Journal articles: 'Single Molecule Fluorescence Resonance Energy Transfer (smFRET)'

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Yang, Ziyu, Haiqi Xu, Jiayu Wang, Wei Chen, and Meiping Zhao. "Single-Molecule Fluorescence Techniques for Membrane Protein Dynamics Analysis." Applied Spectroscopy 75, no. 5 (April 20, 2021): 491–505. http://dx.doi.org/10.1177/00037028211009973.

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Fluorescence-based single-molecule techniques, mainly including fluorescence correlation spectroscopy (FCS) and single-molecule fluorescence resonance energy transfer (smFRET), are able to analyze the conformational dynamics and diversity of biological macromolecules. They have been applied to analysis of the dynamics of membrane proteins, such as membrane receptors and membrane transport proteins, due to their superior ability in resolving spatio-temporal heterogeneity and the demand of trace amounts of analytes. In this review, we first introduced the basic principle involved in FCS and smFRET. Then we summarized the labeling and immobilization strategies of membrane protein molecules, the confocal-based and TIRF-based instrumental configuration, and the data processing methods. The applications to membrane protein dynamics analysis are described in detail with the focus on how to select suitable fluorophores, labeling sites, experimental setup, and analysis methods. In the last part, the remaining challenges to be addressed and further development in this field are also briefly discussed.

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Sengupta, Bhaswati, and Mai Huynh. "Contribution of smFRET to Chromatin Research." Biophysica 3, no. 1 (February 8, 2023): 93–108. http://dx.doi.org/10.3390/biophysica3010007.

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Chromatins are structural components of chromosomes and consist of DNA and histone proteins. The structure, dynamics, and function of chromatins are important in regulating genetic processes. Several different experimental and theoretical tools have been employed to understand chromatins better. In this review, we will focus on the literatures engrossed in understanding of chromatins using single-molecule Förster resonance energy transfer (smFRET). smFRET is a single-molecule fluorescence microscopic technique that can furnish information regarding the distance between two points in space. This has been utilized to efficiently unveil the structural details of chromatins.

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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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Li, Maodong, Tanlin Sun, Fan Jin, Daqi Yu, and Zhirong Liu. "Dimension conversion and scaling of disordered protein chains." Molecular BioSystems 12, no. 9 (2016): 2932–40. http://dx.doi.org/10.1039/c6mb00415f.

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To extract protein dimension and energetics information from single-molecule fluorescence resonance energy transfer spectroscopy (smFRET) data, it is essential to establish the relationship between the distributions of the radius of gyration (R_g) and the end-to-end (donor-to-acceptor) distance (R_ee).

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Yukhnovets, Olessya, Henning Höfig, Nuno Bustorff, Alexandros Katranidis, and Jörg Fitter. "Impact of Molecule Concentration, Diffusion Rates and Surface Passivation on Single-Molecule Fluorescence Studies in Solution." Biomolecules 12, no. 3 (March 18, 2022): 468. http://dx.doi.org/10.3390/biom12030468.

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For single-molecule studies in solution, very small concentrations of dye-labelled molecules are employed in order to achieve single-molecule sensitivity. In typical studies with confocal microscopes, often concentrations in the pico-molar regime are required. For various applications that make use of single-molecule Förster resonance energy transfer (smFRET) or two-color coincidence detection (TCCD), the molecule concentration must be set explicitly to targeted values and furthermore needs to be stable over a period of several hours. As a consequence, specific demands must be imposed on the surface passivation of the cover slides during the measurements. The aim of having only one molecule in the detection volume at the time is not only affected by the absolute molecule concentration, but also by the rate of diffusion. Therefore, we discuss approaches to control and to measure absolute molecule concentrations. Furthermore, we introduce an approach to calculate the probability of chance coincidence events and demonstrate that measurements with challenging smFRET samples require a strict limit of maximal sample concentrations in order to produce meaningful results.

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Hu, Jinyong, Meiyan Wu, Li Jiang, Zhensheng Zhong, Zhangkai Zhou, Thitima Rujiralai, and Jie Ma. "Combining gold nanoparticle antennas with single-molecule fluorescence resonance energy transfer (smFRET) to study DNA hairpin dynamics." Nanoscale 10, no. 14 (2018): 6611–19. http://dx.doi.org/10.1039/c7nr08397a.

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Girodat, Dylan, Avik K. Pati, Daniel S. Terry, Scott C. Blanchard, and Karissa Y. Sanbonmatsu. "Quantitative comparison between sub-millisecond time resolution single-molecule FRET measurements and 10-second molecular simulations of a biosensor protein." PLOS Computational Biology 16, no. 11 (November 5, 2020): e1008293. http://dx.doi.org/10.1371/journal.pcbi.1008293.

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Molecular Dynamics (MD) simulations seek to provide atomic-level insights into conformationally dynamic biological systems at experimentally relevant time resolutions, such as those afforded by single-molecule fluorescence measurements. However, limitations in the time scales of MD simulations and the time resolution of single-molecule measurements have challenged efforts to obtain overlapping temporal regimes required for close quantitative comparisons. Achieving such overlap has the potential to provide novel theories, hypotheses, and interpretations that can inform idealized experimental designs that maximize the detection of the desired reaction coordinate. Here, we report MD simulations at time scales overlapping with in vitro single-molecule Förster (fluorescence) resonance energy transfer (smFRET) measurements of the amino acid binding protein LIV-BPSS at sub-millisecond resolution. Computationally efficient all-atom structure-based simulations, calibrated against explicit solvent simulations, were employed for sampling multiple cycles of LIV-BPSS clamshell-like conformational changes on the time scale of seconds, examining the relationship between these events and those observed by smFRET. The MD simulations agree with the smFRET measurements and provide valuable information on local dynamics of fluorophores at their sites of attachment on LIV-BPSS and the correlations between fluorophore motions and large-scale conformational changes between LIV-BPSS domains. We further utilize the MD simulations to inform the interpretation of smFRET data, including Förster radius (R0) and fluorophore orientation factor (κ2) determinations. The approach we describe can be readily extended to distinct biochemical systems, allowing for the interpretation of any FRET system conjugated to protein or ribonucleoprotein complexes, including those with more conformational processes, as well as those implementing multi-color smFRET.

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Yang, Jie, Sarah Perrett, and Si Wu. "Single Molecule Characterization of Amyloid Oligomers." Molecules 26, no. 4 (February 11, 2021): 948. http://dx.doi.org/10.3390/molecules26040948.

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The misfolding and aggregation of polypeptide chains into β-sheet-rich amyloid fibrils is associated with a wide range of neurodegenerative diseases. Growing evidence indicates that the oligomeric intermediates populated in the early stages of amyloid formation rather than the mature fibrils are responsible for the cytotoxicity and pathology and are potentially therapeutic targets. However, due to the low-populated, transient, and heterogeneous nature of amyloid oligomers, they are hard to characterize by conventional bulk methods. The development of single molecule approaches provides a powerful toolkit for investigating these oligomeric intermediates as well as the complex process of amyloid aggregation at molecular resolution. In this review, we present an overview of recent progress in characterizing the oligomerization of amyloid proteins by single molecule fluorescence techniques, including single-molecule Förster resonance energy transfer (smFRET), fluorescence correlation spectroscopy (FCS), single-molecule photobleaching and super-resolution optical imaging. We discuss how these techniques have been applied to investigate the different aspects of amyloid oligomers and facilitate understanding of the mechanism of amyloid aggregation.

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Verma, Awadhesh Kumar, Ashab Noumani, Amit K. Yadav, and Pratima R. Solanki. "FRET Based Biosensor: Principle Applications Recent Advances and Challenges." Diagnostics 13, no. 8 (April 8, 2023): 1375. http://dx.doi.org/10.3390/diagnostics13081375.

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Förster resonance energy transfer (FRET)-based biosensors are being fabricated for specific detection of biomolecules or changes in the microenvironment. FRET is a non-radiative transfer of energy from an excited donor fluorophore molecule to a nearby acceptor fluorophore molecule. In a FRET-based biosensor, the donor and acceptor molecules are typically fluorescent proteins or fluorescent nanomaterials such as quantum dots (QDs) or small molecules that are engineered to be in close proximity to each other. When the biomolecule of interest is present, it can cause a change in the distance between the donor and acceptor, leading to a change in the efficiency of FRET and a corresponding change in the fluorescence intensity of the acceptor. This change in fluorescence can be used to detect and quantify the biomolecule of interest. FRET-based biosensors have a wide range of applications, including in the fields of biochemistry, cell biology, and drug discovery. This review article provides a substantial approach on the FRET-based biosensor, principle, applications such as point-of-need diagnosis, wearable, single molecular FRET (smFRET), hard water, ions, pH, tissue-based sensors, immunosensors, and aptasensor. Recent advances such as artificial intelligence (AI) and Internet of Things (IoT) are used for this type of sensor and challenges.

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Durham, Ryan J., Nabina Paudyal, Elisa Carrillo, Nidhi Kaur Bhatia, David M. Maclean, Vladimir Berka, Drew M. Dolino, Alemayehu A. Gorfe, and Vasanthi Jayaraman. "Conformational spread and dynamics in allostery of NMDA receptors." Proceedings of the National Academy of Sciences 117, no. 7 (February 3, 2020): 3839–47. http://dx.doi.org/10.1073/pnas.1910950117.

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Allostery can be manifested as a combination of repression and activation in multidomain proteins allowing for fine tuning of regulatory mechanisms. Here we have used single molecule fluorescence resonance energy transfer (smFRET) and molecular dynamics simulations to study the mechanism of allostery underlying negative cooperativity between the two agonists glutamate and glycine in the NMDA receptor. These data show that binding of one agonist leads to conformational flexibility and an increase in conformational spread at the second agonist site. Mutational and cross-linking studies show that the dimer–dimer interface at the agonist-binding domain mediates the allostery underlying the negative cooperativity. smFRET on the transmembrane segments shows that they are tightly coupled in the unliganded and single agonist-bound form and only upon binding both agonists the transmembrane domain explores looser packing which would facilitate activation.

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Barth, Anders, Oleg Opanasyuk, Thomas-Otavio Peulen, Suren Felekyan, Stanislav Kalinin, Hugo Sanabria, and Claus A. M. Seidel. "Unraveling multi-state molecular dynamics in single-molecule FRET experiments. I. Theory of FRET-lines." Journal of Chemical Physics 156, no. 14 (April 14, 2022): 141501. http://dx.doi.org/10.1063/5.0089134.

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Conformational dynamics of biomolecules are of fundamental importance for their function. Single-molecule studies of Förster Resonance Energy Transfer (smFRET) between a tethered donor and acceptor dye pair are a powerful tool to investigate the structure and dynamics of labeled molecules. However, capturing and quantifying conformational dynamics in intensity-based smFRET experiments remains challenging when the dynamics occur on the sub-millisecond timescale. The method of multiparameter fluorescence detection addresses this challenge by simultaneously registering fluorescence intensities and lifetimes of the donor and acceptor. Together, two FRET observables, the donor fluorescence lifetime τD and the intensity-based FRET efficiency E, inform on the width of the FRET efficiency distribution as a characteristic fingerprint for conformational dynamics. We present a general framework for analyzing dynamics that relates average fluorescence lifetimes and intensities in two-dimensional burst frequency histograms. We present parametric relations of these observables for interpreting the location of FRET populations in E–τ D diagrams, called FRET-lines. To facilitate the analysis of complex exchange equilibria, FRET-lines serve as reference curves for a graphical interpretation of experimental data to (i) identify conformational states, (ii) resolve their dynamic connectivity, (iii) compare different kinetic models, and (iv) infer polymer properties of unfolded or intrinsically disordered proteins. For a simplified graphical analysis of complex kinetic networks, we derive a moment-based representation of the experimental data that decouples the motion of the fluorescence labels from the conformational dynamics of the biomolecule. Importantly, FRET-lines facilitate exploring complex dynamic models via easily computed experimental observables. We provide extensive computational tools to facilitate applying FRET-lines.

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Klostermeier, Dagmar. "Single-molecule FRET reveals nucleotide-driven conformational changes in molecular machines and their link to RNA unwinding and DNA supercoiling." Biochemical Society Transactions 39, no. 2 (March 22, 2011): 611–16. http://dx.doi.org/10.1042/bst0390611.

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Many complex cellular processes in the cell are catalysed at the expense of ATP hydrolysis. The enzymes involved bind and hydrolyse ATP and couple ATP hydrolysis to the catalysed process via cycles of nucleotide-driven conformational changes. In this review, I illustrate how smFRET (single-molecule fluorescence resonance energy transfer) can define the underlying conformational changes that drive ATP-dependent molecular machines. The first example is a DEAD-box helicase that alternates between two different conformations in its catalytic cycle during RNA unwinding, and the second is DNA gyrase, a topoisomerase that undergoes a set of concerted conformational changes during negative supercoiling of DNA.

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Song, Chun-Xiao, Jiajie Diao, Axel T. Brunger, and Stephen R. Quake. "Simultaneous single-molecule epigenetic imaging of DNA methylation and hydroxymethylation." Proceedings of the National Academy of Sciences 113, no. 16 (March 28, 2016): 4338–43. http://dx.doi.org/10.1073/pnas.1600223113.

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The modifications 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are the two major DNA epigenetic modifications in mammalian genomes and play crucial roles in development and pathogenesis. Little is known about the colocalization or potential correlation of these two modifications. Here we present an ultrasensitive single-molecule imaging technology capable of detecting and quantifying 5hmC and 5mC from trace amounts of DNA. We used this approach to perform single-molecule fluorescence resonance energy transfer (smFRET) experiments which measure the proximity between 5mC and 5hmC in the same DNA molecule. Our results reveal high levels of adjacent and opposing methylated and hydroxymethylated CpG sites (5hmC/5mCpGs) in mouse genomic DNA across multiple tissues. This identifies the previously undetectable and unappreciated 5hmC/5mCpGs as one of the major states for 5hmC in the mammalian genome and suggest that they could function in promoting gene expression.

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Greenfeld, Max, Sergey V. Solomatin, and Daniel Herschlag. "Removal of Covalent Heterogeneity Reveals Simple Folding Behavior for P4-P6 RNA." Journal of Biological Chemistry 286, no. 22 (April 8, 2011): 19872–79. http://dx.doi.org/10.1074/jbc.m111.235465.

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RNA folding landscapes have been described alternately as simple and as complex. The limited diversity of RNA residues and the ability of RNA to form stable secondary structures prior to adoption of a tertiary structure would appear to simplify folding relative to proteins. Nevertheless, there is considerable evidence for long-lived misfolded RNA states, and these observations have suggested rugged energy landscapes. Recently, single molecule fluorescence resonance energy transfer (smFRET) studies have exposed heterogeneity in many RNAs, consistent with deeply furrowed rugged landscapes. We turned to an RNA of intermediate complexity, the P4-P6 domain from the Tetrahymena group I intron, to address basic questions in RNA folding. P4-P6 exhibited long-lived heterogeneity in smFRET experiments, but the inability to observe exchange in the behavior of individual molecules led us to probe whether there was a non-conformational origin to this heterogeneity. We determined that routine protocols in RNA preparation and purification, including UV shadowing and heat annealing, cause covalent modifications that alter folding behavior. By taking measures to avoid these treatments and by purifying away damaged P4-P6 molecules, we obtained a population of P4-P6 that gave near-uniform behavior in single molecule studies. Thus, the folding landscape of P4-P6 lacks multiple deep furrows that would trap different P4-P6 molecules in different conformations and contrasts with the molecular heterogeneity that has been seen in many smFRET studies of structured RNAs. The simplicity of P4-P6 allowed us to reliably determine the thermodynamic and kinetic effects of metal ions on folding and to now begin to build more detailed models for RNA folding behavior.

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Kaur, Anisa, Roaa Mahmoud, Anoja Megalathan, Sydney Pettit, and Soma Dhakal. "Multiplexed smFRET Nucleic Acid Sensing Using DNA Nanotweezers." Biosensors 13, no. 1 (January 10, 2023): 119. http://dx.doi.org/10.3390/bios13010119.

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The multiplexed detection of disease biomarkers is part of an ongoing effort toward improving the quality of diagnostic testing, reducing the cost of analysis, and accelerating the treatment processes. Although significant efforts have been made to develop more sensitive and rapid multiplexed screening methods, such as microarrays and electrochemical sensors, their limitations include their intricate sensing designs and semi-quantitative detection capabilities. Alternatively, fluorescence resonance energy transfer (FRET)-based single-molecule counting offers great potential for both the sensitive and quantitative detection of various biomarkers. However, current FRET-based multiplexed sensing typically requires the use of multiple excitation sources and/or FRET pairs, which complicates labeling schemes and the post-analysis of data. We present a nanotweezer (NT)-based sensing strategy that employs a single FRET pair and is capable of detecting multiple targets. Using DNA mimics of miRNA biomarkers specific to triple-negative breast cancer (TNBC), we demonstrated that the developed sensors are sensitive down to the low picomolar range (≤10 pM) and can discriminate between targets with a single-base mismatch. These simple hybridization-based sensors hold great promise for the sensitive detection of a wider spectrum of nucleic acid biomarkers.

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Zhang, Yiming, Zongzhou Ji, Xin Wang, Yi Cao, and Hai Pan. "Single–Molecule Study of DNAzyme Reveals Its Intrinsic Conformational Dynamics." International Journal of Molecular Sciences 24, no. 2 (January 7, 2023): 1212. http://dx.doi.org/10.3390/ijms24021212.

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DNAzyme is a class of DNA molecules that can perform catalytic functions with high selectivity towards specific metal ions. Due to its potential applications for biosensors and medical therapeutics, DNAzyme has been extensively studied to characterize the relationships between its biochemical properties and functions. Similar to protein enzymes and ribozymes, DNAzymes have been found to undergo conformational changes in a metal–ion–dependent manner for catalysis. Despite the important role the conformation plays in the catalysis process, such structural and dynamic information might not be revealed by conventional approaches. Here, by using the single–molecule fluorescence resonance energy transfer (smFRET) technique, we were able to investigate the detailed conformational dynamics of a uranyl–specific DNAzyme 39E. We observed conformation switches of 39E to a folded state with the addition of Mg2+ and to an extended state with the addition of UO22+. Furthermore, 39E can switch to a more compact configuration with or without divalent metal ions. Our findings reveal that 39E can undergo conformational changes spontaneously between different configurations.

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Sapkota, Kaur, Megalathan, Donkoh-Moore, and Dhakal. "Single-Step FRET-Based Detection of Femtomoles DNA." Sensors 19, no. 16 (August 9, 2019): 3495. http://dx.doi.org/10.3390/s19163495.

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Sensitive detection of nucleic acids and identification of single nucleotide polymorphism (SNP) is crucial in diagnosis of genetic diseases. Many strategies have been developed for detection and analysis of DNA, including fluorescence, electrical, optical, and mechanical methods. Recent advances in fluorescence resonance energy transfer (FRET)-based sensing have provided a new avenue for sensitive and quantitative detection of various types of biomolecules in simple, rapid, and recyclable platforms. Here, we report single-step FRET-based DNA sensors designed to work via a toehold-mediated strand displacement (TMSD) process, leading to a distinct change in the FRET efficiency upon target binding. Using single-molecule FRET (smFRET), we show that these sensors can be regenerated in situ, and they allow detection of femtomoles DNA without the need for target amplification while still using a dramatically small sample size (fewer than three orders of magnitude compared to the typical sample size of bulk fluorescence). In addition, these single-molecule sensors exhibit a dynamic range of approximately two orders of magnitude. Using one of the sensors, we demonstrate that the single-base mismatch sequence can be discriminated from a fully matched DNA target, showing a high specificity of the method. These sensors with simple and recyclable design, sensitive detection of DNA, and the ability to discriminate single-base mismatch sequences may find applications in quantitative analysis of nucleic acid biomarkers.

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Du, Jinxi, Ricky Dartawan, William Rice, Forrest Gao, Joseph H. Zhou, and Jia Sheng. "Fluorescent Platforms for RNA Chemical Biology Research." Genes 13, no. 8 (July 27, 2022): 1348. http://dx.doi.org/10.3390/genes13081348.

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Efficient detection and observation of dynamic RNA changes remain a tremendous challenge. However, the continuous development of fluorescence applications in recent years enhances the efficacy of RNA imaging. Here we summarize some of these developments from different aspects. For example, single-molecule fluorescence in situ hybridization (smFISH) can detect low abundance RNA at the subcellular level. A relatively new aptamer, Mango, is widely applied to label and track RNA activities in living cells. Molecular beacons (MBs) are valid for quantifying both endogenous and exogenous mRNA and microRNA (miRNA). Covalent binding enzyme labeling fluorescent group with RNA of interest (ROI) partially overcomes the RNA length limitation associated with oligonucleotide synthesis. Forced intercalation (FIT) probes are resistant to nuclease degradation upon binding to target RNA and are used to visualize mRNA and messenger ribonucleoprotein (mRNP) activities. We also summarize the importance of some fluorescence spectroscopic techniques in exploring the function and movement of RNA. Single-molecule fluorescence resonance energy transfer (smFRET) has been employed to investigate the dynamic changes of biomolecules by covalently linking biotin to RNA, and a focus on dye selection increases FRET efficiency. Furthermore, the applications of fluorescence assays in drug discovery and drug delivery have been discussed. Fluorescence imaging can also combine with RNA nanotechnology to target tumors. The invention of novel antibacterial drugs targeting non-coding RNAs (ncRNAs) is also possible with steady-state fluorescence-monitored ligand-binding assay and the T-box riboswitch fluorescence anisotropy assay. More recently, COVID-19 tests using fluorescent clustered regularly interspaced short palindromic repeat (CRISPR) technology have been demonstrated to be efficient and clinically useful. In summary, fluorescence assays have significant applications in both fundamental and clinical research and will facilitate the process of RNA-targeted new drug discovery, therefore deserving further development and updating.

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LeVine, Michael V., Daniel S. Terry, George Khelashvili, Zarek S. Siegel, Matthias Quick, Jonathan A. Javitch, Scott C. Blanchard, and Harel Weinstein. "The allosteric mechanism of substrate-specific transport in SLC6 is mediated by a volumetric sensor." Proceedings of the National Academy of Sciences 116, no. 32 (July 19, 2019): 15947–56. http://dx.doi.org/10.1073/pnas.1903020116.

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Neurotransmitter:sodium symporters (NSSs) in the SLC6 family terminate neurotransmission by coupling the thermodynamically favorable transport of ions to the thermodynamically unfavorable transport of neurotransmitter back into presynaptic neurons. Results from many structural, functional, and computational studies on LeuT, a bacterial NSS homolog, have provided critical insight into the mechanism of sodium-coupled transport, but the mechanism underlying substrate-specific transport rates is still not understood. We present a combination of molecular dynamics simulations, single-molecule fluorescence resonance energy transfer (smFRET) imaging, and measurements of Na+ binding and substrate transport that reveals an allosteric substrate specificity mechanism. In this mechanism, residues F259 and I359 in the substrate binding pocket couple the binding of substrate to Na+ release from the Na2 site by allosterically modulating the stability of a partially open, inward-facing state. We propose a model for transport selectivity in which residues F259 and I359 act as a volumetric sensor that inhibits the transport of bulky amino acids.

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Edwards, Devin T., Marc-Andre LeBlanc, and Thomas T. Perkins. "Modulation of a protein-folding landscape revealed by AFM-based force spectroscopy notwithstanding instrumental limitations." Proceedings of the National Academy of Sciences 118, no. 12 (March 15, 2021): e2015728118. http://dx.doi.org/10.1073/pnas.2015728118.

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Single-molecule force spectroscopy is a powerful tool for studying protein folding. Over the last decade, a key question has emerged: how are changes in intrinsic biomolecular dynamics altered by attachment to μm-scale force probes via flexible linkers? Here, we studied the folding/unfolding of α3D using atomic force microscopy (AFM)–based force spectroscopy. α3D offers an unusual opportunity as a prior single-molecule fluorescence resonance energy transfer (smFRET) study showed α3D’s configurational diffusion constant within the context of Kramers theory varies with pH. The resulting pH dependence provides a test for AFM-based force spectroscopy’s ability to track intrinsic changes in protein folding dynamics. Experimentally, however, α3D is challenging. It unfolds at low force (<15 pN) and exhibits fast-folding kinetics. We therefore used focused ion beam–modified cantilevers that combine exceptional force precision, stability, and temporal resolution to detect state occupancies as brief as 1 ms. Notably, equilibrium and nonequilibrium force spectroscopy data recapitulated the pH dependence measured using smFRET, despite differences in destabilization mechanism. We reconstructed a one-dimensional free-energy landscape from dynamic data via an inverse Weierstrass transform. At both neutral and low pH, the resulting constant-force landscapes showed minimal differences (∼0.2 to 0.5 kBT) in transition state height. These landscapes were essentially equal to the predicted entropic barrier and symmetric. In contrast, force-dependent rates showed that the distance to the unfolding transition state increased as pH decreased and thereby contributed to the accelerated kinetics at low pH. More broadly, this precise characterization of a fast-folding, mechanically labile protein enables future AFM-based studies of subtle transitions in mechanoresponsive proteins.

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Wu, Si, Liu Hong, Yuqing Wang, Jieqiong Yu, Jie Yang, Jie Yang, Hong Zhang, and Sarah Perrett. "Kinetics of the conformational cycle of Hsp70 reveals the importance of the dynamic and heterogeneous nature of Hsp70 for its function." Proceedings of the National Academy of Sciences 117, no. 14 (March 20, 2020): 7814–23. http://dx.doi.org/10.1073/pnas.1914376117.

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Hsp70 is a conserved molecular chaperone that plays an indispensable role in regulating protein folding, translocation, and degradation. The conformational dynamics of Hsp70 and its regulation by cochaperones are vital to its function. Using bulk and single-molecule fluorescence resonance energy transfer (smFRET) techniques, we studied the interdomain conformational distribution of human stress-inducible Hsp70A1 and the kinetics of conformational changes induced by nucleotide and the Hsp40 cochaperone Hdj1. We found that the conformations between and within the nucleotide- and substrate-binding domains show heterogeneity. The conformational distribution in the ATP-bound state can be induced by Hdj1 to form an “ADP-like” undocked conformation, which is an ATPase-stimulated state. Kinetic measurements indicate that Hdj1 binds to monomeric Hsp70 as the first step, then induces undocking of the two domains and closing of the substrate-binding cleft. Dimeric Hdj1 then facilitates dimerization of Hsp70 and formation of a heterotetrameric Hsp70–Hsp40 complex. Our results provide a kinetic view of the conformational cycle of Hsp70 and reveal the importance of the dynamic nature of Hsp70 for its function.

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Sapkota, Kumar, and Soma Dhakal. "FRET-Based Aptasensor for the Selective and Sensitive Detection of Lysozyme." Sensors 20, no. 3 (February 9, 2020): 914. http://dx.doi.org/10.3390/s20030914.

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Lysozyme is a conserved antimicrobial enzyme and has been cited for its role in immune modulation. Increase in lysozyme concentration in body fluids is also regarded as an early warning of some diseases such as Alzheimer’s, sarcoidosis, Crohn’s disease, and breast cancer. Therefore, a method for a sensitive and selective detection of lysozyme can benefit many different areas of research. In this regard, several aptamers that are specific to lysozyme have been developed, but there is still a lack of a detection method that is sensitive, specific, and quantitative. In this work, we demonstrated a single-molecule fluorescence resonance energy transfer (smFRET)-based detection of lysozyme using an aptamer sensor (also called aptasensor) in which the binding of lysozyme triggers its conformational switch from a low-FRET to high-FRET state. Using this strategy, we demonstrated that the aptasensor is sensitive down to 2.3 picomoles (30 nM) of lysozyme with a dynamic range extending to ~2 µM and has little to no interference from similar biomolecules. The smFRET approach used here requires a dramatically small amount of aptasensor (~3000-fold less as compared to typical bulk fluorescence methods), and it is cost effective compared to enzymatic and antibody-based approaches. Additionally, the aptasensor can be readily regenerated in situ via a process called toehold mediated strand displacement (TMSD). The FRET-based aptasensing of lysozyme that we developed here could be implemented to detect other protein biomarkers by incorporating protein-specific aptamers without the need for changing fluorophore-labeled DNA strands.

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Gonzalez, Cuauhtemoc U., Elisa Carrillo, Vladimir Berka, and Vasanthi Jayaraman. "Structural Arrangement Produced by Concanavalin A Binding to Homomeric GluK2 Receptors." Membranes 11, no. 8 (August 11, 2021): 613. http://dx.doi.org/10.3390/membranes11080613.

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Kainate receptors are members of the ionotropic glutamate receptor family. They form cation-specific transmembrane channels upon binding glutamate that desensitize in the continued presence of agonists. Concanavalin A (Con-A), a lectin, stabilizes the active open-channel state of the kainate receptor and reduces the extent of desensitization. In this study, we used single-molecule fluorescence resonance energy transfer (smFRET) to investigate the conformational changes underlying kainate receptor modulation by Con-A. These studies showed that Con-A binding to GluK2 homomeric kainate receptors resulted in closer proximity of the subunits at the dimer–dimer interface at the amino-terminal domain as well as between the subunits at the dimer interface at the agonist-binding domain. Additionally, the modulation of receptor functions by monovalent ions, which bind to the dimer interface at the agonist-binding domain, was not observed in the presence of Con-A. Based on these results, we conclude that Con-A modulation of kainate receptor function is mediated by a shift in the conformation of the kainate receptor toward a tightly packed extracellular domain.

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Basak, Sujit, Nabanita Saikia, David Kwun, Ucheor B. Choi, Feng Ding, and Mark E. Bowen. "Different Forms of Disorder in NMDA-Sensitive Glutamate Receptor Cytoplasmic Domains Are Associated with Differences in Condensate Formation." Biomolecules 13, no. 1 (December 20, 2022): 4. http://dx.doi.org/10.3390/biom13010004.

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The N-methyl-D-aspartate (NMDA)-sensitive glutamate receptor (NMDAR) helps assemble downstream signaling pathways through protein interactions within the postsynaptic density (PSD), which are mediated by its intracellular C-terminal domain (CTD). The most abundant NMDAR subunits in the brain are GluN2A and GluN2B, which are associated with a developmental switch in NMDAR composition. Previously, we used single molecule fluorescence resonance energy transfer (smFRET) to show that the GluN2B CTD contained an intrinsically disordered region with slow, hop-like conformational dynamics. The CTD from GluN2B also undergoes liquid–liquid phase separation (LLPS) with synaptic proteins. Here, we extend these observations to the GluN2A CTD. Sequence analysis showed that both subunits contain a form of intrinsic disorder classified as weak polyampholytes. However, only GluN2B contained matched patterning of arginine and aromatic residues, which are linked to LLPS. To examine the conformational distribution, we used discrete molecular dynamics (DMD), which revealed that GluN2A favors extended disordered states containing secondary structures while GluN2B favors disordered globular states. In contrast to GluN2B, smFRET measurements found that GluN2A lacked slow conformational dynamics. Thus, simulation and experiments found differences in the form of disorder. To understand how this affects protein interactions, we compared the ability of these two NMDAR isoforms to undergo LLPS. We found that GluN2B readily formed condensates with PSD-95 and SynGAP, while GluN2A failed to support LLPS and instead showed a propensity for colloidal aggregation. That GluN2A fails to support this same condensate formation suggests a developmental switch in LLPS propensity.

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Fuertes, Gustavo, Niccolò Banterle, Kiersten M. Ruff, Aritra Chowdhury, Davide Mercadante, Christine Koehler, Michael Kachala, et al. "Decoupling of size and shape fluctuations in heteropolymeric sequences reconciles discrepancies in SAXS vs. FRET measurements." Proceedings of the National Academy of Sciences 114, no. 31 (July 17, 2017): E6342—E6351. http://dx.doi.org/10.1073/pnas.1704692114.

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

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Pei, Kai, Jie Zhang, Tingting Zou, and Zhu Liu. "AimR Adopts Preexisting Dimer Conformations for Specific Target Recognition in Lysis-Lysogeny Decisions of Bacillus Phage phi3T." Biomolecules 11, no. 9 (September 7, 2021): 1321. http://dx.doi.org/10.3390/biom11091321.

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A bacteriophage switches between lytic and lysogenic life cycles. The AimR-AimP-AimX communication system is responsible for phage lysis-lysogeny decisions during the infection of Bacillus subtilis. AimX is a regulator biasing phage lysis, AimR is a transcription factor activating AimX expression, and AimP is an arbitrium peptide that determines phage lysogeny by deactivating AimR. A strain-specific mechanism for the lysis-lysogeny decisions is proposed in SPbeta and phi3T phages. That is, the arbitrium peptide of the SPbeta phage stabilizes the SPbeta AimR (spAimR) dimer, whereas the phi3T-derived peptide disassembles the phi3T AimR (phAimR) dimer into a monomer. Here, we find that phAimR does not undergo dimer-to-monomer conversion upon arbitrium peptide binding. Gel-filtration, static light scattering (SLS) and analytical ultracentrifugation (AUC) results show that phAimR is dimeric regardless of the presence of arbitrium peptide. Small-angle X-ray scattering (SAXS) reveals that the arbitrium peptide binding makes an extended dimeric conformation. Single-molecule fluorescence resonance energy transfer (smFRET) analysis reveals that the phAimR dimer fluctuates among two distinct conformational states, and each preexisting state is selectively recognized by the arbitrium peptide or the target DNA, respectively. Collectively, our biophysical characterization of the phAimR dynamics underlying specific target recognition provides new mechanistic insights into understanding lysis-lysogeny decisions in Bacillus phage phi3T.

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Planer, William, Zhiwei Chen, Mathivanan Chinnaraj, Xiaobing Zuo, Vittorio Pengo, Paolo Macor, Francesco Tedesco, and Nicola Pozzi. "X-Ray Crystallographic and Single-Molecule Fluorescence Studies of Beta-2 Glycoprotein I Reveal an Alternative Mechanism of Autoantibody Recognition." Blood 134, Supplement_1 (November 13, 2019): 91. http://dx.doi.org/10.1182/blood-2019-122064.

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Background. Antiphospholipid antibodies (aPL) recognizing an epitope comprising residues R39-R43 in the N-terminal domain, Domain I (DI), of beta-2 glycoprotein I (b2GPI) are considered among the most pathogenic in patients with Antiphospholipid Syndrome (APS). How such autoantibodies engage b2GPI at the molecular level remains incompletely understood. Aim. To better understand how pathogenic anti-DI antibodies engage b2GPI at the molecular level. Results. Under physiological conditions, b2GPI is believed to adopt a closed conformation featuring an intramolecular interaction between DI and DV with amino acids R39 and R43 in DI being masked by DV. This conformation is therefore predicted to be immunologically inert, incapable of reacting against pathogenic anti-DI antibodies. Once bound to the membranes, however, b2GPI is believed to undergo a dramatic conformational change which liberates DI to the solvent. To get a better grasp of the molecular flexibility of b2GPI under conditions relevant to physiology, we expressed and purified fully-glycosylated human recombinant b2GPI (hr-b2GPI) from HEK293 cells at high yield and purity suitable for structural biology and biophysical studies. After native purification, we found that the recombinant protein bound to heparin and negatively charged phospholipids with affinities comparable to those obtained for b2GPI that was purified from plasma using the perchloric acid method (p-b2GPI); hr-b2GPI also displayed similar reactivity against anti-b2GPI immunoglobulin G antibodies that were isolated from 5 APS patients. Surprisingly, hr-b2GPI and p-b2GPI were structurally similar, too. The X-ray crystal structures of hr-b2GPI and p-b2GPI solved at 2.6 and 2.4 Å resolution were superimposable documenting a J-shaped elongated conformation of the molecule in which DI was located > 90 Å away from the C-terminal DV. Both structures were characterized by 22 oxidized cysteine residues forming 11 disulfide bonds, 4 N-glycosylations, and an intact yet flexible phospholipid-binding loop in DV. Since crystallization occurred at high salt concentrations, validation of the crystal structure of hr-b2GPI in solution was obtained by single-molecule Förster Resonance Energy Transfer (smFRET) and small-angle X-ray scattering (SAXS), while surface plasmon resonance (SPR) was used to probe the binding of a recently developed monoclonal anti-DI antibody, i.e., MBBS, to hr-b2GPI and p-b2GPI in solution. In keeping with the X-ray structural data, donor and acceptor fluorophores incorporated at positions 13/312 in DI and DV and 112/312 in DII and DV reported no measurable energy transfer whereas probes located at positions 13/112 in DI and DII displayed very high energy transfer. Likewise, the scattering profiles of the recombinant and plasma purified proteins returned similar hydrodynamic radii characteristic of elongated, flexible protein structures, and not circular. Notably, both hr-b2GPI and p-b2GPI in the elongated conformation were capable of interacting with MBBS without the need of phospholipids, even though addition of negatively charged phospholipids decreased the apparent dissociation affinity constant due to a reduction of the dissociation rate constant and a remarkable time-dependent accumulation of b2GPI onto the lipid surface, suggestive of a phospholipid-induced oligomerization mechanism. Conclusions. This study demonstrates that human b2GPI can adopt an elongated conformation in solution that is primed for phospholipid, heparin, and autoantibodies binding with DI constitutively exposed to the solvent. The fact that phospholipid-bound b2GPI is a better antigen for anti-DI autoantibody under physiological conditions as compared to the elongated form in solution can be explained by the relatively low affinity and bivalency of such autoantibodies that likely recognize a peptide motif pattern rather than a specific sequence of residues. Whether other context-dependent conformational changes occur after binding of the protein to the lipid surface, thus facilitating aPL binding, remain to be established. Since our studies failed to detect the closed form of b2GPI previously documented by electron and atomic force microscopy studies, it is possible that this conformation may arise from chemical and/or posttranslational modifications that occur in vivo while the protein circulates in the plasma. Disclosures No relevant conflicts of interest to declare.

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Qiao, Yi, Yuhan Luo, Naiyun Long, Yi Xing, and Jing Tu. "Single-Molecular Förster Resonance Energy Transfer Measurement on Structures and Interactions of Biomolecules." Micromachines 12, no. 5 (April 27, 2021): 492. http://dx.doi.org/10.3390/mi12050492.

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Single-molecule Förster resonance energy transfer (smFRET) inherits the strategy of measurement from the effective “spectroscopic ruler” FRET and can be utilized to observe molecular behaviors with relatively high throughput at nanometer scale. The simplicity in principle and configuration of smFRET make it easy to apply and couple with other technologies to comprehensively understand single-molecule dynamics in various application scenarios. Despite its widespread application, smFRET is continuously developing and novel studies based on the advanced platforms have been done. Here, we summarize some representative examples of smFRET research of recent years to exhibit the versatility and note typical strategies to further improve the performance of smFRET measurement on different biomolecules.

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Huynh, Mai, and Bhaswati Sengupta. "Analysis of Enzyme Conformation Dynamics Using Single-Molecule Förster Resonance Energy Transfer (smFRET)." Biophysica 2, no. 2 (June 6, 2022): 123–34. http://dx.doi.org/10.3390/biophysica2020014.

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Single-molecule Förster resonance energy transfer (smFRET) enables the deconvolution of various conformational substates of biomolecules. Over the past two decades, it has been widely used to understand the conformational dynamics of enzymes. Commonly, enzymes undergo reversible transitions between active and inactive states in solution. Using smFRET, the details of these transitions and the effect of ligands on these dynamics have been determined. In this mini-review, we discuss the various works focused on the investigation of enzyme conformational dynamics using smFRET.

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Huynh, Mai, and Bhaswati Sengupta. "Analysis of Enzyme Conformation Dynamics Using Single-Molecule Förster Resonance Energy Transfer (smFRET)." Biophysica 2, no. 2 (June 6, 2022): 123–34. http://dx.doi.org/10.3390/biophysica2020014.

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Single-molecule Förster resonance energy transfer (smFRET) enables the deconvolution of various conformational substates of biomolecules. Over the past two decades, it has been widely used to understand the conformational dynamics of enzymes. Commonly, enzymes undergo reversible transitions between active and inactive states in solution. Using smFRET, the details of these transitions and the effect of ligands on these dynamics have been determined. In this mini-review, we discuss the various works focused on the investigation of enzyme conformational dynamics using smFRET.

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31

Ha, Taekjip. "Single-Molecule Fluorescence Resonance Energy Transfer." Methods 25, no. 1 (September 2001): 78–86. http://dx.doi.org/10.1006/meth.2001.1217.

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Metskas, Lauren Ann, and Elizabeth Rhoades. "Single-Molecule FRET of Intrinsically Disordered Proteins." Annual Review of Physical Chemistry 71, no. 1 (April 20, 2020): 391–414. http://dx.doi.org/10.1146/annurev-physchem-012420-104917.

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Intrinsically disordered proteins (IDPs) are now widely recognized as playing critical roles in a broad range of cellular functions as well as being implicated in diverse diseases. Their lack of stable secondary structure and tertiary interactions, coupled with their sensitivity to measurement conditions, stymies many traditional structural biology approaches. Single-molecule Förster resonance energy transfer (smFRET) is now widely used to characterize the physicochemical properties of these proteins in isolation and is being increasingly applied to more complex assemblies and experimental environments. This review provides an overview of confocal diffusion-based smFRET as an experimental tool, including descriptions of instrumentation, data analysis, and protein labeling. Recent papers are discussed that illustrate the unique capability of smFRET to provide insight into aggregation-prone IDPs, protein–protein interactions involving IDPs, and IDPs in complex experimental milieus.

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Meiser, Nathalie, Christin Fuks, and Martin Hengesbach. "Cooperative Analysis of Structural Dynamics in RNA-Protein Complexes by Single-Molecule Förster Resonance Energy Transfer Spectroscopy." Molecules 25, no. 9 (April 28, 2020): 2057. http://dx.doi.org/10.3390/molecules25092057.

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RNA-protein complexes (RNPs) are essential components in a variety of cellular processes, and oftentimes exhibit complex structures and show mechanisms that are highly dynamic in conformation and structure. However, biochemical and structural biology approaches are mostly not able to fully elucidate the structurally and especially conformationally dynamic and heterogeneous nature of these RNPs, to which end single molecule Förster resonance energy transfer (smFRET) spectroscopy can be harnessed to fill this gap. Here we summarize the advantages of strategic smFRET studies to investigate RNP dynamics, complemented by structural and biochemical data. Focusing on recent smFRET studies of three essential biological systems, we demonstrate that investigation of RNPs on a single molecule level can answer important functional questions that remained elusive with structural or biochemical approaches alone: The complex structural rearrangements throughout the splicing cycle, unwinding dynamics of the G-quadruplex (G4) helicase RHAU, and aspects in telomere maintenance regulation and synthesis.

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Beckers, M., F. Drechsler, T. Eilert, J. Nagy, and J. Michaelis. "Quantitative structural information from single-molecule FRET." Faraday Discussions 184 (2015): 117–29. http://dx.doi.org/10.1039/c5fd00110b.

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Single-molecule studies can be used to study biological processes directly and in real-time. In particular, the fluorescence energy transfer between reporter dye molecules attached to specific sites on macromolecular complexes can be used to infer distance information. When several measurements are combined, the information can be used to determine the position and conformation of certain domains with respect to the complex. However, data analysis schemes that include all experimental uncertainties are highly complex, and the outcome depends on assumptions about the state of the dye molecules. Here, we present a new analysis algorithm using Bayesian parameter estimation based on Markov Chain Monte Carlo sampling and parallel tempering termed Fast-NPS that can analyse large smFRET networks in a relatively short time and yields the position of the dye molecules together with their respective uncertainties. Moreover, we show what effects different assumptions about the dye molecules have on the outcome. We discuss the possibilities and pitfalls in structure determination based on smFRET using experimental data for an archaeal transcription pre-initiation complex, whose architecture has recently been unravelled by smFRET measurements.

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Clamme, Jean-Pierre, and Ashok A. Deniz. "Three-Color Single-Molecule Fluorescence Resonance Energy Transfer." ChemPhysChem 6, no. 1 (January 14, 2005): 74–77. http://dx.doi.org/10.1002/cphc.200400261.

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Hohng, Sungchul, and Taekjip Ha. "Single-Molecule Quantum-Dot Fluorescence Resonance Energy Transfer." ChemPhysChem 6, no. 5 (May 13, 2005): 956–60. http://dx.doi.org/10.1002/cphc.200400557.

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Ariunbold, G. O., G. S. Agarwal, Z. Wang, M. O. Scully, and H. Walther. "Nanosecond Dynamics of Single-Molecule Fluorescence Resonance Energy Transfer." Journal of Physical Chemistry A 108, no. 13 (April 2004): 2402–4. http://dx.doi.org/10.1021/jp037609h.

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Sasmal, Dibyendu K., Laura E. Pulido, Shan Kasal, and Jun Huang. "Single-molecule fluorescence resonance energy transfer in molecular biology." Nanoscale 8, no. 48 (2016): 19928–44. http://dx.doi.org/10.1039/c6nr06794h.

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Orte, Angel, Richard W. Clarke, and David Klenerman. "Fluorescence Coincidence Spectroscopy for Single-Molecule Fluorescence Resonance Energy-Transfer Measurements." Analytical Chemistry 80, no. 22 (November 15, 2008): 8389–97. http://dx.doi.org/10.1021/ac8009092.

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Li, Chen-chen, Ying Li, Yan Zhang, and Chun-yang Zhang. "Single-molecule fluorescence resonance energy transfer and its biomedical applications." TrAC Trends in Analytical Chemistry 122 (January 2020): 115753. http://dx.doi.org/10.1016/j.trac.2019.115753.

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Zhao, Rui, and David Rueda. "RNA folding dynamics by single-molecule fluorescence resonance energy transfer." Methods 49, no. 2 (October 2009): 112–17. http://dx.doi.org/10.1016/j.ymeth.2009.04.017.

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Lu, Maolin. "Single-Molecule FRET Imaging of Virus Spike–Host Interactions." Viruses 13, no. 2 (February 21, 2021): 332. http://dx.doi.org/10.3390/v13020332.

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As a major surface glycoprotein of enveloped viruses, the virus spike protein is a primary target for vaccines and anti-viral treatments. Current vaccines aiming at controlling the COVID-19 pandemic are mostly directed against the SARS-CoV-2 spike protein. To promote virus entry and facilitate immune evasion, spikes must be dynamic. Interactions with host receptors and coreceptors trigger a cascade of conformational changes/structural rearrangements in spikes, which bring virus and host membranes in proximity for membrane fusion required for virus entry. Spike-mediated viral membrane fusion is a dynamic, multi-step process, and understanding the structure–function-dynamics paradigm of virus spikes is essential to elucidate viral membrane fusion, with the ultimate goal of interventions. However, our understanding of this process primarily relies on individual structural snapshots of endpoints. How these endpoints are connected in a time-resolved manner, and the order and frequency of conformational events underlying virus entry, remain largely elusive. Single-molecule Förster resonance energy transfer (smFRET) has provided a powerful platform to connect structure–function in motion, revealing dynamic aspects of spikes for several viruses: SARS-CoV-2, HIV-1, influenza, and Ebola. This review focuses on how smFRET imaging has advanced our understanding of virus spikes’ dynamic nature, receptor-binding events, and mechanism of antibody neutralization, thereby informing therapeutic interventions.

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Bao, Shuying, Guangcun Shan, and Xinghai Zhao. "RNA Dynamics Probed by Single-Molecule Fluorescence Resonance Energy Transfer Studies." Journal of Computational and Theoretical Nanoscience 8, no. 4 (April 1, 2011): 664–69. http://dx.doi.org/10.1166/jctn.2011.1737.

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Kim, Sung-Hyun, Don-Seong Choi, and Do-Seok Kim. "Single-molecule Detection of Fluorescence Resonance Energy Transfer Using Confocal Microscopy." Journal of the Optical Society of Korea 12, no. 2 (June 25, 2008): 107–11. http://dx.doi.org/10.3807/josk.2008.12.2.107.

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Schröder, Gunnar F., and Helmut Grubmüller. "Maximum likelihood trajectories from single molecule fluorescence resonance energy transfer experiments." Journal of Chemical Physics 119, no. 18 (November 8, 2003): 9920–24. http://dx.doi.org/10.1063/1.1616511.

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Wang, Dong, and Eitan Geva. "Protein Structure and Dynamics from Single-Molecule Fluorescence Resonance Energy Transfer." Journal of Physical Chemistry B 109, no. 4 (February 2005): 1626–34. http://dx.doi.org/10.1021/jp0478864.

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Sekatskii, S. K., G. Dietler, and V. S. Letokhov. "Single molecule fluorescence resonance energy transfer scanning near-field optical microscopy." Chemical Physics Letters 452, no. 1-3 (February 2008): 220–24. http://dx.doi.org/10.1016/j.cplett.2007.12.064.

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Li, H., L. Ying, X. Ren, S. Balasubramanian, and D. Klenerman. "Fluorescence studies of single biomolecules." Biochemical Society Transactions 32, no. 5 (October 26, 2004): 753–56. http://dx.doi.org/10.1042/bst0320753.

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Single-molecule fluorescence has the capability to detect properties buried in ensemble measurements and, hence, provides new insights about biological processes. Ratiometric methods are normally used to reduce the effects of excitation beam inhomogeneity. Fluorescence resonance energy transfer is widely used but there are problems in inserting the fluorophores in the correct position on the biomolecule, particularly if the structure is not known. We have recently developed two-colour coincidence single-molecule fluorescence that addresses this problem. This method can be used to determine quantitatively the multimerization states of biomolecules, in solution without separation. The future prospects of single-molecule fluorescence as applied to biological molecules are discussed.

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Rahmanseresht, Sheema, Peker Milas, Kieran P. Ramos, Ben D. Gamari, and Lori S. Goldner. "Single-molecule-sensitive fluorescence resonance energy transfer in freely-diffusing attoliter droplets." Applied Physics Letters 106, no. 19 (May 11, 2015): 194107. http://dx.doi.org/10.1063/1.4921202.

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Keller, Aaron M., Matthew S. DeVore, Dominik G. Stich, Dung M. Vu, Timothy Causgrove, and James H. Werner. "Multicolor Three-Dimensional Tracking for Single-Molecule Fluorescence Resonance Energy Transfer Measurements." Analytical Chemistry 90, no. 10 (April 19, 2018): 6109–15. http://dx.doi.org/10.1021/acs.analchem.8b00244.

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Journal articles on the topic 'Single Molecule Fluorescence Resonance Energy Transfer (smFRET)'

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