Single-molecule Fluorescence Measurements Research Articles

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

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.

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

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.

Bleaching-resistant, Near-continuous Single-molecule Fluorescence and FRET Based on Fluorogenic and Transient DNA Binding.

Photobleaching of fluorescent probes limits the observation span of typical single-molecule fluorescence measurements and hinders observation of dynamics at long timescales. Here, we present a general strategy to circumvent photobleaching by replenishing fluorescent probes via transient binding of fluorogenic DNAs to complementary DNA strands attached to a target molecule. Our strategy allows observation of near-continuous single-molecule fluorescence for more than an hour, a timescale two orders of magnitude longer than the typical photobleaching time of single fluorophores under our conditions. Using two orthogonal sequences, we show that our method is adaptable to Förster Resonance Energy Transfer (FRET) and that can be used to study the conformational dynamics of dynamic structures, such as DNA Holliday junctions, for extended periods. By adjusting the temporal resolution and observation span, our approach enables capturing the conformational dynamics of proteins and nucleic acids over a wide range of timescales.

The energetics of silent translocation through a biological nanopore.

Electrophoretically driving molecules through the lumen of a nanopore can produce transient channel conductance fluctuations by occlusion of ionic flow. However, many molecules do not possess the necessary size, shape, charge, or binding affinity to produce a detectable electrical signature. For cases where transport is quick (e.g., <20 μs), or transport is exceedingly rare, or critical interaction with the pore is lacking, the resulting current recordings appear silent. Within the performance range of common electrophysiological amplifiers, these recordings show no excess noise and no momentary electrical blockage. In general, there is no electrical way to discern if translocation occurs. Here, we report on the electrically silent passage of fluorescent dye molecules through the alpha hemolysin (αHL) nanopore. Simultaneous single-molecule fluorescence and electrical measurements in bilayers formed on microcavity electrode arrays (MECA-opto chips) demonstrate that translocation does, indeed, take place, albeit infrequently. We describe the methodology for detecting fluorophore transport, report observations of silent translocations as a function of time, dye concentration, and nanopore population in the bilayer. Lastly, measurements of the translocation rate as a function of applied potential permit estimation of the energy barrier for transport through the pore, as well as the effective charge on the dye. The energetics of silent translocation can only be revealed using measurement techniques that complement traditional electrical recording.

The nucleosome unwrapping free energy landscape defines distinct regions of transcription factor accessibility and kinetics.

Transcription factors (TF) require access to target sites within nucleosomes to initiate transcription. The target site position within the nucleosome significantly influences TF occupancy, but how is not quantitatively understood. Using ensemble and single-molecule fluorescence measurements, we investigated the targeting and occupancy of the transcription factor, Gal4, at different positions within the nucleosome. We observe a dramatic decrease in TF occupancy to sites extending past 30 base pairs (bp) into the nucleosome which cannot be explained by changes in the TF dissociation rate or binding site orientation. Instead, the nucleosome unwrapping free energy landscape is the primary determinant of Gal4 occupancy by reducing the Gal4 binding rate. The unwrapping free energy landscape defines two distinct regions of accessibility and kinetics with a boundary at 30 bp into the nucleosome where the inner region is over 100-fold less accessible. The Gal4 binding rate in the inner region no longer depends on its concentration because it is limited by the nucleosome unwrapping rate, while the frequency of nucleosome rewrapping decreases because Gal4 exchanges multiple times before the nucleosome rewraps. Our findings highlight the importance of the nucleosome unwrapping free energy landscape on TF occupancy and dynamics that ultimately influences transcription initiation.

Surface Preparation for Single-Molecule Fluorescence Imaging in Organic Solvents.

The development of single-molecule techniques provides opportunities to investigate the properties and heterogeneities of individual molecules, which are almost impossible to be obtained in ensemble measurements. Recently, single-molecule fluorescence microscopy is being applied more and more to study chemical reactions in organic solvents. However, little has been done to optimize the surface preparation procedures for single-molecule fluorescence imaging in organic solvents. In this work, we developed a method to prepare the surface for single-molecule fluorescence imaging in organic solvents with a well-controlled surface density of chemically immobilized dye molecules and a low density of nonspecifically adsorbed impurities. We also compared the surfaces prepared by two different procedures and studied the impacts of the polarities of the solvent and the surface functionality on the quality of prepared surface. We found that higher polarities of both the solvent and the surface functionality provided better control of the surface density of chemically immobilized dyes and helped reduce the nonspecific adsorption of both dyes and fluorescent impurities in organic solvents. We further performed single-molecule fluorescence imaging in DMF and investigated the photophysical properties of dyes and fluorescent impurities, which could be used to filter out false counts in single-molecule fluorescence measurements.

Multiplexing single-molecule fluorescence measurements with spectroscopically diverse DNA-based FRET constructs

A fundamental challenge for single-molecule fluorescence techniques is simultaneous measurement of multiple analyte species with unique fluorescent labels. Although structurally different fluorophores could be used to create a set of labels with unique photophysical properties, they would also vary greatly in their chemical compatibility, and families of chemically similar fluorophores are limited in number. We have engineered a series of nanoscale DNA-FRET constructs that position the same donor-acceptor fluorophores (internal Cy3 / Cy5 integrated into the phosphate backbone) with different spacing and sequence contexts, which can be used as a large set of fluorescent labels to enable multiplexing of single-molecule fluorescence measurements.

Autophosphorylation of the KaiC-like protein ArlH inhibits oligomerization and interaction with ArlI, the motor ATPase of the archaellum.

Motile archaea are propelled by the archaellum, whose motor complex consists of the membrane protein ArlJ, the ATPase ArlI, and the ATP-binding protein ArlH. Despite its essential function and the existence of structural and biochemical data on ArlH, the role of ArlH in archaellum assembly and function remains elusive. ArlH is a structural homolog of KaiC, the central component of the cyanobacterial circadian clock. Since autophosphorylation and dephosphorylation of KaiC are central properties for the function of KaiC, we asked whether autophosphorylation is also a property of ArlH proteins. We observed that both ArlH from the euryarchaeon Pyrococcus furiosus (PfArlH) and from the crenarchaeon Sulfolobus acidocaldarius (SaArlH) have autophosphorylation activity. Using a combination of single-molecule fluorescence measurements and biochemical assays, we show that autophosphorylation of ArlH is closely linked to its oligomeric state when bound to hexameric ArlI. These experiments also strongly suggest that ArlH is a hexamer in its ArlI-bound state. Mutagenesis of the putative catalytic residue (Glu-57 in SaArlH) in ArlH results in a reduced autophosphorylation activity and abolished archaellation and motility in S. acidocaldarius, indicating that optimum phosphorylation activity of ArlH is essential for archaellation and motility.

Modern Kinetics and Mechanism of Protein Folding: A Retrospective.

Modern experimental kinetics of protein folding began in the early 1990s with the introduction of nanosecond laser pulses to trigger the folding reaction, providing an almost 106-fold improvement in time resolution over the stopped-flow method being employed at the time. These experiments marked the beginning of the “fast-folding” subfield that enabled investigation of the kinetics of formation of secondary structural elements and disordered loops for the first time, as well as the fastest folding proteins. When I started to work on this subject, a fast folding protein was one that folded in milliseconds. There were, moreover, no analytical theoretical models and no atomistic or coarse-grained molecular dynamics simulations to describe the mechanism. Two of the most important discoveries from my lab since then are a protein that folds in hundreds of nanoseconds, as determined from nanosecond laser temperature experiments, and the discovery that the theoretically predicted barrier crossing time is about the same for proteins that differ in folding rates by 104-fold, as determined from single molecule fluorescence measurements. We also developed what has been called the “Hückel model” of protein folding, which quantitatively explains a wide range of equilibrium and kinetic measurements. This retrospective traces the history of contributions to the “fast folding” subfield from my lab until about 3 years ago, when I left protein folding to spend the rest of my research career trying to discover an inexpensive drug for treating sickle cell disease.

Hopping and Flipping of RNA Polymerase on DNA during Recycling for Reinitiation after Intrinsic Termination in Bacterial Transcription.

Two different molecular mechanisms, sliding and hopping, are employed by DNA-binding proteins for their one-dimensional facilitated diffusion on nonspecific DNA regions until reaching their specific target sequences. While it has been controversial whether RNA polymerases (RNAPs) use one-dimensional diffusion in targeting their promoters for transcription initiation, two recent single-molecule studies discovered that post-terminational RNAPs use one-dimensional diffusion for their reinitiation on the same DNA molecules. Escherichia coli RNAP, after synthesizing and releasing product RNA at intrinsic termination, mostly remains bound on DNA and diffuses in both forward and backward directions for recycling, which facilitates reinitiation on nearby promoters. However, it has remained unsolved which mechanism of one-dimensional diffusion is employed by recycling RNAP between termination and reinitiation. Single-molecule fluorescence measurements in this study reveal that post-terminational RNAPs undergo hopping diffusion during recycling on DNA, as their one-dimensional diffusion coefficients increase with rising salt concentrations. We additionally find that reinitiation can occur on promoters positioned in sense and antisense orientations with comparable efficiencies, so reinitiation efficiency depends primarily on distance rather than direction of recycling diffusion. This additional finding confirms that orientation change or flipping of RNAP with respect to DNA efficiently occurs as expected from hopping diffusion.

Solid-phase XRN1 reactions for RNA cleavage: application in single-molecule sequencing.

Modifications in RNA are numerous (∼170) and in higher numbers compared to DNA (∼5) making the ability to sequence an RNA molecule to identify these modifications highly tenuous using next generation sequencing (NGS). The ability to immobilize an exoribonuclease enzyme, such as XRN1, to a solid support while maintaining its activity and capability to cleave both the canonical and modified ribonucleotides from an intact RNA molecule can be a viable approach for single-molecule RNA sequencing. In this study, we report an enzymatic reactor consisting of covalently attached XRN1 to a solid support as the groundwork for a novel RNA exosequencing technique. The covalent attachment of XRN1 to a plastic solid support was achieved using EDC/NHS coupling chemistry. Studies showed that the solid-phase digestion efficiency of model RNAs was 87.6 ± 2.8%, while the XRN1 solution-phase digestion for the same model was 78.3 ± 4.4%. The ability of immobilized XRN1 to digest methylated RNA containing m6A and m5C ribonucleotides was also demonstrated. The processivity and clipping rate of immobilized XRN1 secured using single-molecule fluorescence measurements of a single RNA transcript demonstrated a clipping rate of 26 ± 5 nt s−1 and a processivity of >10.5 kb at 25°C.

Single-molecule fluorescence detection of a tricyclic nucleoside analogue.

Fluorescent nucleobase surrogates capable of Watson–Crick hydrogen bonding are essential probes of nucleic acid structure and dynamics, but their limited brightness and short absorption and emission wavelengths have rendered them unsuitable for single-molecule detection. Aiming to improve on these properties, we designed a new tricyclic pyrimidine nucleoside analogue with a push–pull conjugated system and synthesized it in seven sequential steps. The resulting C-linked 8-(diethylamino)benzo[b][1,8]naphthyridin-2(1H)-one nucleoside, which we name ABN, exhibits ε442 = 20 000 M−1 cm−1 and Φem,540 = 0.39 in water, increasing to Φem = 0.50–0.53 when base paired with adenine in duplex DNA oligonucleotides. Single-molecule fluorescence measurements of ABN using both one-photon and two-photon excitation demonstrate its excellent photostability and indicate that the nucleoside is present to > 95% in a bright state with count rates of at least 15 kHz per molecule. This new fluorescent nucleobase analogue, which, in duplex DNA, is the brightest and most red-shifted known, is the first to offer robust and accessible single-molecule fluorescence detection capabilities.

Dependence of Fluorescence Quenching of CY3 Oligonucleotide Conjugates on the Oxidation Potential of the Stacking Base Pair.

To understand the complex fluorescence properties of astraphloxin (CY3)-labelled oligonucleotides, it is necessary to take into account the redox properties of the nucleobases. In oligonucleotide hybrids, we observed a dependence of the fluorescence intensity on the oxidation potential of the neighbouring base pair. For the series I < A < G < 8-oxoG, the extent of fluorescence quenching follows the trend of decreasing oxidation potentials. In a series of 7 nt hybrids, stacking interactions of CY3 with perfect match and mismatch base pairs were found to stabilise the hybrid by 7–8 kJ/mol. The fluorescence measurements can be explained by complex formation resulting in fluorescence quenching that prevails over the steric effect of a reduced excited state trans-cis isomerisation, which was expected to increase the fluorescence efficiency of the dye when stacking to a base pair. This can be explained by the fact that, in a double strand, base pairing and stacking cause a dramatic change in the oxidation potential of the nucleobases. In single-molecule fluorescence measurements, the oxidation of G to 8-oxoG was observed as a result of photoinduced electron transfer and subsequent chemical reactions. Our results demonstrate that covalently linked CY3 is a potent oxidant towards dsDNA. Sulfonated derivatives should be used instead.

Quantitative comparison between sub-millisecond time resolution single-molecule FRET measurements and 10-second molecular simulations of a biosensor protein.

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

Structural basis for the transition from translation initiation to elongation by an 80S-eIF5B complex

Recognition of a start codon by the initiator aminoacyl-tRNA determines the reading frame of messenger RNA (mRNA) translation by the ribosome. In eukaryotes, the GTPase eIF5B collaborates in the correct positioning of the initiator Met-tRNAiMet on the ribosome in the later stages of translation initiation, gating entrance into elongation. Leveraging the long residence time of eIF5B on the ribosome recently identified by single-molecule fluorescence measurements, we determine the cryoEM structure of the naturally long-lived ribosome complex with eIF5B and Met-tRNAiMet immediately before transition into elongation. The structure uncovers an unexpected, eukaryotic specific and dynamic fidelity checkpoint implemented by eIF5B in concert with components of the large ribosomal subunit.

Extending the photobleaching lifetime in the presence of an optical tweezers by wavefront engineering

Here we lay the foundations for a novel approach to simultaneously performing optical force spectroscopy and single-molecule fluorescence measurements by adapting techniques from wavefront engineering. Our method relies upon the axial displacement (i.e. along the direction of propagation of the trap laser) of an appropriately constructed Laguerre-Gaussian (LG), or ‘donut’ beam, to generate an optical force on a surface tethered microsphere. With this geometry, one maintains a region of near-infrared (IR) light below the microsphere, where a biomolecule of interest can be placed, that is low enough in intensity to not significantly interfere with fluorescence measurements. We are thus able to separate the fluorescence illumination from the trap light without having to physically separate the two by several micrometers, or employ long DNA linker molecules as handles. As a proof of principal, we show that within the region of low IR intensity, directly below the trap center, the photobleaching lifetime of the organic dye Alexa-647, which is highly sensitive to the intense trap light, is significantly extended.

Electron Transfer in a Naphthalene Diimide System Studied by Single-Molecule Delayed Fluorescence

Electron transfer (ET) is a key chemical reaction in nature and has been extensively studied in bulk systems, but remains challenging to investigate at the single-molecule level. A previously reported naphthalene diimide (NDI)-based system (Higginbotham et al., Chem. Commun. 2013, 49, 5061–5063) displays delayed fluorescence with good quantum yield (~0.5) and long-lived (nanoseconds) prompt and delayed fluorescence lifetimes, providing an opportunity to interrogate the underlying ET processes in single molecules. Time-resolved single-molecule fluorescence measurements enabled forward and reverse ET rate constants to be calculated for 45 individual molecules embedded in poly(methylmethacrylate) (PMMA) film. Interpretation of the results within the framework of Marcus–Hush theory for ET demonstrates that variation in both the electronic coupling and the driving force for ET is occurring from molecule to molecule within the PMMA film and over time for individual molecules.

Enzymatic Labeling of Bacterial Proteins for Super-resolution Imaging in Live Cells

Methods that enable the super-resolution imaging of intracellular proteins in live bacterial cells provide powerful tools for the study of prokaryotic cell biology. Photoswitchable organic dyes exhibit many of the photophysical properties needed for super-resolution imaging, including high brightness, photostability, and photon output, but most such dyes require organisms to be fixed and permeabilized if intracellular targets are to be labeled. We recently reported a general strategy for the chemoenzymatic labeling of bacterial proteins with azide-bearing fatty acids in live cells using the eukaryotic enzyme N-myristoyltransferase. Here we demonstrate the labeling of proteins in live Escherichia coli using cell-permeant bicyclononyne-functionalized photoswitchable rhodamine spirolactams. Single-molecule fluorescence measurements on model rhodamine spirolactam salts show that these dyes emit hundreds of photons per switching event. Super-resolution imaging was performed on bacterial chemotaxis proteins Tar and CheA and cell division proteins FtsZ and FtsA. High-resolution imaging of Tar revealed a helical pattern; imaging of FtsZ yielded banded patterns dispersed throughout the cell. The precision of radial and axial localization in reconstructed images approaches 15 and 30 nm, respectively. The simplicity of the method, which does not require redox imaging buffers, should make this approach broadly useful for imaging intracellular bacterial proteins in live cells with nanometer resolution.

Supported Solid Lipid Bilayers as a Platform for Single-Molecule Force Measurements.

Biocompatible surfaces are important for basic and applied research in life science with experiments ranging from the organismal to the single-molecule level. For the latter, examples include the translocation of kinesin motor proteins along microtubule cytoskeletal filaments or the study of DNA-protein interactions. Such experiments often employ single-molecule fluorescence or force microscopy. In particular for force measurements, a key requirement is to prevent nonspecific interactions of biomolecules and force probes with the surface, while providing specific attachments that can sustain loads. Common approaches to reduce nonspecific interactions include supported lipid bilayers or PEGylated surfaces. However, fluid lipid bilayers do not support loads and PEGylation may require harsh chemical surface treatments and have limited reproducibility. Here, we developed and applied a supported solid lipid bilayer (SSLB) as a platform for specific, load bearing attachments with minimal nonspecific interactions. Apart from single-molecule fluorescence measurements, anchoring molecules to lipids in the solid phase enabled us to perform force measurements of molecular motors and overstretch DNA. Furthermore, using a heating laser, we could switch the SSLB to its fluid state allowing for manipulation of anchoring points. The assay had little nonspecific interactions, was robust, reproducible, and time-efficient, and required less hazardous and toxic chemicals for preparation. In the long term, we expect that SSLBs can be widely employed for single-molecule fluorescence microscopy, force spectroscopy, and cellular assays in mechanobiology.

Excitonic Processes in a Conjugated Polyelectrolyte Complex

In aqueous solution, a di-sulfonated phenylenevinylene polymer (DPS-PPV) forms a complex with non-ionic poly(vinyl alcohol) (PVA) leading to absorption spectroscopic shifts and a dramatic (6-fold) increase in DPS-PPV fluorescence intensity. Spectroscopic investigations demonstrate that the complexation with PVA and other neutral polymers results in conformational changes in the DPS-PPV chains that lead to the removal of non-fluorescent energy traps and results in the observed increase in fluorescence in the bulk solution. Single molecule fluorescence measurements of DPS-PPV chains dispersed on glass and in PVA films confirm that efficient exciton energy transfer occurs within each photo-excited DPS-PPV chain and that the observed increase in fluorescence intensity in the PVA film environment is also associated with fewer quenching sites. The results highlight the importance of conjugated polyelectrolyte conformation on exciton relaxation pathways.