical Journal 476, no. 23 (December 12, 2019): 3661–85. http://dx.doi.org/10.1042/bcj20190532.

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In this study, we investigate the ability of ethanol-inducible CYP2E1 to interact with other cytochrome P450 species and affect the metabolism of their substrates. As a model system, we used CYP2E1-enriched human liver microsomes (HLM) obtained by the incorporation of purified CYP2E1. Using a technique based on homo-FRET in oligomers of CYP2E1 labeled with BODIPY 577/618 maleimide we demonstrated that the interactions of CYP2E1 with HLM result in the formation of its mixed oligomers with other P450 species present in the microsomal membrane. Incorporation of CYP2E1 results in a multifold increase in the rate of metabolism of CYP2E1-specific substrates p-Nitrophenol and Chlorzaxozone. The rate of their oxidation remains proportional to the amount of incorporated CYP2E1 up to the content of 0.3–0.4 nmol/mg protein (or ∼50% CYP2E1 in the P450 pool). The incorporated CYP2E1 becomes a fully functional member of the P450 ensemble and do not exhibit any detectable functional differences with the endogenous CYP2E1. Enrichment of HLM with CYP2E1 results in pronounced changes in the metabolism of 7-ethoxy-4-cyanocoumarin (CEC), the substrate of CYP2C19 and CYP1A2 suggesting an increase in the involvement of the latter in its metabolism. This effect goes together with an augmentation of the rate of dealkylation of CYP1A2-specific substrate 7-ethoxyresorufin. Furthermore, probing the interactions of CYP2E1 with model microsomes containing individual P450 enzymes we found that CYP2E1 efficiently interacts with CYP1A2, but lacks any ability to form complexes with CYP2C19. This finding goes inline with CYP2E1-induced redirection of the main route of CEC metabolism from CYP2C19 to CYP1A2.

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40

Brigham, Benjamin S., Jonathan P. Kitzrow, Joshua-Paolo C. Reyes, Karin Musier-Forsyth, and James B. Munro. "Intrinsic conformational dynamics of the HIV-1 genomic RNA 5′UTR." Proceedings of the National Academy of Sciences 116, no. 21 (May 8, 2019): 10372–81. http://dx.doi.org/10.1073/pnas.1902271116.

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The highly conserved 5′ untranslated region (5′UTR) of the HIV-1 RNA genome is central to the regulation of virus replication. NMR and biochemical experiments support a model in which the 5′UTR can transition between at least two conformational states. In one state the genome remains a monomer, as the palindromic dimerization initiation site (DIS) is sequestered via base pairing to upstream sequences. In the second state, the DIS is exposed, and the genome is competent for kissing loop dimerization and packaging into assembling virions where an extended dimer is formed. According to this model the conformation of the 5′UTR determines the fate of the genome. In this work, the dynamics of this proposed conformational switch and the factors that regulate it were probed using multiple single-molecule and in-gel ensemble FRET assays. Our results show that the HIV-1 5′UTR intrinsically samples conformations that are stabilized by both viral and host factor binding. Annealing of tRNALys3, the primer for initiation of reverse transcription, can promote the kissing dimer but not the extended dimer. In contrast, HIV-1 nucleocapsid (NC) promotes formation of the extended dimer in both the absence and presence of tRNALys3. Our data are consistent with an ordered series of events that involves primer annealing, genome dimerization, and virion assembly.

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41

Sun, Xiao Wei, and Hong Bo Zhou. "An Empirical Evaluation of Boosting-BAN and Boosting-MultiTAN." Applied Mechanics and Materials 513-517 (February 2014): 506–9. http://dx.doi.org/10.4028/www.scientific.net/amm.513-517.506.

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An ensemble consists of a set of independently trained classifiers whose predictions are combined when classifying novel instances. Previous research has shown that an ensemble as a whole is often more accurate than any of the single classifiers in the ensemble. Boosting-BAN classifier is considered stronger than Boosting-MultiTAN on noise-free data. However, there are strong empirical indications that Boosting-MultiTAN is much more robust than Boosting-BAN in noisy settings. For this reason, in this paper we built an ensemble using a voting methodology of Boosting-BAN and Boosting-MultiTAN ensembles with 10 sub-classifiers in each one. We performed a comparison with Boosting-BAN and Boosting-MultiTAN ensembles with 25 sub-classifiers on standard benchmark datasets and the proposed technique was the most accurate.

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42

Pérez, Iván, Thomas Heitkamp, and Michael Börsch. "Mechanism of ADP-Inhibited ATP Hydrolysis in Single Proton-Pumping FoF1-ATP Synthase Trapped in Solution." International Journal of Molecular Sciences 24, no. 9 (May 8, 2023): 8442. http://dx.doi.org/10.3390/ijms24098442.

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FoF1-ATP synthases in mitochondria, in chloroplasts, and in most bacteria are proton-driven membrane enzymes that supply the cells with ATP made from ADP and phosphate. Different control mechanisms exist to monitor and prevent the enzymes’ reverse chemical reaction of fast wasteful ATP hydrolysis, including mechanical or redox-based blockade of catalysis and ADP inhibition. In general, product inhibition is expected to slow down the mean catalytic turnover. Biochemical assays are ensemble measurements and cannot discriminate between a mechanism affecting all enzymes equally or individually. For example, all enzymes could work more slowly at a decreasing substrate/product ratio, or an increasing number of individual enzymes could be completely blocked. Here, we examined the effect of increasing amounts of ADP on ATP hydrolysis of single Escherichia coli FoF1-ATP synthases in liposomes. We observed the individual catalytic turnover of the enzymes one after another by monitoring the internal subunit rotation using single-molecule Förster resonance energy transfer (smFRET). Observation times of single FRET-labeled FoF1-ATP synthases in solution were extended up to several seconds using a confocal anti-Brownian electrokinetic trap (ABEL trap). By counting active versus inhibited enzymes, we revealed that ADP inhibition did not decrease the catalytic turnover of all FoF1-ATP synthases equally. Instead, increasing ADP in the ADP/ATP mixture reduced the number of remaining active enzymes that operated at similar catalytic rates for varying substrate/product ratios.

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43

Bédard, Joël, Mark Buehner, Jean-François Caron, Seung-Jong Baek, and Luc Fillion. "Practical Ensemble-Based Approaches to Estimate Atmospheric Background Error Covariances for Limited-Area Deterministic Data Assimilation." Monthly Weather Review 146, no. 11 (October 26, 2018): 3717–33. http://dx.doi.org/10.1175/mwr-d-18-0145.1.

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Abstract High-resolution flow-dependent background error covariances can allow for a better usage of dense observation networks in applications of data assimilation for numerical weather prediction. The generation of high-resolution ensembles, however, can be computationally cost prohibitive. In this study, practical and low-cost ensemble generation methods are presented and compared against both global and regional ensemble Kalman filters (G-EnKF and R-EnKF, respectively). The goal is to provide limited-area deterministic assimilation schemes with higher-resolution flow-dependent background error covariances that perform at least as well as those from the G-EnKF when assimilating the same observations. The low-cost methods are based on short-range regional ensemble forecasts initialized from 1) deterministic analysis plus balanced perturbations (filter free approach) and 2) a simplified ensemble square root filter (S-EnSRF), centered on deterministic analyses. The resulting ensembles from the different approaches are used within a 4D ensemble–variational (4D-EnVar) assimilation system covering most of Canada and the northern United States. Diagnostic results show that the mean is an important component of the ensembles. Results also show that the persistence of the homogeneous characteristics of the perturbations in the filter free approach makes this method unsuited for short assimilation time windows since some error structures take longer to develop. The S-EnSRF approach overcomes this limitation by recycling part of the prior perturbations. Results from 1-month assimilation experiments show that the S-EnSRF and R-EnKF experiments provide forecasts of similar quality to those from G-EnKF. Furthermore, results from precipitation verification indicate that the R-EnKF experiment provides the best precipitation accumulation predictions over 24-h periods.

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44

Yin, Lei, and Defu Hou. "s-Wave holographic superconductor in different ensembles and its thermodynamic consistency." International Journal of Modern Physics D 26, no. 04 (February 17, 2017): 1750027. http://dx.doi.org/10.1142/s0218271817500274.

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In this paper, we analytically study the consistency between the Ginzburg–Landau theory of the holographic superconductor in different ensembles and the fundamental thermodynamic relation, we derive the equation of motion of the scalar field which depicts the superconducting phase in canonical ensemble (CE) and a consistent formula to connect the holographic order-parameter to the Ginzburg–Landau coefficients in different thermodynamic ensembles, and we also study the spatially nonuniform Helmholtz free energy.

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45

Morrison, Steven J., Mark Montemayor, and Eric S. Wiltshire. "The Effect of a Recorded Model on Band Students' Performance Self-Evaluations, Achievement, and Attitude." Journal of Research in Music Education 52, no. 2 (July 2004): 116–29. http://dx.doi.org/10.2307/3345434.

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In this study, we examined effectiveness of recorded models in the context of ensemble rehearsals. Over a 5-week treatment period, directors of three middle/junior high and two high school bands systematically included professional recordings as part of their preparation of selected pieces. Students completed weekly self-evaluation reports assessing their individual progress and their ensembles' progress on model and no-model pieces. Using numerical and free-response formats, students evaluated self-achievement and ensemble achievement on notes/rhythms, articulation/dynamics, tuning, and balance. Expert evaluations revealed no difference in achievement between model and no-model pieces on pretreatment and posttreatment performance recordings. Student evaluations showed more modest achievement gains for model pieces. High school students demonstrated more positive self-evaluations for their own versus their ensembles' performance and greater overall differentiation in their evaluations across time. Middle school/junior high students were significantly more positive toward the model pieces. All students provided a greater number of free-response comments for model pieces.

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46

Kurowski, Marcin J., Kay Suselj, Wojciech W. Grabowski, and Joao Teixeira. "Shallow-to-Deep Transition of Continental Moist Convection: Cold Pools, Surface Fluxes, and Mesoscale Organization." Journal of the Atmospheric Sciences 75, no. 12 (November 8, 2018): 4071–90. http://dx.doi.org/10.1175/jas-d-18-0031.1.

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Abstract Large-eddy simulation is used to investigate the effects of cold pools driven by rain evaporation on the shallow-to-deep convection transition over land. The physically consistent methodologies are developed to obtain a time-dependent reference ensemble without cold pools and to apply interactive surface heat fluxes without modeling of surface energy and water budgets. Three different simulation ensembles are contrasted. The reference ensemble, in the spirit of one-dimensional single-column models, eliminates cold pools by horizontally homogenizing negative buoyancy production due to rain evaporation. The additional ensembles complement the reference cold-pool-free ensemble by including cold pools and by applying either interactive or prescribed surface fluxes. Contrasting these ensembles suggests possible improvements of convection parameterization in large-scale models of weather and climate. Without cold pools, the reference ensemble preserves key features of buoyancy-driven cellular convection associated with a field of convective plumes, as assumed in a typical convection parameterization. With cold pools, a significant enhancement of surface heat and moisture fluxes and about an hour delay of their daily maximum is simulated. Cold pools enhance near-surface temperature and moisture standard deviations as well as maxima of the near-surface updraft velocity. They also lead to the reduction of cloud lateral entrainment, deeper vertical development of the cloud layer, and a few-times-larger accumulated surface precipitation. Interactive surface fluxes provide a damping mechanism that noticeably suppresses all these effects. Perhaps surprisingly, cold pools do not significantly change the cloud-base convective mass flux that approximately follows the evolution of surface heat fluxes.

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47

Lin, Shawn H., Dacheng Zhao, Vivian Deng, Veronica K. Birdsall, Suzanne Ho, Olga Buzovetsky, Candice M. Etson, and Ishita Mukerji. "Integration Host Factor Binds DNA Holliday Junctions." International Journal of Molecular Sciences 24, no. 1 (December 29, 2022): 580. http://dx.doi.org/10.3390/ijms24010580.

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Integration host factor (IHF) is a nucleoid-associated protein involved in DNA packaging, integration of viral DNA and recombination. IHF binds with nanomolar affinity to duplex DNA containing a 13 bp consensus sequence, inducing a bend of ~160° upon binding. We determined that IHF binds to DNA Four-way or Holliday junctions (HJ) with high affinity regardless of the presence of the consensus sequence, signifying a structure-based mechanism of recognition. Junctions, important intermediates in DNA repair and homologous recombination, are dynamic and can adopt either an open or stacked conformation, where the open conformation facilitates branch migration and strand exchange. Using ensemble and single molecule Förster resonance energy transfer (FRET) methods, we investigated IHF-induced changes in the population distribution of junction conformations and determined that IHF binding shifts the population to the open conformation. Further analysis of smFRET dynamics revealed that even in the presence of protein, the junctions remain dynamic as fast transitions are observed for the protein-bound open state. Protein binding alters junction conformational dynamics, as cross correlation analyses reveal the protein slows the transition rate at 1 mM Mg2+ but accelerates the transition rate at 10 mM Mg2+. Stopped flow kinetic experiments provide evidence for two binding steps, a rapid, initial binding step followed by a slower step potentially associated with a conformational change. These measurements also confirm that the protein remains bound to the junction during the conformer transitions and further suggest that the protein forms a partially dissociated state that allows junction arms to be dynamic. These findings, which demonstrate that IHF binds HJs with high affinity and stabilizes junctions in the open conformation, suggest that IHF may play multiple roles in the processes of integration and recombination in addition to stabilizing bacterial biofilms.

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48

ROUSSEL, HAROLD. "SOLUTION OF MATRIX MODELS IN THE D III GENERATOR ENSEMBLE." International Journal of Modern Physics A 09, no. 19 (July 30, 1994): 3339–51. http://dx.doi.org/10.1142/s0217751x9400131x.

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In this work we solve two new matrix models using standard and new techniques. The two models are based on matrix ensembles not previously considered, namely the D III generator ensemble. It is shown that, in the double scaling limit, their free energy has the same behavior as previous models describing oriented and unoriented surfaces. We also found an additional solution for one of the models.

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49

Candille, Guillem, Stéphane Beauregard, and Normand Gagnon. "Bias Correction and Multiensemble in the NAEFS Context or How to Get a “Free Calibration” through a Multiensemble Approach." Monthly Weather Review 138, no. 11 (November 1, 2010): 4268–81. http://dx.doi.org/10.1175/2010mwr3349.1.

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Abstract Previous studies have shown that the raw combination (i.e., the combination of the direct output model without any postprocessing procedure) of the National Centers for Environmental Prediction (NCEP) and Meteorological Service of Canada (MSC) ensemble prediction systems (EPS) improves the probabilistic forecast both in terms of reliability and resolution. This combination palliates the lack of reliability of the NCEP EPS because of the too small dispersion of the predicted ensemble and the lack of probabilistic resolution of the MSC EPS. Such a multiensemble, called the North American Ensemble Forecast System (NAEFS), especially shows bias reductions and dispersion improvements that could only come from the combination of different forecast errors. It is then legitimate to wonder whether these improvements in terms of biases and dispersions, and by extension the skill improvements, are only due to the balancing between opposite model errors. In the NAEFS framework, bias corrections “on the fly,” where the bias is updated over time, are applied to the operational EPSs. Each model of the EPS components (NCEP/MSC) is individually bias corrected against its own analysis with the same process. The bias correction improves the reliability of each EPS component. It also slightly improves the accuracy of the predicted ensembles and thus the probabilistic resolution of the forecasts. Once the EPSs are combined, the improvements due to the bias correction are not so obvious, tending to show that the success of the multiensemble method does not only come from the cancellation of different biases. This study also shows that the combination of the raw EPS components (NAEFS) is generally better than either the bias corrected NCEP or MSC ensembles.

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50

Caldararu, Octav, Vilhelm Ekberg, Derek T. Logan, Esko Oksanen, and Ulf Ryde. "Exploring ligand dynamics in protein crystal structures with ensemble refinement." Acta Crystallographica Section D Structural Biology 77, no. 8 (July 29, 2021): 1099–115. http://dx.doi.org/10.1107/s2059798321006513.

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Understanding the dynamics of ligands bound to proteins is an important task in medicinal chemistry and drug design. However, the dominant technique for determining protein–ligand structures, X-ray crystallography, does not fully account for dynamics and cannot accurately describe the movements of ligands in protein binding sites. In this article, an alternative method, ensemble refinement, is used on six protein–ligand complexes with the aim of understanding the conformational diversity of ligands in protein crystal structures. The results show that ensemble refinement sometimes indicates that the flexibility of parts of the ligand and some protein side chains is larger than that which can be described by a single conformation and atomic displacement parameters. However, since the electron-density maps are comparable and R free values are slightly increased, the original crystal structure is still a better model from a statistical point of view. On the other hand, it is shown that molecular-dynamics simulations and automatic generation of alternative conformations in crystallographic refinement confirm that the flexibility of these groups is larger than is observed in standard refinement. Moreover, the flexible groups in ensemble refinement coincide with groups that give high atomic displacement parameters or non-unity occupancy if optimized in standard refinement. Therefore, the conformational diversity indicated by ensemble refinement seems to be qualitatively correct, indicating that ensemble refinement can be an important complement to standard crystallographic refinement as a tool to discover which parts of crystal structures may show extensive flexibility and therefore are poorly described by a single conformation. However, the diversity of the ensembles is often exaggerated (probably partly owing to the rather poor force field employed) and the ensembles should not be trusted in detail.

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