Article

Computational Tool Photon-HDF5: An Open File Format for Timestamp-Based Single-Molecule Fluorescence Experiments

January 2016
Biophysical Journal 110(1):26-33

January 2016
110(1):26-33

DOI:10.1016/j.bpj.2015.11.013

Authors:

Antonino Ingargiola

University of California, Los Angeles

Ted A Laurence

Lawrence Livermore National Laboratory

Robert Boutelle

University of California, Los Angeles

Shimon Weiss

University of California, Los Angeles

Show all 5 authorsHide

We introduce Photon-HDF5, an open and efficient file format to simplify exchange and long-term accessibility of data from single-molecule fluorescence experiments based on photon-counting detectors such as single-photon avalanche diode, photomultiplier tube, or arrays of such detectors. The format is based on HDF5, a widely used platform- and language-independent hierarchical file format for which user-friendly viewers are available. Photon-HDF5 can store raw photon data (timestamp, channel number, etc.) from any acquisition hardware, but also setup and sample description, information on provenance, authorship and other metadata, and is flexible enough to include any kind of custom data. The format specifications are hosted on a public website, which is open to contributions by the biophysics community. As an initial resource, the website provides code examples to read Photon-HDF5 files in several programming languages and a reference Python library (phconvert), to create new Photon-HDF5 files and convert several existing file formats into Photon-HDF5. To encourage adoption by the academic and commercial communities, all software is released under the MIT open source license.

FRET-based dynamic structural biology: Challenges, perspectives and an appeal for open-science practices

Article

Full-text available

Mar 2021
eLife

Single-molecule FRET (smFRET) has become a mainstream technique for studying biomolecular structural dynamics. The rapid and wide adoption of smFRET experiments by an ever-increasing number of groups has generated significant progress in sample preparation, measurement procedures, data analysis, algorithms and documentation. Several labs that employ smFRET approaches have joined forces to inform the smFRET community about streamlining how to perform experiments and analyze results for obtaining quantitative information on biomolecular structure and dynamics. The recent efforts include blind tests to assess the accuracy and the precision of smFRET experiments among different labs using various procedures. These multi-lab studies have led to the development of smFRET procedures and documentation, which are important when submitting entries into the archiving system for integrative structure models, PDB-Dev. This position paper describes the current ‘state of the art’ from different perspectives, points to unresolved methodological issues for quantitative structural studies, provides a set of ‘soft recommendations’ about which an emerging consensus exists, and lists openly available resources for newcomers and seasoned practitioners. To make further progress, we strongly encourage ‘open science’ practices.

S1 File

Data

Full-text available

Apr 2017

Supporting Information for the manuscript “Multispot single-molecule FRET: towards high-throughput analysis of freely diffusing molecules”. (PDF)

Systematic Assessment of Burst Impurity in Confocal-Based Single-Molecule Fluorescence Detection Using Brownian Motion Simulations

Article

Full-text available

Jul 2019
MOLECULES

Single-molecule fluorescence detection (SMFD) experiments are useful in distinguishing sub-populations of molecular species when measuring heterogeneous samples. One experimental platform for SMFD is based on a confocal microscope, where molecules randomly traverse an effective detection volume. The non-uniformity of the excitation profile and the random nature of Brownian motion, produce fluctuating fluorescence signals. For these signals to be distinguished from the background, burst analysis is frequently used. Yet, the relation between the results of burst analyses and the underlying information of the diffusing molecules is still obscure and requires systematic assessment. In this work we performed three-dimensional Brownian motion simulations of SMFD, and tested the positions at which molecules emitted photons that passed the burst analysis criteria for different values of burst analysis parameters. The results of this work verify which of the burst analysis parameters and experimental conditions influence both the position of molecules in space when fluorescence is detected and taken into account, and whether these bursts of photons arise purely from single molecules, or not entirely. Finally, we show, as an example, the effect of bursts that are not purely from a single molecule on the accuracy in single-molecule Förster resonance energy transfer measurements.

48-spot single-molecule FRET setup with periodic acceptor excitation

Article

Full-text available

Jun 2017

Single-molecule FRET (smFRET) allows measuring distances between donor and acceptor fluorophores on the 3-10 nm range. Solution-based smFRET allows measurement of binding-unbinding events or conformational changes of dye-labeled biomolecules without ensemble averaging and free from surface perturbations. When employing dual (or multi) laser exci-tation, smFRET allows resolving the number of fluorescent labels on each molecule, greatly enhancing the ability to study heterogeneous samples. A major drawback to solution-based smFRET is the low throughput, which renders repetitive measurements expensive and hin-ders the ability to study kinetic phenomena in real-time. Here we demonstrate a high-throughput smFRET system which multiplexes acquisition by using 48 excitation spots and two 48-pixel SPAD array detectors. The system employs two excitation lasers allowing separation of species with one or two active fluorophores. The performance of the system is demonstrated on a set of doubly-labeled double-stranded DNA oligonucleotides with different distances between donor and acceptor dyes along the DNA duplex. We show that the acquisition time for accurate subpopulation identification is reduced from several minutes to seconds, opening the way to high-throughput screening applications and real-time kinetics studies of enzymatic reactions such as DNA transcription by bacterial RNA polymerase.

The substrate-binding domains of the osmoregulatory ABC importer OpuA transiently interact

Article

Full-text available

May 2024
eLife

Bacteria utilize various strategies to prevent internal dehydration during hypertonic stress. A common approach to countering the effects of the stress is to import compatible solutes such as glycine betaine, leading to simultaneous passive water fluxes following the osmotic gradient. OpuA from Lactococcus lactis is a type I ABC-importer that uses two substrate-binding domains (SBDs) to capture extracellular glycine betaine and deliver the substrate to the transmembrane domains for subsequent transport. OpuA senses osmotic stress via changes in the internal ionic strength and is furthermore regulated by the 2nd messenger cyclic-di-AMP. We now show, by means of solution-based single-molecule FRET and analysis with multi-parameter photon-by-photon hidden Markov modeling, that the SBDs transiently interact in an ionic strength-dependent manner. The smFRET data are in accordance with the apparent cooperativity in transport and supported by new cryo-EM data of OpuA. We propose that the physical interactions between SBDs and cooperativity in substrate delivery are part of the transport mechanism.

Diameter Dependence of Transport through Nuclear Pore Complex Mimics Studied Using Optical Nanopores

Preprint

Full-text available

Dec 2023

The nuclear pore complex (NPC) regulates the selective transport of large biomolecules through the nuclear envelope. As a model system for nuclear transport, we construct NPC mimics by functionalizing the pore walls of freestanding palladium zero-mode waveguides with the FG-nucleoporin Nsp1. This approach enables the measurement of single-molecule translocations through individual pores using optical detection. We probe the selectivity of Nsp1-coated pores by quantitatively comparing the translocation rates of the nuclear transport receptor Kap95 to the inert probe BSA over a wide range of pore sizes from 35 nm to 160 nm. Pores below 55 ± 5 nm show significant selectivity that gradually decreases for larger pores. This finding is corroborated by coarse-grained molecular-dynamics simulations of the Nsp1 mesh within the pore, which suggest that leakage of BSA occurs by diffusion through transient openings within the dynamic mesh. Furthermore, we experimentally observe a modulation of the BSA permeation when varying the concentration of Kap95. The results demonstrate the potential of single-molecule fluorescence measurements on biomimetic NPCs to elucidate the principles of nuclear transport.

The substrate-binding domains of the osmoregulatory ABC importer OpuA physically interact

Preprint

Full-text available

Jul 2023

Bacteria utilize various strategies to prevent internal dehydration during hypertonic stress. A common approach to countering the effects of the stress is to import compatible solutes such as glycine betaine, leading to simultaneous passive water fluxes following the osmotic gradient. OpuA from Lactococcus lactis is a type I ABC-importer that uses two substrate-binding domains (SBDs) to capture extracellular glycine betaine and deliver the substrate to the transmembrane domains for subsequent transport. OpuA senses osmotic stress via changes in the internal ionic strength and is furthermore regulated by the 2nd messenger cyclic-di-AMP. We now show, by means of solution-based single-molecule FRET and analysis with multi-parameter photon-by-photon hidden Markov modeling, that the SBDs interact in an ionic strength-dependent manner. The smFRET data are in accordance with the apparent cooperativity in transport and supported by new cryo-EM data of OpuA. We propose that physical interactions between SBDs and cooperativity in substrate delivery could be more widespread than recognized thus far.

Structural and dynamic insights into α-synuclein dimer conformations

Article

Feb 2023
STRUCTURE

Parkinson disease is associated with the aggregation of the protein α-synuclein. While α-synuclein can exist in multiple oligomeric states, the dimer has been a subject of extensive debates. Here, using an array of biophysical approaches, we demonstrate that α-synuclein in vitro exhibits primarily a monomer-dimer equilibrium in nanomolar concentrations and up to a few micromolars. We then use spatial information from hetero-isotopic cross-linking mass spectrometry experiments as restrains in discrete molecular dynamics simulations to obtain the ensemble structure of dimeric species. Out of eight structural sub-populations of dimers, we identify one that is compact, stable, abundant, and exhibits partially exposed β-sheet structures. This compact dimer is the only one where the hydroxyls of tyrosine 39 are in proximity that may promote dityrosine covalent linkage upon hydroxyl radicalization, which is implicated in α-synuclein amyloid fibrils. We propose that this α-synuclein dimer features etiological relevance to Parkinson disease.

Flexible Target Recognition of the Intrinsically Disordered DNA-Binding Domain of CytR Monitored by Single-Molecule Fluorescence Spectroscopy

Article

Full-text available

Aug 2022

The intrinsically disordered DNA-binding domain of cytidine repressor (CytR-DBD) folds in the presence of target DNA and regulates the expression of multiple genes in E. coli. To explore the conformational rearrangements in the unbound state and the target recognition mechanisms of CytR-DBD, we carried out single-molecule Förster resonance energy transfer (smFRET) measurements. The smFRET data of CytR-DBD in the absence of DNA show one major and one minor population assignable to an expanded unfolded state and a compact folded state, respectively. The population of the folded state increases and decreases upon titration with salt and denaturant, respectively, in an apparent two-state manner. The peak FRET efficiencies of both the unfolded and folded states change continuously with denaturant concentration, demonstrating the intrinsic flexibility of the DNA-binding domain and the deviation from a strict two-state transition. Remarkably, the CytR-DBD exhibits a compact structure when bound to both the specific and nonspecific DNA; however, the peak FRET efficiencies of the two structures are slightly but consistently different. The observed conformational heterogeneity highlights the potential structural changes required for CytR to bind variably spaced operator sequences.

A user-friendly tool to convert photon counting data to the open-source Photon-HDF5 file format

Preprint

Full-text available

Mar 2022

Photon-HDF5 is an open-source and open file format for storing photon-counting data from single molecule microscopy experiments, introduced to simplify data exchange and increase the reproducibility of data analysis. Part of the Photon-HDF5 ecosystem, is phconvert, an extensible python library that allows converting proprietary formats into Photon-HDF5 files. However, its use requires some proficiency with command line instructions, the python programming language, and the YAML markup format. This creates a significant barrier for potential users without that expertise, but who want to benefit from the advantages of releasing their files in an open format. In this work, we present a GUI that lowers this barrier, thus simplifying the use of Photon-HDF5. This tool uses the phconvert python library to convert data files originally saved in proprietary data formats to Photon-HDF5 files, without users having to write a single line of code. Because reproducible analyses depend on essential experimental information, such as laser power or sample description, the GUI also includes (currently limited) functionality to associate valid metadata with the converted file, without having to write any YAML. Finally, the GUI includes several productivity-enhancing features such as whole-directory batch conversion and the ability to re-run a failed batch, only converting the files that could not be converted in the previous run.

Structural and Dynamic Insights Into α-Synuclein Dimer Conformations

Preprint

Full-text available

Dec 2021

Parkinson's disease is associated with the aggregation of the protein α-synuclein. While α-synuclein can exist in multiple oligomeric states, the dimer has been a subject of extensive debates. Here, using an array of biophysical approaches, we demonstrate α-synuclein in vitro exhibits primarily a monomer-dimer equilibrium in nanomolar concentrations and up to a few micromolars. We then use spatial information from hetero-isotopic cross-linking mass spectrometry experiments as restrains in discrete molecular dynamics simulations to obtain the ensemble structure of dimeric species. Out of eight structural sub-populations of dimers we identify one that is compact, stable, abundant, and exhibits partially exposed β-sheet structures. This compact dimer is the only one where the hydroxyls of tyrosine 39 are in a proximity that can promote dityrosine covalent linkage implicated in amyloidogenesis. We propose that this α-synuclein dimer features etiological relevance to Parkinson's disease.

Utilizing Time-Resolved Protein-Induced Fluorescence Enhancement to Identify Stable Local Conformations One α-Synuclein Monomer at a Time

Article

May 2021
JoVE

Using spectroscopic rulers to track multiple conformations of single biomolecules and their dynamics have revolutionized the understanding of structural dynamics and its contributions to biology. While the FRET-based ruler reports on inter-dye distances in the 3-10 nm range, other spectroscopic techniques, such as protein-induced fluorescence enhancement (PIFE), report on the proximity between a dye and a protein surface in the shorter 0-3 nm range. Regardless of the method of choice, its use in measuring freely-diffusing biomolecules one at a time retrieves histograms of the experimental parameter yielding separate centrally-distributed sub-populations of biomolecules, where each sub-population represents either a single conformation that stayed unchanged within milliseconds, or multiple conformations that interconvert much faster than milliseconds, and hence an averaged-out sub-population. In single-molecule FRET, where the reported parameter in histograms is the inter-dye FRET efficiency, an intrinsically disordered protein, such as the α-Synuclein monomer in buffer, was previously reported as exhibiting a single averaged-out sub-population of multiple conformations interconverting rapidly. While these past findings depend on the 3-10 nm range of the FRET-based ruler, we sought to put this protein to the test using single-molecule PIFE, where we track the fluorescence lifetime of site-specific sCy3-labeled α-Synuclein proteins one at a time. Interestingly, using this shorter range spectroscopic proximity sensor, sCy3-labeled α-Synuclein exhibits several lifetime sub-populations with distinctly different mean lifetimes that interconvert in 10-100 ms. These results show that while α-Synuclein might be disordered globally, it nonetheless attains stable local structures. In summary, in this work we highlight the advantage of using different spectroscopic proximity sensors that track local or global structural changes one biomolecule at a time.

OME-NGFF: scalable format strategies for interoperable bioimaging data

Preprint

Full-text available

Apr 2021

Biological imaging is one of the most innovative fields in the modern biological sciences. New imaging modalities, probes, and analysis tools appear every few months and often prove decisive for enabling new directions in scientific discovery. One feature of this dynamic field is the need to capture new types of data and data structures. While there is a strong drive to make scientific data Findable, Accessible, Interoperable and Reproducible (FAIR, ¹ ), the rapid rate of innovation in imaging impedes the unification and adoption of standardized data formats. Despite this, the opportunities for sharing and integrating bioimaging data and, in particular, linking these data to other “omics” datasets have never been greater; therefore, to every extent possible, increasing “FAIRness” of bioimaging data is critical for maximizing scientific value, as well as for promoting openness and integrity. In the absence of a common, FAIR format, two approaches have emerged to provide access to bioimaging data: translation and conversion. On-the-fly translation produces a transient representation of bioimage metadata and binary data but must be repeated on each use. In contrast, conversion produces a permanent copy of the data, ideally in an open format that makes the data more accessible and improves performance and parallelization in reads and writes. Both approaches have been implemented successfully in the bioimaging community but both have limitations. At cloud-scale, those shortcomings limit scientific analysis and the sharing of results. We introduce here next-generation file formats (NGFF) as a solution to these challenges.

Palladium zero-mode waveguides for optical single molecule detection with nanopores

Article

Full-text available

Feb 2021
NANOTECHNOLOGY

Holes in metal films block any transmitting light if the wavelength is much larger than the hole diameter, establishing such nanopores as so-called Zero Mode Waveguides (ZMWs). Molecules on the other hand, can still passage through these holes. We use this to detect individual fluorophore-labelled molecules as they travel through a ZMW and thereby traverse from the dark region to the illuminated side, upon which they emit fluorescent light. This is beneficial both for background suppression and to prevent premature bleaching. We use palladium as a novel metal-film material for ZMWs, which is advantageous compared to conventionally used metals. We demonstrate that it is possible to simultaneously detect translocations of individual free fluorophores of different colors. Labeled DNA and protein biomolecules can be detected as well at the single-molecule level with a high signal-to-noise ratio and at high bandwidth, which opens the door to a variety of single-molecule biophysics studies.

The smfBox is an open-source platform for single-molecule FRET

Article

Full-text available

Nov 2020

Single-molecule Förster Resonance Energy Transfer (smFRET) is a powerful technique capable of resolving both relative and absolute distances within and between structurally dynamic biomolecules. High instrument costs, and a lack of open-source hardware and acquisition software have limited smFRET’s broad application by non-specialists. Here, we present the smfBox, a cost-effective confocal smFRET platform, providing detailed build instructions, open-source acquisition software, and full validation, thereby democratising smFRET for the wider scientific community.

Distortion of the bilayer and dynamics of the BAM complex in lipid nanodiscs

Article

Full-text available

Dec 2020

The β-barrel assembly machinery (BAM) catalyses the folding and insertion of β-barrel outer membrane proteins (OMPs) into the outer membranes of Gram-negative bacteria by mechanisms that remain unclear. Here, we present an ensemble of cryoEM structures of the E. coli BamABCDE (BAM) complex in lipid nanodiscs, determined using multi-body refinement techniques. These structures, supported by single-molecule FRET measurements, describe a range of motions in the BAM complex, mostly localised within the periplasmic region of the major subunit BamA. The β-barrel domain of BamA is in a ‘lateral open’ conformation in all of the determined structures, suggesting that this is the most energetically favourable species in this bilayer. Strikingly, the BAM-containing lipid nanodisc is deformed, especially around BAM’s lateral gate. This distortion is also captured in molecular dynamics simulations, and provides direct structural evidence for the lipid ‘disruptase’ activity of BAM, suggested to be an important part of its functional mechanism.

Inter-domain dynamics in the chaperone SurA and multi-site binding to its outer membrane protein clients

Article

Full-text available

May 2020

The periplasmic chaperone SurA plays a key role in outer membrane protein (OMP) biogenesis. E. coli SurA comprises a core domain and two peptidylprolyl isomerase domains (P1 and P2), but its mechanisms of client binding and chaperone function have remained unclear. Here, we use chemical cross-linking, hydrogen-deuterium exchange mass spectrometry, single-molecule FRET and molecular dynamics simulations to map the client binding site(s) on SurA and interrogate the role of conformational dynamics in OMP recognition. We demonstrate that SurA samples an array of conformations in solution in which P2 primarily lies closer to the core/P1 domains than suggested in the SurA crystal structure. OMP binding sites are located primarily in the core domain, and OMP binding results in conformational changes between the core/P1 domains. Together, the results suggest that unfolded OMP substrates bind in a cradle formed between the SurA domains, with structural flexibility between domains assisting OMP recognition, binding and release. The chaperone SurA is involved in outer membrane protein (OMP) biogenesis in Gram-negative bacteria, but its mechanism of action is not fully understood. Combining mass spectrometric, biophysical and computational approaches, the authors here show how the conformational dynamics of SurA facilitate OMP binding.

The smfBox: an open-source platform for single-molecule FRET

Preprint

Full-text available

Dec 2019

Single-molecule Förster Resonance Energy Transfer (smFRET) is a powerful technique capable of resolving both relative and absolute distances within and between structurally dynamic biomolecules. High instrument costs, and a lack of open-source hardware and acquisition software have limited smFRET's broad application by non-specialists. Here, we present the smfBox, a cost-effective confocal smFRET platform, providing detailed build instructions, open-source acquisition software, and full validation, thereby democratising smFRET for the wider scientific community.

High-throughput smFRET analysis of freely diffusing nucleic acid molecules and associated proteins

Article

Full-text available

May 2019

Single-molecule Förster resonance energy transfer (smFRET) is a powerful technique for nanometer-scale studies of single molecules. Solution-based smFRET, in particular, can be used to study equilibrium intra- and intermolecular conformations, binding/unbinding events and conformational changes under biologically relevant conditions without ensemble averaging. However, single-spot smFRET measurements in solution are slow. Here, we detail a high-throughput smFRET approach that extends the traditional single-spot confocal geometry to a multispot one. The excitation spots are optically conjugated to two custom silicon single photon avalanche diode (SPAD) arrays. Two-color excitation is implemented using a periodic acceptor excitation (PAX), allowing distinguishing between singly- and doubly-labeled molecules. We demonstrate the ability of this setup to rapidly and accurately determine FRET efficiencies and population stoichiometries by pooling the data collected independently from the multiple spots. We also show how the high throughput of this approach can be used to increase the temporal resolution of single-molecule FRET population characterization from minutes to seconds. Combined with microfluidics, this high-throughput approach will enable simple real-time kinetic studies as well as powerful molecular screening applications.

High-throughput smFRET analysis of freely diffusing nucleic acid molecules and associated proteins

Preprint

Full-text available

May 2019

An Expanded Conformation of an Antibody Fab Region by X-Ray Scattering, Molecular Dynamics and smFRET Identifies an Aggregation Mechanism

Article

Full-text available

Feb 2019

Protein aggregation is the underlying cause of many diseases, and also limits the usefulness of many natural and engineered proteins in biotechnology. Better mechanistic understanding and characterization of aggregation-prone states, is needed to guide protein engineering, formulation, and drug-targeting strategies that prevent aggregation. While several final aggregated states - notably amyloids - have been characterized structurally, very little is known about the native structural conformers that initiate aggregation. We used a novel combination of small-angle X-ray scattering (SAXS), atomistic molecular dynamics (MD) simulations, single-molecule FRET (smFRET), and aggregation-prone region (APR) predictions, to characterize structural changes in a native humanized Fab A33 antibody fragment, that correlated with the experimental aggregation kinetics. SAXS revealed increases in the native state radius of gyration, Rg, of 2.2% to 4.1%, at pH 5.5 and below, concomitant with accelerated aggregation. In a cutting-edge approach, we fitted the SAXS data to full molecular dynamics simulations from the same conditions, and located the conformational changes in the native state to the constant domain of the light chain (CL). This CL displacement was independently confirmed using smFRET measurements with two dual-labeled Fabs. These conformational changes were also found to increase the solvent exposure of a predicted aggregation-prone region (APR), suggesting a likely mechanism through which they promote aggregation. Our findings provide a means by which aggregation-prone conformational states can be readily determined experimentally, and thus potentially used to guide protein engineering, or ligand binding strategies, with the aim of stabilizing the protein against aggregation.

Monte-Carlo Diffusion-Enhanced Photon Inference: Distance Distributions And Conformational Dynamics In Single-Molecule FRET

Preprint

Full-text available

Aug 2018

Single-molecule Förster Resonance Energy Transfer (smFRET) is utilized to study the structure and dynamics of many bio-molecules, such as proteins, DNA and their various complexes. The structural assessment is based on the well-known Förster relationship between the measured efficiency of energy transfer between a donor (D) and an acceptor (A) dye and the distance between them. Classical smFRET analysis methods called photon distribution analysis (PDA) take into account photon shot-noise, D-A distance distribution and, more recently, interconversion between states in order to extract accurate distance information. It is known that rapid D-A distance fluctuations on the order of the D lifetime (or shorter) can increase the measured mean FRET efficiency and thus decrease the estimated D-A distance. Nonetheless, this effect has been so far neglected in smFRET experiments, potentially leading to biases in estimated distances. Here we introduce a PDA approach dubbed Monte-Carlo-diffusion-enhanced photon inference (MC-DEPI). MC-DEPI recolor detected photons of smFRET experiments taking into account dynamics of D-A distance fluctuations, multiple interconverting states and photo-blinking. Using this approach, we show how different underlying conditions may yield identical FRET histograms and how the additional information from fluorescence decays helps distinguishing between the different conditions. We also introduce a machine learning fitting approach for retrieving the D-A distance distribution, decoupled from the above-mentioned effects. We show that distance interpretation of smFRET experiments of even the simplest dsDNA is nontrivial and requires decoupling the effects of rapid D-A distance fluctuations on FRET in order to avoid systematic biases in the estimation of the D-A distance distribution.

Dynamic action of the Sec machinery during initiation, protein translocation and termination

Article

Full-text available

Jun 2018
eLife

Protein translocation across cell membranes is a ubiquitous process required for protein secretion and membrane protein insertion. In bacteria, this is mostly mediated by the conserved SecYEG complex, driven through rounds of ATP hydrolysis by the cytoplasmic SecA, and the trans-membrane proton motive force. We have used single molecule techniques to explore SecY pore dynamics on multiple timescales in order to dissect the complex reaction pathway. The results show that SecA, both the signal sequence and mature components of the pre-protein, and ATP hydrolysis each have important and specific roles in channel unlocking, opening and priming for transport. After channel opening, translocation proceeds in two phases: a slow phase independent of substrate length, and a length-dependent transport phase with an intrinsic translocation rate of ~40 amino acids per second for the proOmpA substrate. Broad translocation rate distributions reflect the stochastic nature of polypeptide transport.

Characterizing highly dynamic conformational states: The transcription bubble in RNAP-promoter open complex as an example

Article

Dec 2017
J CHEM PHYS

Bio-macromolecules carry out complicated functions through structural changes. To understand their mechanism of action, the structure of each step has to be characterized. While classical structural biology techniques allow the characterization of a few “structural snapshots” along the enzymatic cycle (usually of stable conformations), they do not cover all (and often fast interconverting) structures in the ensemble, where each may play an important functional role. Recently, several groups have demonstrated that structures of different conformations in solution could be solved by measuring multiple distances between different pairs of residues using single-molecule Förster resonance energy transfer (smFRET) and using them as constrains for hybrid/integrative structural modeling. However, this approach is limited in cases where the conformational dynamics is faster than the technique’s temporal resolution. In this study, we combine existing tools that elucidate sub-millisecond conformational dynamics together with hybrid/integrative structural modeling to study the conformational states of the transcription bubble in the bacterial RNA polymerase-promoter open complex (RPo). We measured microsecond alternating laser excitation-smFRET of differently labeled lacCONS promoter dsDNA constructs. We used a combination of burst variance analysis, photon-by-photon hidden Markov modeling, and the FRET-restrained positioning and screening approach to identify two conformational states for RPo. The experimentally derived distances of one conformational state match the known crystal structure of bacterial RPo. The experimentally derived distances of the other conformational state have characteristics of a scrunched RPo. These findings support the hypothesis that sub-millisecond dynamics in the transcription bubble are responsible for transcription start site selection.

Multispot single-molecule FRET: High-throughput analysis of freely diffusing molecules

Article

Full-text available

Apr 2017
PLOS ONE

We describe an 8-spot confocal setup for high-throughput smFRET assays and illustrate its performance with two characteristic experiments. First, measurements on a series of freely diffusing doubly-labeled dsDNA samples allow us to demonstrate that data acquired in multiple spots in parallel can be properly corrected and result in measured sample characteristics consistent with those obtained with a standard single-spot setup. We then take advantage of the higher throughput provided by parallel acquisition to address an outstanding question about the kinetics of the initial steps of bacterial RNA transcription. Our real-time kinetic analysis of promoter escape by bacterial RNA polymerase confirms results obtained by a more indirect route, shedding additional light on the initial steps of transcription. Finally, we discuss the advantages of our multispot setup, while pointing potential limitations of the current single laser excitation design, as well as analysis challenges and their solutions.

Diameter dependence of transport through nuclear pore complex mimics studied using optical nanopores

Article

Full-text available

Feb 2024
eLife

The nuclear pore complex (NPC) regulates the selective transport of large biomolecules through the nuclear envelope. As a model system for nuclear transport, we construct NPC mimics by functionalizing the pore walls of freestanding palladium zero-mode waveguides with the FG-nucleoporin Nsp1. This approach enables the measurement of single-molecule translocations through individual pores using optical detection. We probe the selectivity of Nsp1-coated pores by quantitatively comparing the translocation rates of the nuclear transport receptor Kap95 to the inert probe BSA over a wide range of pore sizes from 35 nm to 160 nm. Pores below 55 ± 5 nm show significant selectivity that gradually decreases for larger pores. This finding is corroborated by coarse-grained molecular dynamics simulations of the Nsp1 mesh within the pore, which suggest that leakage of BSA occurs by diffusion through transient openings within the dynamic mesh. Furthermore, we experimentally observe a modulation of the BSA permeation when varying the concentration of Kap95. The results demonstrate the potential of single-molecule fluorescence measurements on biomimetic NPCs to elucidate the principles of nuclear transport.

The substrate-binding domains of the osmoregulatory ABC importer OpuA physically interact

Preprint

Dec 2023
eLife

Bacteria utilize various strategies to prevent internal dehydration during hypertonic stress. A common approach to countering the effects of the stress is to import compatible solutes such as glycine betaine, leading to simultaneous passive water fluxes following the osmotic gradient. OpuA from Lactococcus lactis is a type I ABC-importer that uses two substrate-binding domains (SBDs) to capture extracellular glycine betaine and deliver the substrate to the transmembrane domains for subsequent transport. OpuA senses osmotic stress via changes in the internal ionic strength and is furthermore regulated by the 2nd messenger cyclic-di-AMP. We now show, by means of solution-based single-molecule FRET and analysis with multi- parameter photon-by-photon hidden Markov modeling, that the SBDs interact in an ionic strength-dependent manner. The smFRET data are in accordance with the apparent cooperativity in transport and supported by new cryo-EM data of OpuA. We propose that physical interactions between SBDs and cooperativity in substrate delivery could be more widespread than recognized thus far.

The substrate-binding domains of the osmoregulatory ABC importer OpuA physically interact

Preprint

Full-text available

Dec 2023

Bacteria utilize various strategies to prevent internal dehydration during hypertonic stress. A common approach to countering the effects of the stress is to import compatible solutes such as glycine betaine, leading to simultaneous passive water fluxes following the osmotic gradient. OpuA from Lactococcus lactis is a type I ABC-importer that uses two substrate-binding domains (SBDs) to capture extracellular glycine betaine and deliver the substrate to the transmembrane domains for subsequent transport. OpuA senses osmotic stress via changes in the internal ionic strength and is furthermore regulated by the 2nd messenger cyclic-di-AMP. We now show, by means of solution-based single-molecule FRET and analysis with multi- parameter photon-by-photon hidden Markov modeling, that the SBDs interact in an ionic strength-dependent manner. The smFRET data are in accordance with the apparent cooperativity in transport and supported by new cryo-EM data of OpuA. We propose that physical interactions between SBDs and cooperativity in substrate delivery could be more widespread than recognized thus far.

Diameter Dependence of Transport through Nuclear Pore Complex Mimics Studied Using Optical Nanopores

Preprint

Full-text available

Jun 2023

Diameter Dependence of Transport through Nuclear Pore Complex Mimics Studied Using Optical Nanopores

Preprint

Full-text available

Jun 2023
eLife

Diameter Dependence of Transport through Nuclear Pore Complex Mimics Studied Using Optical Nanopores

Preprint

Full-text available

Feb 2023

Making Precise and Accurate Single-Molecule FRET Measurements using the Open-Source smfBox

Article

Jul 2021
JoVE

From squiggle to sequence: bioinformatics in the era of single-molecule biopolymer analysis

Thesis

Jan 2022

Carlos de Lannoy

A user-friendly tool to convert photon counting data to the open-source Photon-HDF5 file format

Article

Mar 2022
Proceedings of SPIE

Data structure designing of the internal component libraries for learning experiment environment

Article

Full-text available

Apr 2022

Tetyana Neroda

It has been substantiated that the layout of the scheme of the educational experiment and further research of the subject area by the authorized user of educational space is hindered by the lack of common tools and standards for maintaining a suffi cient and relevant nomenclature of components. It is shown that the available solutions do not fully provide a hierarchical ordering of component attributes in the data structure of the internal library. A unifi ed specifi cation of the packed fi le format of the internal library for diff erent types of components has been designed to provide data fl ow support for software developed within the virtual platform of learning research. The hierarchical format is based on the ANSI-description of the subject area components and is suitable for constructing a ramifi ed contour of experimental scheme and further transitional processes studying. Formulation of the problem. Computerized environments of design works and of subject area modeling use fl exible fi le formats for interaction and data exchange. The need for adequate data structures for specifi c subject areas is common in many scientifi c branches. Most representatives of technically oriented community operate with standardized fi le formats from LabVIEW, Micro-Cap, MATLAB, etc. If these or other common specifi cations are used in original experimental research environments, it may take a long time to process all the available tags. Taking into account the multidisciplinary character of such educational environments, the scheme of a particular experiment uses only a small part of the specifi cation, depending on the subject area. Also, these formats are protected by patent law with trade secrets elements and their data structures do not take into account a number of research nuances in qualifi ed professionals training within the industry educational institution. Therefore, the analysis of fi le specifi cations in order to fi nd adequate for components attributes preserve of subject area is necessary for eff ective deployment of virtual platform environment of learning experiment, which is organically integrated into academic information space. Analysis of recent research and publications. Scientists from world educational institutions pay attention to the problems of data selection and formatting in context

Multi-parameter photon-by-photon hidden Markov modeling

Article

Full-text available

Feb 2022

Single molecule Förster resonance energy transfer (smFRET) is a unique biophysical approach for studying conformational dynamics in biomacromolecules. Photon-by-photon hidden Markov modeling (H2MM) is an analysis tool that can quantify FRET dynamics of single biomolecules, even if they occur on the sub-millisecond timescale. However, dye photophysical transitions intertwined with FRET dynamics may cause artifacts. Here, we introduce multi-parameter H2MM (mpH2MM), which assists in identifying FRET dynamics based on simultaneous observation of multiple experimentally-derived parameters. We show the importance of using mpH2MM to decouple FRET dynamics caused by conformational changes from photophysical transitions in confocal-based smFRET measurements of a DNA hairpin, the maltose binding protein, MalE, and the type-III secretion system effector, YopO, from Yersinia species, all exhibiting conformational dynamics ranging from the sub-second to microsecond timescales. Overall, we show that using mpH2MM facilitates the identification and quantification of biomolecular sub-populations and their origin. In this work, the authors demonstrate the application of multi-parameter photon-by-photon hidden Markov modeling (mpH2MM) on alternating laser excitation (ALEX)-based smFRET measurements. The utility of mpH2MM in identifying and quantifying dynamic biomolecular sub-populations is demonstrated in three different systems.

Making Precise and Accurate Single-Molecule FRET Measurements using the Open-Source smfBox

Article

Jul 2021
JoVE

The smfBox is a recently developed cost-effective, open-source instrument for single-molecule Förster Resonance Energy Transfer (smFRET), which makes measurements on freely diffusing biomolecules more accessible. This overview includes a step-by-step protocol for using this instrument to make measurements of precise FRET efficiencies in duplex DNA samples, including details of the sample preparation, instrument setup and alignment, data acquisition, and complete analysis routines. The presented approach, which includes how to determine all the correction factors required for accurate FRET-derived distance measurements, builds on a large body of recent collaborative work across the FRET Community, which aims to establish standard protocols and analysis approaches. This protocol, which is easily adaptable to a range of biomolecular systems, adds to the growing efforts in democratising smFRET for the wider scientific community.

FRETboard: Semisupervised classification of FRET traces

Article

Jul 2021
BIOPHYS J

Förster resonance energy transfer (FRET) is a useful phenomenon in biomolecular investigations, as it can be leveraged for nano-scale measurements. The optical signals produced by such experiments can be analyzed by fitting a statistical model. Several software tools exist to fit such models in an unsupervised manner, but lack the flexibility to adapt to different experimental setups and require local installations. Here we propose to fit models to optical signals more intuitively by adopting a semi-supervised approach, in which the user interactively guides the model to fit a given dataset, and introduce FRETboard, a web tool that allows users to provide such guidance. We show that our approach is able to closely reproduce ground truth FRET statistics in a wide range of simulated single-molecule scenarios, and correctly estimate parameters for up to eleven states. On in vitro data we retrieve parameters identical to those obtained by laborious manual classification in a fraction of the required time. Moreover, we designed FRETboard to be easily extendable to other models, allowing it to adapt to future developments in FRET measurement and analysis.

The structural heterogeneity of α-synuclein is governed by several distinct subpopulations with interconversion times slower than milliseconds

Article

May 2021
STRUCTURE

α-Synuclein plays an important role in synaptic functions by interacting with synaptic vesicle membrane, while its oligomers and fibrils are associated with several neurodegenerative diseases. The specific monomer structures that promote its membrane binding and self-association remain elusive due to its transient nature as an intrinsically disordered protein. Here, we use inter-dye distance distributions from bulk time-resolved Förster resonance energy transfer as restraints in discrete molecular dynamics simulations to map the conformational space of the α-synuclein monomer. We further confirm the generated conformational ensemble in orthogonal experiments utilizing far-UV circular dichroism and cross-linking mass spectrometry. Single-molecule protein-induced fluorescence enhancement measurements show that within this conformational ensemble, some of the conformations of α-synuclein are surprisingly stable, exhibiting conformational transitions slower than milliseconds. Our comprehensive analysis of the conformational ensemble reveals essential structural properties and potential conformations that promote its various functions in membrane interaction or oligomer and fibril formation.

Dynamics of Membrane Monitored by Single-Molecule Fluorescence Across Multiple Timescales

Chapter

Jan 2020

Single-molecule techniques provide insights into the heterogeneity and dynamics of ensembles and enable the extraction of mechanistic information that is complementary to high-resolution structural techniques. Here, we describe the application of single-molecule Förster resonance energy transfer to study the dynamics of integral membrane protein complexes on timescales spanning sub-milliseconds to minutes (10⁻⁹–10² s).

Multispot single-molecule FRET: high-throughput analysis of freely diffusing molecules

Preprint

Full-text available

Jan 2017

We describe an 8-spot confocal setup for high-throughput smFRET assays and illustrate its performance with two characteristic experiments. First, measurements on a series of freely diffusing doubly-labeled dsDNA samples allow us to demonstrate that data acquired in multiple spots in parallel can be properly corrected and result in measured sample characteristics identical to those obtained with a standard single-spot setup. We then take advantage of the higher throughput provided by parallel acquisition to address an outstanding question about the kinetics of the initial steps of bacterial RNA transcription. Our real-time kinetic analysis of promoter escape by bacterial RNA polymerase confirms results obtained by a more indirect route, shedding additional light on the initial steps of transcription. Finally, we discuss the advantages of our multispot setup, while pointing potential limitations of the current single laser excitation design, as well as analysis challenges and their solutions.

The structural heterogeneity of α-synuclein is governed by several distinct subpopulations with interconversion times slower than milliseconds

Preprint

Full-text available

Nov 2020

The intrinsically disordered protein, α-synuclein, implicated in synaptic vesicle homeostasis and neurotransmitter release, is also associated with several neurodegenerative diseases. The different roles of α-synuclein are characterized by distinct structural states (membrane-bound, dimer, tetramer, oligomer, and fibril), which are originated from its various monomeric conformations. The pathological states, determined by the ensemble of α-synuclein monomer conformations and dynamic pathways of interconversion between dominant states, remain elusive due to their transient nature. Here, we use inter-dye distance distributions from bulk time-resolved Forster resonance energy transfer as restraints in discrete molecular dynamics simulations to map the conformational space of the α-synuclein monomer. We further confirm the generated conformational ensemble in orthogonal experiments utilizing far-UV circular dichroism and cross-linking mass spectrometry. Single-molecule protein-induced fluorescence enhancement measurements show that within this conformational ensemble, some of the conformations of α-synuclein are surprisingly stable, exhibiting conformational transitions slower than milliseconds. Our comprehensive analysis of the conformational ensemble reveals essential structural properties and potential conformations that promote its various functions in membrane interaction or oligomer and fibril formation.

FRETboard: semi-supervised classification of FRET traces

Preprint

Full-text available

Aug 2020

A bstract Förster resonance energy transfer (FRET) is a useful phenomenon in biomolecular investigations, as it can be leveraged for nano-scale measurements. The optical signals produced by such experiments can be analyzed by fitting a statistical model. Several software tools exist to fit such models in an unsupervised manner, but their operating system-dependent installation requirements and lack of flexibility impede wide-spread adoption. Here we propose to fit such models more efficiently and intuitively by adopting a semi-supervised approach, in which the user interactively guides the model to fit a given dataset, and introduce FRETboard, a web tool that allows users to provide such guidance. We show that our approach is able to closely reproduce ground truth FRET statistics in a wide range of simulated single-molecule scenarios, and correctly estimate parameters for up to eleven states. On in vitro data we retrieve parameters identical to those obtained by laborious manual classification in a fraction of the required time. Moreover, we designed FRETboard to be easily extendable to other models, allowing it to adapt to future developments in FRET measurement and analysis. Availability source code is available at https://github.com/cvdelannoy/FRETboard . The FRETboard classification tool is also available as a browser application at https://www.bioinformatics.nl/FRETboard .

The FRET-based structural dynamics challenge -- community contributions to consistent and open science practices

Article

Full-text available

Jun 2020

Single-molecule Förster resonance energy transfer (smFRET) has become a mainstream technique for probing biomolecular structural dynamics. The rapid and wide adoption of the technique by an ever-increasing number of groups has generated many improvements and variations in the technique itself, in methods for sample preparation and characterization, in analysis of the data from such experiments, and in analysis codes and algorithms. Recently, several labs that employ smFRET have joined forces to try to bring the smFRET community together in adopting a consensus on how to perform experiments and analyze results for achieving quantitative structural information. These recent efforts include multi-lab blind-tests to assess the accuracy and precision of smFRET between different labs using different procedures, the formal assembly of the FRET community and development of smFRET procedures to be considered for entries in the wwPDB. Here we delve into the different approaches and viewpoints in the field. This position paper describes the current "state-of-the field", points to unresolved methodological issues for quantitative structural studies, provides a set of 'soft recommendations' about which an emerging consensus exists, and a list of resources that are openly available. To make further progress, we strongly encourage 'open science' practices. We hope that this position paper will provide a roadmap for newcomers to the field, as well as a reference for seasoned practitioners.

flimview : A software framework to handle, visualize and analyze FLIM data

Article

Full-text available

Jun 2020

flimview is a bio-imaging Python software package to read, explore, manage and visualize Fluorescence-Lifetime Imaging Microscopy (FLIM) images. It can open the standard FLIM data file conventions (e.g., sdt and ptu) and processes them from the raw format to a more readable and manageable binned and fitted format. It allows customized kernels for binning the data as well as user defined masking operations for pre-processing the images. It also allows customized fluorescence decay fitting functions and preserves all of the metadata generated for provenance and reproducibility. Outcomes from the analysis are lossless compressed and stored in an efficient way providing the necessary open-source tools to access and explore the data. flimview is open source and includes example data, example Jupyter notebooks and tutorial documentation. The package, test data and documentation are available on Github .

The FRET-based structural dynamics challenge -- community contributions to consistent and open science practices

Preprint

Full-text available

Jun 2020

Single-molecule F\"{o}rster resonance energy transfer (smFRET) has become a mainstream technique for probing biomolecular structural dynamics. The rapid and wide adoption of the technique by an ever-increasing number of groups has generated many improvements and variations in the technique itself, in methods for sample preparation and characterization, in analysis of the data from such experiments, and in analysis codes and algorithms. Recently, several labs that employ smFRET have joined forces to try to bring the smFRET community together in adopting a consensus on how to perform experiments and analyze results for achieving quantitative structural information. These recent efforts include multi-lab blind-tests to assess the accuracy and precision of smFRET between different labs using different procedures, the formal assembly of the FRET community and development of smFRET procedures to be considered for entries in the wwPDB. Here we delve into the different approaches and viewpoints in the field. This position paper describes the current "state-of-the field", points to unresolved methodological issues for quantitative structural studies, provides a set of 'soft recommendations' about which an emerging consensus exists, and a list of resources that are openly available. To make further progress, we strongly encourage 'open science' practices. We hope that this position paper will provide a roadmap for newcomers to the field, as well as a reference for seasoned practitioners.

Inter-domain dynamics in the chaperone SurA and multi-site binding to its unfolded outer membrane protein clients

Preprint

Full-text available

Dec 2019

The periplasmic chaperone SurA plays a key role in outer membrane protein (OMP) biogenesis. E. coli SurA comprises a core domain and two peptidylprolyl isomerase domains (P1 and P2), but how it binds its OMP clients and the mechanism(s) of its chaperone action remain unclear. Here, we have used chemical cross-linking, hydrogen-deuterium exchange, single-molecule FRET and molecular dynamics simulations to map the client binding site(s) on SurA and to interrogate the role of conformational dynamics of the chaperone's domains in OMP recognition. We demonstrate that SurA samples a broad array of conformations in solution in which P2 primarily lies closer to the core/P1 domains than suggested by its crystal structure. Multiple binding sites for OMPs are located primarily in the core domain, with binding of the unfolded OMP resulting in conformational changes between the core/P1 domains. Together, the results portray a model in which unfolded OMP substrates bind in a cradle formed between the SurA domains, with structural flexibility between its domains assisting OMP recognition, binding and release.

Methods and Technologies for Research- and Metadata Management in Collaborative Experimental Research

Article

Full-text available

Nov 2018

Newly developed technologies and methods for the purpose of controlling uncertainty in technical systems must be proven and validated against reliable experimental studies. The availability of descriptive metadata is mandatory to enable long term usability and sharing of such experimental research data. This article introduces a concept for a software independent solution for managing data in collaborative research environments. The proposed approach leverages the advantages of capturing metadata in a uniform, modular data structure and providing software independent access to a centralized data repository as well as its contents by means of a web-application. The article presents a prototype implementation of the proposed approach and discusses its application on the demonstrator test rig of a collaborative research centre in the field of mechanical engineering.

Toward dynamic structural biology: Two decades of single-molecule Förster resonance energy transfer

Article

Jan 2018
SCIENCE

Watching single molecules in motion Structural techniques such as x-ray crystallography and electron microscopy give insight into how macromolecules function by providing snapshots of different conformational states. Function also depends on the path between those states, but to see that path involves watching single molecules move. This became possible with the advent of single-molecule Förster resonance energy transfer (smFRET), which was first implemented in 1996. Lerner et al. review how smFRET has been used to study macromolecules in action, providing mechanistic insights into processes such as DNA repair, transcription, and translation. They also describe current limitations of the approach and suggest how future developments may expand the applications of smFRET. Science , this issue p. eaan1133

48-spot single-molecule FRET setup with periodic acceptor excitation

Article

Nov 2017

Single-molecule Förster resonance energy transfer (smFRET) allows measuring distances between donor and acceptor fluorophores on the 3–10 nm range. Solution-based smFRET allows measurement of binding-unbinding events or conformational changes of dye-labeled biomolecules without ensemble averaging and free from surface perturbations. When employing dual (or multi) laser excitation, smFRET allows resolving the number of fluorescent labels on each molecule, greatly enhancing the ability to study heterogeneous samples. A major drawback to solution-based smFRET is the low throughput, which renders repetitive measurements expensive and hinders the ability to study kinetic phenomena in real-time. Here we demonstrate a high-throughput smFRET system that multiplexes acquisition by using 48 excitation spots and two 48-pixel single-photon avalanche diode array detectors. The system employs two excitation lasers allowing separation of species with one or two active fluorophores. The performance of the system is demonstrated on a set of doubly labeled double-stranded DNA oligonucleotides with different distances between donor and acceptor dyes along the DNA duplex. We show that the acquisition time for accurate subpopulation identification is reduced from several minutes to seconds, opening the way to high-throughput screening applications and real-time kinetics studies of enzymatic reactions such as DNA transcription by bacterial RNA polymerase.

Single-molecule dataset (SMD): A generalized storage format for raw and processed single-molecule data

Article

Full-text available

Jan 2015
BMC BIOINFORMATICS

Background Single-molecule techniques have emerged as incisive approaches for addressing a wide range of questions arising in contemporary biological research [ 1-4]. The analysis and interpretation of raw single-molecule data benefits greatly from the ongoing development of sophisticated statistical analysis tools that enable accurate inference at the low signal-to-noise ratios frequently associated with these measurements. While a number of groups have released analysis toolkits as open source software [5-14], it remains difficult to compare analysis for experiments performed in different labs due to a lack of standardization.ResultsHere we propose a standardized single-molecule dataset (SMD) file format. SMD is designed to accommodate a wide variety of computer programming languages, single-molecule techniques, and analysis strategies. To facilitate adoption of this format we have made two existing data analysis packages that are used for single-molecule analysis compatible with this format.Conclusion Adoption of a common, standard data file format for sharing raw single-molecule data and analysis outcomes is a critical step for the emerging and powerful single-molecule field, which will benefit both sophisticated users and non-specialists by allowing standardized, transparent, and reproducible analysis practices.

Silicon Photon-Counting Avalanche Diodes for Single-Molecule Fluorescence Spectroscopy

Article

Full-text available

Jul 2014

Solution-based single-molecule fluorescence spectroscopy is a powerful experimental tool with applications in cell biology, biochemistry and biophysics. The basic feature of this technique is to excite and collect light from a very small volume and work in a low concentration regime resulting in rare burst-like events corresponding to the transit of a single molecule. Detecting photon bursts is a challenging task: the small number of emitted photons in each burst calls for high detector sensitivity. Bursts are very brief, requiring detectors with fast response time and capable of sustaining high count rates. Finally, many bursts need to be accumulated to achieve proper statistical accuracy, resulting in long measurement time unless parallelization strategies are implemented to speed up data acquisition. In this paper we will show that silicon single-photon avalanche diodes (SPADs) best meet the needs of single-molecule detection. We will review the key SPAD parameters and highlight the issues to be addressed in their design, fabrication and operation. After surveying the state-of-the-art SPAD technologies, we will describe our recent progress towards increasing the throughput of single-molecule fluorescence spectroscopy in solution using parallel arrays of SPADs. The potential of this approach is illustrated with single-molecule Förster resonance energy transfer measurements.

Single Molecule Analysis Research Tool (SMART): An Integrated Approach for Analyzing Single Molecule Data

Article

Full-text available

Feb 2012
PLOS ONE

Single molecule studies have expanded rapidly over the past decade and have the ability to provide an unprecedented level of understanding of biological systems. A common challenge upon introduction of novel, data-rich approaches is the management, processing, and analysis of the complex data sets that are generated. We provide a standardized approach for analyzing these data in the freely available software package SMART: Single Molecule Analysis Research Tool. SMART provides a format for organizing and easily accessing single molecule data, a general hidden Markov modeling algorithm for fitting an array of possible models specified by the user, a standardized data structure and graphical user interfaces to streamline the analysis and visualization of data. This approach guides experimental design, facilitating acquisition of the maximal information from single molecule experiments. SMART also provides a standardized format to allow dissemination of single molecule data and transparency in the analysis of reported data.

Fluorescence Spectroscopy of Single Biomolecules

Article

Full-text available

Mar 1999

Shimon Weiss

Recent advances in single-molecule detection and single-molecule spectroscopy at room temperature by laser-induced fluorescence offer new tools for the study of individual macromolecules under physiological conditions. These tools relay conformational states, conformational dynamics, and activity of single biological molecules to physical observables, unmasked by ensemble averaging. Distributions and time trajectories of these observables can therefore be measured during a reaction without the impossible need to synchronize all the molecules in the ensemble. The progress in applying these tools to biological studies with the use of fluorophores that are site-specifically attached to macromolecules is reviewed.

Single Molecule Dynamics Studied by Polarization Modulation

Article

Full-text available

Dec 1996
PHYS REV LETT

We observed and made unambiguous distinctions between abrupt photophysical events of single molecules: a rotational jump of a single dipole, a transition to a dark state (reversible and irreversible photobleaching), and a spectral jump. The study was performed in the far field by modulating the excitation polarization and monitoring the fluorescence in time. This technique also allowed us to measure the in-plane dipole orientation of stationary single molecular dipoles with subdegree accuracy and to resolve desorption and readsorption of fluorophores from and onto a glass surface. In one case, clear evidence was obtained for rapid rotation of the dipole after a desorption process. {copyright} {ital 1996 The American Physical Society.}

Single-Molecule Measurement of Protein Folding Kinetics

Article

Full-text available

Sep 2003
SCIENCE

In order to investigate the behavior of single molecules under conditions far from equilibrium, we have coupled a microfabricated laminar-flow mixer to a confocal optical system. This combination enables time-resolved measurement of Förster resonance energy transfer after an abrupt change in solution conditions. Observations of a small protein show the evolution of the intramolecular distance distribution as folding progresses. This technique can expose subpopulations, such as unfolded protein under conditions favoring the native structure, that would be obscured in equilibrium experiments.

Protein Conformational Dynamics Probed by Single-Molecule Electron Transfer

Article

Full-text available

Nov 2003
SCIENCE

Electron transfer is used as a probe for angstrom-scale structural changes in single protein molecules. In a flavin reductase, the fluorescence of flavin is quenched by a nearby tyrosine residue by means of photo-induced electron transfer. By probing the fluorescence lifetime of the single flavin on a photon-by-photon basis, we were able to observe the variation of flavin-tyrosine distance over time. We could then determine the potential of mean force between the flavin and the tyrosine, and a correlation analysis revealed conformational fluctuation at multiple time scales spanning from hundreds of microseconds to seconds. This phenomenon suggests the existence of multiple interconverting conformers related to the fluctuating catalytic reactivity.

Berman, H, Henrick, K and Nakamura, H. Announcing the worldwide Protein Data Bank. Nat Struct Biol 10: 980

Article

Full-text available

Jan 2004
Nat Struct Biol

mentation will be kept publicly available and the distribution sites will mirror the PDB archive using identical contents and subdirec- tory structure. However, each member of the wwPDB will be able to develop its own web site, with a unique view of the primary data, providing a variety of tools and resources for the global community. An Advisory Board consisting of appointees from the wwPDB, the International Union of Crystallography and the International Council on Magnetic Resonance in Biological Systems will provide guidance through annual meetings with the wwPDB consortium. This board is responsible for reviewing and deter- mining policy as well as providing a forum for resolving issues related to the wwPDB. Specific details about the Advisory Board can be found in the wwPDB charter, available on the wwPDB web site. The RCSB is the 'archive keeper' of wwPDB. It has sole write access to the PDB archive and control over directory structure and contents, as well as responsibility for dis- tributing new PDB identifiers to all deposi- tion sites. The PDB archive is a collection of flat files in the legacy PDB file format 3 and in the mmCIF 4 format that follows the PDB exchange dictionary (http://deposit.pdb.org/ mmcif/). This dictionary describes the syntax and semantics of PDB data that are processed and exchanged during the process of data annotation. It was designed to provide consis- tency in data produced in structure laborato- ries, processed by the wwPDB members and used in bioinformatics applications. The PDB archive does not include the websites, browsers, software and database query engines developed by researchers worldwide. The members of the wwPDB will jointly agree to any modifications or extensions to the PDB exchange dictionary. As data tech- nology progresses, other data formats (such as XML) and delivery methods may be included in the official PDB archive if all the wwPDB members concur on the alteration. Any new formats will follow the naming and description conventions of the PDB exchange dictionary. In addition, the legacy PDB for- mat would not be modified unless there is a compelling reason for a change. Should such a situation occur, all three wwPDB members would have to agree on the changes and give the structural biology community 90 days advance notice. The creation of the wwPDB formalizes the international character of the PDB and ensures that the archive remains single and uniform. It provides a mechanism to ensure consistent data for software developers and users world- wide. We hope that this will encourage individ- ual creativity in developing tools for presenting structural data, which could benefit the scien- tific research community in general.

Kapanidis, AN, Lee, NK, Laurence, TA, Doose, S, Margeat, E and Weiss, S. Fluorescence-aided molecule sorting: analysis of structure and interactions by alternating-laser excitation of single molecules. Proc Natl Acad Sci USA 101: 8936-8941

Article

Full-text available

Jul 2004

We use alternating-laser excitation to achieve fluorescence-aided molecule sorting (FAMS) and enable simultaneous analysis of biomolecular structure and interactions at the level of single molecules. This was performed by labeling biomolecules with fluorophores that serve as donor–acceptor pairs for Förster resonance energy transfer, and by using alternating-laser excitation to excite directly both donors and acceptors present in single diffusing molecules. Emissions were reduced to the distance-dependent ratio E, and a distance-independent, stoichiometry-based ratio S. Histograms of E and S sorted species based on the conformation and association status of each species. S was sensitive to the stoichiometry and relative brightness of fluorophores in single molecules, observables that can monitor oligomerization and local-environment changes, respectively. FAMS permits equilibrium and kinetic analysis of macromolecule-ligand interactions; this was validated by measuring equilibrium and kinetic dissociation constants for the interaction of Escherichia coli catabolite activator protein with DNA. FAMS is a general platform for ratiometric measurements that report on structure, dynamics, stoichiometries, environment, and interactions of diffusing or immobilized molecules, thus enabling detailed mechanistic studies and ultrasensitive diagnostics. • single-molecule fluorescence spectroscopy • Förster resonance energy transfer • biomolecular interactions • catabolite activator protein • protein–DNA interactions

Probing structural heterogeneities and fluctuations of nucleic acids and denatured proteins

Article

Full-text available

Dec 2005

We study protein and nucleic acid structure and dynamics using single-molecule FRET and alternating-laser excitation. Freely diffusing molecules are sorted into subpopulations based on single-molecule signals detected within 100 μs to 1 ms. Distance distributions caused by fluctuations faster than 100 μs are studied within these subpopulations by using time-correlated single-photon counting. Measured distance distributions for dsDNA can be accounted for by considering fluorophore linkers and fluorophore rotational diffusion, except that we find smaller fluctuations for internally labeled dsDNA than DNA with one of the fluorophores positioned at a terminal site. We find that the electrostatic portion of the persistence length of short single-stranded poly(dT) varies approximately as the ionic strength (I) to the –1/2 power (I –1/2), and that the average contribution to the contour length per base is 0.40–0.45 nm. We study unfolded chymotrypsin inhibitor 2 (CI2) and unfolded acyl-CoA binding protein (ACBP) even under conditions where folded and unfolded subpopulations coexist (contributions from folded proteins are excluded by using alternating-laser excitation). At lower denaturant concentrations, unfolded CI2 and ACBP are more compact and display larger fluctuations than at higher denaturant concentrations where only unfolded proteins are present. The experimentally measured fluctuations are larger than the fluctuations predicted from a Gaussian chain model or a wormlike chain model. We propose that the larger fluctuations may indicate transient residual structure in the unfolded state. • conformational dynamics • protein folding • single-molecule fluorescence spectroscopy • nucleic acid structure • fluorescence resonance energy transfer

The Worldwide Protein Data Bank

Chapter

Apr 2012

The Protein Data Bank (PDB) is the single, freely available, global archive of structural data for biological macromolecules. It is maintained by the wwPDB consortium consisting of the Research Collaboratory for Structural Bioinformatics (RCSB PDB), the Protein Data Bank in Europe (PDBe), the PDB Japan (PDBj) and the BioMagResBank (BMRB). This chapter describes the organization of the wwPDB, the systems in place for data deposition, annotation and distribution, and a summary of the services provided by the wwPDB member sites. Keywords: Protein Data Bank

Profile of Eric Betzig, Stefan Hell, and W. E. Moerner, 2014 Nobel Laureates in Chemistry

Article

Feb 2015

Jennifer Lippincott-Schwartz

Scientists once believed that the laws of physics would prevent them from peering into the structures of the cell. The story of the winners of the 2014 Nobel Prize in Chemistry is about how three imaginative scientists pioneered ways to work around these supposed limits, transforming microscopy into nanoscopy.

Interactive notebooks: Sharing the code

Article

Nov 2014

Helen Shen

The free IPython notebook makes data analysis easier to record, understand and reproduce.

Fluorescence Correlation Spectroscopy at Micromolar Concentrations without Optical Nanoconfinement

Article

Jul 2014

Fluorescence Correlation Spectroscopy (FCS) is an important technique for studying biochemical interactions dynamically that may be used in vitro and in cell-based studies. It is generally claimed that FCS may only be used at nM concentrations. We show that this general consensus is incorrect and that the limitation to nM concentrations is not fundamental, but due to detector limits as well as laser fluctuations. With a high count rate detector system and applying laser fluctuation corrections, we demonstrate FCS measurements up to 38 μM with the same signal to noise as at lower concentrations. Optical nanoconfinement approaches previously used to increase the concentration range of FCS are not necessary, and further increases above 38 μM may be expected using detectors and detector arrays with higher saturation rates and better laser fluctuation corrections. This approach greatly widens the possibilities of dynamic measurements of biochemical interactions using FCS at physiological concentrations.

Empirical Bayes Methods Enable Advanced Population-Level Analyses of Single-Molecule FRET Experiments

Article

Mar 2014
BIOPHYS J

Many single-molecule experiments aim to characterize biomolecular processes in terms of kinetic models that specify the rates of transition between conformational states of the biomolecule. Estimation of these rates often requires analysis of a population of molecules, in which the conformational trajectory of each molecule is represented by a noisy, time-dependent signal trajectory. Although hidden Markov models (HMMs) may be used to infer the conformational trajectories of individual molecules, estimating a consensus kinetic model from the population of inferred conformational trajectories remains a statistically difficult task, as inferred parameters vary widely within a population. Here, we demonstrate how a recently developed empirical Bayesian method for HMMs can be extended to enable a more automated and statistically principled approach to two widely occurring tasks in the analysis of single-molecule fluorescence resonance energy transfer (smFRET) experiments: 1), the characterization of changes in rates across a series of experiments performed under variable conditions; and 2), the detection of degenerate states that exhibit the same FRET efficiency but differ in their rates of transition. We apply this newly developed methodology to two studies of the bacterial ribosome, each exemplary of one of these two analysis tasks. We conclude with a discussion of model-selection techniques for determination of the appropriate number of conformational states. The code used to perform this analysis and a basic graphical user interface front end are available as open source software.

Multipixel single-photon avalanche diode array for parallel photon counting applications

Article

Jan 2009

In life science, optical techniques for the characterization of biological processes are well established and widely used. In most of them, to obtain the best performances, detectors with single-photon detection capability are required. Moreover, the growing demand for this type of information is pushing the technology towards a parallelization of the analysis. These requirements make it very challenging to develop new detection heads, because state-of-the-art detectors, such as photomultiplier tubes (PMTs) and charged coupled devices (CCDs), have some drawbacks. For example, in fluorescence correlation spectroscopy (FCS) fluorescence fluctuations must be monitored on a short time scale because typical time constants range from hundreds of nanoseconds to milliseconds. In this case, developing parallel modules for this application is very challenging. In fact, PMTs are bulky and they cannot be integrated, while the use of imaging detectors, such as CCDs and electron multiplying CCDs, is strongly limited by the read time that determines the minimum correlation time detectable by FCS analysis. We present here a multichannel photon counting module that exploits a monolithic array of single-photon avalanche diodes (SPADs). The detector array consists of eight 50 µm diameter SPADs featuring low dark counting rate and high photon detection efficiency (50% at 550 nm); inter-pixel crosstalk probability is as low as 2 × 10. The use of highly integrated active quenching circuits makes it possible to design a very compact read-out circuit, yet providing eight fully independent counting channels operating at high count rate (up to 30 million counts per second). Parallel-mode operation, high count rate and low-cost make this module a potential breakthrough for a widespread diffusion of FCS analysis.

Identification of Single Molecules in Aqueous Solution by Time-Resolved Fluorescence Anisotropy

Article

Dec 1998

Using a confocal epi-illuminated microscope with a polarizing beam splitter and dual-channel detection of single-molecule fluorescence induced by pulsed laser excitation, a new application of the three-dimensional, real-time spectroscopic technique BIFL (burst integrated fluorescence lifetime) is introduced. BIFL allows simultaneous registration of fluorescence intensity, lifetime, and anisotropy. It is shown to be well-suited to identify the freely diffusing fluorescent molecule Rhodamine 123 and the Enhanced Yellow Fluorescent Protein via their characteristic fluorescence anisotropy using a time-resolved analysis. Furthermore, data analysis is discussed and rotational correlation times of single molecules are determined. Applications for multidimensional single-molecule identification are outlined.

Quantitative Identification of Different Single Molecules by Selective Time-Resolved Confocal Fluorescence Spectroscopy

Article

Jul 1998

Using a confocal epi-illuminated microscope together with a pulsed laser, new applications of the recently developed, real-time spectroscopic technique BIFL (burst integrated fluorescence lifetime) are introduced. BIFL registers two different types of information on every detected photon with regard to the macroscopic time scale of a measurement and to the fluorescence lifetime. Thus, it is shown to be well suited to identify freely diffusing single dye molecules via their characteristic fluorescence lifetime. This allows for selective counting of dye molecules in an open volume element and opens up the possibility to quantify the relative concentration of the dye molecules, using a recently derived theoretical model, which analyzes the obtained burst size distribution of a sample survey. A closed theory is presented to calculate the probability of a specific dye to cause a fluorescence burst containing a certain number of detected photons. It considers the distribution of the excitation irradiance over the detection volume together with saturation effects of the fluorescence and of the detection electronics, the probability of different transit times through the detection volume, and the probability of multimolecule events. Using BIFL together with selective counting, the concentration of two dyes, Rhodamine B and Rhodamine 6G, in separate solutions and in a mixture were determined. The obtained results are consistent with the applied dye concentrations and with simultaneous measurements by fluorescence correlation spectroscopy (FCS). The introduced method is an appropriate tool for the complete characterization and quantitative analysis of a highly diluted sample in homogeneous assays.

Immobilization in Surface-Tethered Lipid Vesicles as a New Tool for Single Biomolecule Spectroscopy

Article

Dec 2001

Single-molecule fluorescence studies of functional biomolecule dynamics rely on the ability to provide biologically relevant experimental conditions. Long measurement times on single molecules require their immobilization, which might modify their dynamics through interactions with the trapping medium, e.g., a glass surface or a polymer gel. In an effort to overcome this problem we have devised a new immobilization technique, based on the confinement of single biomolecules inside 100 nm surface-tethered lipid vesicles. The number of molecules in each vesicle can be accurately determined from fluorescence time traces; under our experimental conditions the number distribution of encapsulated molecules obeys a Poisson distribution with an average occupancy of 0.65 molecules per vesicle. It is further shown that the distribution of fluorescence polarization values of trapped molecules can serve as a sensitive probe for their freedom of motion and thus for the environment they sample inside the liposomes. Polarization distributions are obtained for two vesicle-entrapped labeled proteins, bovine serum albumin and adenylate kinase, and compared with distributions measured for the same proteins directly adsorbed on glass. From the significant relative narrowing of the distributions for encapsulated molecules, it is concluded that their motion within the vesicles is quite similar to free solution.

Fluorescence Correlation Spectroscopy with High Count Rate and Low-Background - Analysis of Translational Diffusion

Article

Aug 1993

An epi-illuminated microscope configuration for use in fluorescence correlation spectroscopy in bulk solutions has been analyzed. For determining the effective sample dimensions the spatial distribution of the molecule detection efficiency has been computed and conditions for achieving quasi-cylindrical sample shape have been derived. Model experiments on translational diffusion of rhodamine 6G have been carried out using strong focusing of the laser beam, small pinhole size and an avalanche photodiode in single photon counting mode as the detector. A considerable decrease in background light intensity and measurement time has been observed. The background light is 40 times weaker than the fluorescence signal from one molecule of Rh6G, and the correlation function with signal-to-noise ratio of 150 can be collected in 1 second. The effect of the shape of the sample volume on the autocorrelation function has been discussed.

Probing Single Biomolecules in Solution Using the Anti-Brownian Electrokinetic (ABEL) Trap

Article

May 2012
ACCOUNTS CHEM RES

Single-molecule fluorescence measurements allow researchers to study asynchronous dynamics and expose molecule-to-molecule structural and behavioral diversity, which contributes to the understanding of biological macromolecules. To provide measurements that are most consistent with the native environment of biomolecules, researchers would like to conduct these measurements in the solution phase if possible. However, diffusion typically limits the observation time to approximately 1 ms in many solution-phase single-molecule assays. Although surface immobilization is widely used to address this problem, this process can perturb the system being studied and contribute to the observed heterogeneity.

Ratiometric measurement and identification of single diffusing molecules

Article

Aug 1999
CHEM PHYS

The concept of ratiometric identification and separation of sub-populations of single biomolecules which are labeled with individual fluorophores and are freely diffusing (or flowing) through the laser excitation volume is introduced. The efficacy of the scheme versus the signal and noise level is considered and a general theoretical framework and data analysis schemes are introduced. Experimental results that demonstrate distributions of single molecule fluorescence polarization anisotropy (smFPA) and single pair fluorescence resonance energy transfer (spFRET) in solution are presented. Ultimate resolution for identifying molecular observables such as conformational states, degree of freedom of motion and spectral distribution are discussed.

Electrokinetic trapping at the one nanometer limit

Article

May 2011
P NATL ACAD SCI USA

Anti-Brownian electrokinetic traps have been used to trap and study the free-solution dynamics of large protein complexes and long chains of DNA. Small molecules in solution have thus far proved too mobile to trap by any means. Here we explore the ultimate limits on trapping single molecules. We developed a feedback-based anti-Brownian electrokinetic trap in which classical thermal noise is compensated to the maximal extent allowed by quantum measurement noise. We trapped single fluorophores with a molecular weight of < 1 kDa and a hydrodynamic radius of 6.7 Å for longer than one second, in aqueous buffer at room temperature. This achievement represents an 800-fold decrease in the mass of objects trapped in solution, and opens the possibility to trap and manipulate any soluble molecule that can be fluorescently labeled. To illustrate the use of this trap, we studied the binding of unlabeled RecA to fluorescently labeled single-stranded DNA. Binding of RecA induced changes in the DNA diffusion coefficient, electrophoretic mobility, and brightness, all of which were measured simultaneously and on a molecule-by-molecule basis. This device greatly extends the size range of molecules that can be studied by room temperature feedback trapping, and opens the door to further studies of the binding of unmodified proteins to DNA in free solution.

Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution

Article

May 1997

The present paper describes a new experimental scheme for following diffusion and chemical reaction systems of fluorescently labeled molecules in the nanomolar concentration range by fluorescence correlation analysis. In the dual-color fluorescence cross-correlation spectroscopy provided here, the concentration and diffusion characteristics of two fluorescent species in solution as well as their reaction product can be followed in parallel. By using two differently labeled reaction partners, the selectivity to investigate the temporal evolution of reaction product is significantly increased compared to ordinary one-color fluorescence autocorrelation systems. Here we develop the theoretical and experimental basis for carrying out measurements in a confocal dual-beam fluorescence correlation spectroscopy setup and discuss conditions that are favorable for cross-correlation analysis. The measurement principle is explained for carrying out DNA-DNA renaturation kinetics with two differently labeled complementary strands. The concentration of the reaction product can be directly determined from the cross-correlation amplitude.

Data registration and selective single-molecule analysis using multi-parameter fluorescence detection

Article

May 2001
J BIOTECHNOL

A general strategy to identify and quantify sample molecules in dilute solution employing a new spectroscopic method for data registration and specific burst analysis denoted as multi-parameter fluorescence detection (MFD) was recently developed. While keeping the experimental advantage of monitoring single molecules diffusing through the microscopic open volume element of a confocal epi-illuminated set-up as in experiments of fluorescence correlation spectroscopy, MFD uses pulsed excitation and time-correlated single-photon counting to simultaneously monitor the evolution of the four-dimensional fluorescence information (intensity, F; lifetime, tau; anisotropy, r; and spectral range, lambda(r)) in real time and allows for exclusion of extraneous events for subsequent analysis. In this review, the versatility of this technique in confocal fluorescence spectroscopy will be presented by identifying freely diffusing single dyes via their characteristic fluorescence properties in homogenous assays, resulting in significantly reduced misclassification probabilities. Major improvements in background suppression are demonstrated by time-gated autocorrelation analysis of fluorescence intensity traces extracted from MFD data. Finally, applications of MFD to real-time conformational dynamics studies of fluorescence labeled oligonucleotides will be presented.

Pulsed Interleaved Excitation

Article

Dec 2005

In this article, we demonstrate the new method of pulsed interleaved excitation (PIE), which can be used to extend the capabilities of multiple-color fluorescence imaging, fluorescence cross-correlation spectroscopy (FCCS), and single-pair fluorescence resonance energy transfer (spFRET) measurements. In PIE, multiple excitation sources are interleaved such that the fluorescence emission generated from one pulse is complete before the next excitation pulse arrives. Hence, the excitation source for each detected photon is known. Typical repetition rates used for PIE are between approximately 1 and 50 MHz. PIE has many applications in various fluorescence methods. Using PIE, dual-color measurements can be performed with a single detector. In fluorescence imaging with multicolor detection, spectral cross talk can be removed, improving the contrast of the image. Using PIE with FCCS, we can eliminate spectral cross talk, making the method sensitive to weaker interactions. FCCS measurements with complexes that undergo FRET can be analyzed quantitatively. Under specific conditions, the FRET efficiency can be determined directly from the amplitude of the measured correlation functions without any calibration factors. We also show the application of PIE to spFRET measurements, where complexes that have low FRET efficiency can be distinguished from those that do not have an active acceptor.

Analysis of Single-Molecule FRET Trajectories Using Hidden Markov Modeling

Article

Oct 2006

The analysis of single-molecule fluorescence resonance energy transfer (FRET) trajectories has become one of significant biophysical interest. In deducing the transition rates between various states of a system for time-binned data, researchers have relied on simple, but often arbitrary methods of extracting rates from FRET trajectories. Although these methods have proven satisfactory in cases of well-separated, low-noise, two- or three-state systems, they become less reliable when applied to a system of greater complexity. We have developed an analysis scheme that casts single-molecule time-binned FRET trajectories as hidden Markov processes, allowing one to determine, based on probability alone, the most likely FRET-value distributions of states and their interconversion rates while simultaneously determining the most likely time sequence of underlying states for each trajectory. Together with a transition density plot and Bayesian information criterion we can also determine the number of different states present in a system in addition to the state-to-state transition probabilities. Here we present the algorithm and test its limitations with various simulated data and previously reported Holliday junction data. The algorithm is then applied to the analysis of the binding and dissociation of three RecA monomers on a DNA construct.

Determination of the Fraction and Stoichiometry of Femtomolar Levels of Biomolecular Complexes in an Excess of Monomer Using Single-Molecule, Two-Color Coincidence Detection

Article

Dec 2006

We have extended the method of single-molecule fluorescence, two-color coincidence detection (TCCD) to detect coincident events due to a low fraction of a complex against a background of chance coincident events, due to monomers. We developed two complementary methods to determine the number of chance coincident events using the experimental data and without the need for additional experiments. We show that the subtraction of the chance coincidence level is essential for accurate quantification of the relative number of complexes and their stoichiometry. By performing experiments on model samples made from fluorophore-labeled duplex DNA and free dye, a linear dependence on the fraction of duplex DNA was found, independent of the level or ratio of free dye, with quantification down to a level of 0.5% and 500 fM duplex DNA. The method was then used to measure the equilibrium dissociation constant and offrate of a 9-mer duplex DNA, demonstrating the application of this method to systems with nanomolar dissociation constants. These improvements to the method of TCCD analysis significantly extend the application of TCCD to weakly bound complexes and large multicomponent biomolecular systems.

Optimized Threshold Selection for Single-Molecule Two-Color Fluorescence Coincidence Spectroscopy

Article

May 2007

Two-color coincidence detection is a single-molecule fluorescence technique that is capable of resolving subpopulations of biomolecular complexes at very low concentrations. In this paper, we have developed a method that automatically determines appropriate thresholds for the analysis of sets of two-color coincidence data. This has the distinct advantage of allowing the rapid determination of optimized thresholds in a reproducible fashion. The trade-offs involved in such selections are that the thresholds should be high enough both to exceed the background photon count rates and to ensure a low rate of chance coincident events and that they should be low enough to give reasonably high rates of fluorescence events. Previously, thresholds were selected by judgment to balance these various separate considerations. The method reported in this paper incorporates the three factors into the maximization of a single value derived from the data as a function of the thresholds used in the two channels. The value that is maximized is a ratio of event rates, specifically the rate of coincident events above that expected by chance, divided by the total event rate; this is called the association quotient. In this paper, we demonstrate that maximization of the association quotient selects appropriate thresholds for data derived from dual-labeled duplex DNA samples over a range of concentrations and laser powers. This method should allow the application of two-color coincidence detection to more complex biological systems and cells where the sample concentration and background levels are more variable and where it is not possible to run separate control experiments to determine the latter statistics.

Quantum Dot Photon Statistics Measured by Three-Dimensional Particle Tracking

Article

Dec 2007

We present an instrument for performing correlation spectroscopy on single fluorescent particles while tracking their Brownian motion in three dimensions using real-time feedback. By tracking CdSe/ZnS quantum dots in water (diffusion coefficient approximately 20 microm2/s), we make the first measurements of photon antibunching (at approximately 10 ns) on single fluorophores free in solution and find fluorescence lifetime heterogeneity within a quantum dot sample. In addition, we show that 2-mercaptoethanol suppresses short time-scale intermittency (1 ms to 1 s) in quantum dot fluorescence by reducing time spent in the off-state.

Profile of Eric Betzig, Stefan Hell

Jan 2014
2630-2632

J Lippincott-Schwartz
W E Moerner

Lippincott-Schwartz, J. 2015. Profile of Eric Betzig, Stefan Hell, and W. E. Moerner, 2014 Nobel Laureates in Chemistry. Proc. Natl. Acad. Sci. USA. 112:2630-2632.

Fluorescence-aided molecule sorting: analysis of structure and interactions by alternating-laser excitation of single molecules

Jan 2004
8936-8941

A N Kapanidis
N K Lee
S Weiss

Kapanidis, A. N., N. K. Lee,., S. Weiss. 2004. Fluorescence-aided molecule sorting: analysis of structure and interactions by alternating-laser excitation of single molecules. Proc. Natl. Acad. Sci. USA. 101:8936-8941.

phconvert - Library and converter for Photon-HDF5 files

A Ingargiola

Ingargiola, A. phconvert -Library and converter for Photon-HDF5 files. http://photon-hdf5.github.io/phconvert.

Protein conformational dynamics probed by single-molecule electron transfer

Jan 2003
262-266

H Yang
X S Luo
Xie

Yang, H., G. Luo,., X. S. Xie. 2003. Protein conformational dynamics probed by single-molecule electron transfer. Science. 302:262-266.

Fluorescence correlation spectroscopy at micromolar concentrations without optical nanoconfinement

Jan 2014
J PHYS CHEM B
9662-9667

T A Laurence
M A Ly
Coleman

Laurence, T. A., S. Ly,., M. A. Coleman. 2014. Fluorescence correlation spectroscopy at micromolar concentrations without optical nanoconfinement. J. Phys. Chem. B. 118:9662-9667.

Announcing the worldwide Protein Data Bank

Jan 2003
980

Berman

Berman, H., K. Henrick, and H. Nakamura. 2003. Announcing the worldwide Protein Data Bank. Nat. Struct. Biol. 10:980.

FRETBursts - Burst analysis software for smFRET experiments

A Ingargiola

Ingargiola, A. FRETBursts -Burst analysis software for smFRET experiments. http://tritemio.github.io/FRETBursts/.

smFRET simulatotor using 3D Brownian motion and fluorescence emission under confocal excitation

A Ingargiola
Pybromo

Ingargiola, A. PyBroMo, smFRET simulatotor using 3D Brownian motion and fluorescence emission under confocal excitation. http:// tritemio.github.io/PyBroMo/.

Fluorescence-aided molecule sorting: analysis of structure and interactions by alternating-laser excitation of single molecules

Jan 2004
8936

Kapanidis

Computational Tool Photon-HDF5: An Open File Format for Timestamp-Based Single-Molecule Fluorescence Experiments

Abstract

No full-text available

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