Anti-Brownian ELectrokinetic Research Articles

Quantifying Microsecond Solution-Phase Conformational Dynamics of a DNA Hairpin at the Single-Molecule Level.

Quantifying the rapid conformational dynamics of biological systems is fundamental to understanding the mechanism. However, biomolecules are complex, often containing static and dynamic heterogeneity, thus motivating the use of single-molecule methods, particularly those that can operate in solution. In this study, we measure microsecond conformational dynamics of solution-phase DNA hairpins at the single-molecule level using an anti-Brownian electrokinetic (ABEL) trap. Different conformational states were distinguished by their fluorescence lifetimes, and kinetic parameters describing transitions between these states were determined using two-dimensional fluorescence lifetime correlation (2DFLCS) analysis. Rather than combining fluorescence signals from the entire data set ensemble, long observation times of individual molecules allowed ABEL-2DFLCS to be performed on each molecule independently, yielding the underlying distribution of the system's kinetic parameters. ABEL-2DFLCS on the DNA hairpins resolved an underlying heterogeneity of fluorescence lifetimes and provided signatures of two-state exponential dynamics with rapid (<millisecond) transition times between states without observation of the substantially stretched exponential kinetics that had been observed in previous measurements on diffusing molecules. Numerical simulations were performed to validate the accuracy of this technique and the effects the underlying heterogeneity has on the analysis. Finally, ABEL-2DFLCS was performed on a mixture of hairpins and used to resolve their kinetic data.

Probing single-molecule DNA-protein interactions using ABEL-FRET and ABEL-PIFE.

Single-molecule FRET and PIFE (protein induced fluorescence enhancement) are powerful techniques to probe molecular conformation and binding kinetics of DNA-protein interactions. We combine these techniques with Anti-Brownian Electrokinetic (ABEL) trapping to establish new modalities called ABEL-FRET and ABEL-PIFE. We demonstrate that ABEL-FRET achieves ultrahigh, shot-noise limited resolution of smFRET efficiency in solution without surface tethering. ABEL-PIFE enables simultaneous mapping of binding stoichiometry and sequence context of binding location. We highlight these new capabilities using examples of DNA processing enzymes.

Ratiometric sensing of redox environments inside individual carboxysomes trapped in solution.

The rapid diffusion of biological nanoparticles in solution impedes our ability to continuously monitor individual particles and measure their physical and chemical properties. To overcome this, we previously developed the interferometric scattering anti-Brownian electrokinetic (ISABEL) trap, which uses scattering to localize a particle and applies electrokinetic forces to counteract Brownian motion, thus enabling extended observation. Here we present an improved ISABEL trap that incorporates a near-infrared scatter illumination beam and rapidly interleaves 405- and 488-nm fluorescence excitation beams, and apply this trap to the study of carboxysomes. Carboxysomes are bacterial microcompartments that self-assemble to sequester the enzyme rubisco and provide a favorable chemical environment for the fixation of carbon dioxide. With the ISABEL trap, we monitored the internal redox environment of individual carboxysomes labeled with the ratiometric redox reporter roGFP2. Carboxysomes were observed to vary widely in both redox-dependent fluorescence and scattering contrast (reporting on mass). Furthermore, we used redox sensing to explore the chemical kinetics within intact carboxysomes, where bulk measurements may contain unwanted contributions from aggregates or interfering fluorescent proteins. Overall, we demonstrate the ISABEL trap's ability to sensitively monitor chemical information inside single nanoscale biological objects, enabling new experiments on these systems.

Exploring Masses and Internal Mass Distributions of Single Carboxysomes in Free Solution Using Fluorescence and Interferometric Scattering in an Anti-Brownian Trap.

Carboxysomes are self-assembled bacterial microcompartments that facilitate carbon assimilation by colocalizing the enzymes of CO2 fixation within a protein shell. These microcompartments can be highly heterogeneous in their composition and filling, so measuring the mass and loading of an individual carboxysome would allow for better characterization of its assembly and function. To enable detailed and extended characterizations of single nanoparticles in solution, we recently demonstrated an improved interferometric scattering anti-Brownian electrokinetic (ISABEL) trap, which tracks the position of a single nanoparticle via its scattering of a near-infrared beam and applies feedback to counteract its Brownian motion. Importantly, the scattering signal can be related to the mass of nanoscale proteinaceous objects, whose refractive indices are well-characterized. We calibrate single-particle scattering cross-section measurements in the ISABEL trap and determine individual carboxysome masses in the 50-400 MDa range by analyzing their scattering cross sections with a core-shell model. We further investigate carboxysome loading by combining mass measurements with simultaneous fluorescence reporting from labeled internal components. This method may be extended to other biological objects, such as viruses or extracellular vesicles, and can be combined with orthogonal fluorescence reporters to achieve precise physical and chemical characterization of individual nanoscale biological objects.

Ratiometric Sensing of Redox Environments Inside Individual Carboxysomes Trapped in Solution.

Diffusion of biological nanoparticles in solution impedes our ability to continuously monitor individual particles and measure their physical and chemical properties. To overcome this, we previously developed the interferometric scattering anti-Brownian electrokinetic (ISABEL) trap, which uses scattering to localize a particle and applies electrokinetic forces that counteract Brownian motion, thus enabling extended observation. Here we present an improved ISABEL trap that incorporates a near-infrared scatter illumination beam and rapidly interleaves 405 and 488 nm fluorescence excitation reporter beams. With the ISABEL trap, we monitored the internal redox environment of individual carboxysomes labeled with the ratiometric redox reporter roGFP2. Carboxysomes widely vary in scattering contrast (reporting on size) and redox-dependent ratiometric fluorescence. Furthermore, we used redox sensing to explore the chemical kinetics within intact carboxysomes, where bulk measurements may contain unwanted contributions from aggregates or interfering fluorescent proteins. Overall, we demonstrate the ISABEL trap’s ability to sensitively monitor nanoscale biological objects, enabling new experiments on these systems.

A bottom-up perspective on photodynamics and photoprotection in light-harvesting complexes using anti-Brownian trapping.

Single-molecule fluorescence spectroscopy allows direct, real-time observation of dynamic photophysical changes in light harvesting complexes. The Anti-Brownian ELectrokinetic (ABEL) trap is one such single-molecule method with useful advantages. This approach is particularly well-suited to make detailed spectroscopic measurements of pigment-protein complexes in a solution phase because it enables extended-duration single-molecule observation by counteracting Brownian motion. This Perspective summarizes recent contributions by the authors and others that have utilized the unique capabilities of the ABEL trap to advance our understanding of phycobiliproteins and the phycobilisome complex, the primary light-harvesting apparatus of cyanobacteria. Monitoring the rich spectroscopic data from these measurements, which include brightness, fluorescence lifetime, polarization, and emission spectra, among other measurable parameters, has provided direct characterization of pigments and energy transfer pathways in the phycobilisome, spanning scales from single pigments and monomeric phycobiliproteins to higher order oligomers and protein-protein interactions of the phycobilisome complex. Importantly, new photophysical states and photodynamics were observed to modulate the flow of energy through the phycobilisome and suggest a previously unknown complexity in phycobilisome light harvesting and energy transport with a possible link to photoadaptive or photoprotective functions in cyanobacteria. Beyond deepening our collective understanding of natural light-harvesting systems, these and future discoveries may serve as inspiration for engineering improved artificial light-harvesting technologies.

Classification of DNA-FRET based single-molecule spectroscopic labels in anti-Brownian electrokinetic (ABEL) trap

Fluorescent tags are usually distinguished by their color, making fluorescence multiplexing at the single-molecule level challenging since only a few tags can be distinguished due to the overlap in their emission spectra and the low signal-to-noise ratios inherent in single-molecule measurements. We have engineered a series of spectroscopically distinguishable single-molecule labels whose emission is characterized by unique combinations of photophysical parameters including brightness, lifetime, and emission spectra.

Characterizing physical properties of single carboxysomes in the Interferometric Scattering Anti-Brownian ELectrokinetic trap

Anti-Brownian traps enable extended study of single particles in free solution by closed-loop feedback forces that directly counteract Brownian motion monitored by fluorescence. The longer observation times of individual particles allow for detailed characterization of photophysical properties, dynamics, and infrequent events. We have recently invented the Interferometric Scattering Anti-Brownian ELectrokinetic (ISABEL) trap, which uses scattered light in place of fluorescence to detect the particle position for feedback.

Interferometric Scattering Enables Fluorescence-Free Electrokinetic Trapping of Single Nanoparticles in Free Solution.

Anti-Brownian traps confine single particles in free solution by closed-loop feedback forces that directly counteract Brownian motion. Extended-duration measurements on trapped objects allow detailed characterization of photophysical and transport properties as well as observation of infrequent or rare dynamics. However, this approach has been generally limited to particles that can be tracked by fluorescence emission. Here we present the Interferometric Scattering Anti-Brownian ELectrokinetic (ISABEL) trap, which uses interferometric scattering rather than fluorescence to monitor particle position. By decoupling the ability to track (and therefore trap) a particle from collection of its spectroscopic data, the ISABEL trap enables confinement and extended study of single particles that do not fluoresce, only weakly fluoresce, or exhibit intermittent fluorescence or photobleaching. This new technique significantly expands the range of nanoscale objects that may be investigated at the single-particle level in free solution.

Single-molecule trapping and spectroscopy reveals photophysical heterogeneity of phycobilisomes quenched by Orange Carotenoid Protein

The Orange Carotenoid Protein (OCP) is a cytosolic photosensor that is responsible for non-photochemical quenching (NPQ) of the light-harvesting process in most cyanobacteria. Upon photoactivation by blue-green light, OCP binds to the phycobilisome antenna complex, providing an excitonic trap to thermally dissipate excess energy. At present, both the binding site and NPQ mechanism of OCP are unknown. Using an Anti-Brownian ELectrokinetic (ABEL) trap, we isolate single phycobilisomes in free solution, both in the presence and absence of activated OCP, to directly determine the photophysics and heterogeneity of OCP-quenched phycobilisomes. Surprisingly, we observe two distinct OCP-quenched states, with lifetimes 0.09 ns (6% of unquenched brightness) and 0.21 ns (11% brightness). Photon-by-photon Monte Carlo simulations of exciton transfer through the phycobilisome suggest that the observed quenched states are kinetically consistent with either two or one bound OCPs, respectively, underscoring an additional mechanism for excitation control in this key photosynthetic unit.

Tetherless, Precise and Extended Observation of Single-Molecule FRET in an Anti-Brownian Trap

A comprehensive understanding of biomolecules calls for the ability to observe single-molecule dynamics at the nanometer scale without constraints. Single-molecule Forster resonance energy transfer (smFRET) is a powerful tool for probing nanoscale dynamics, but existing modalities have limitations. Solution based confocal measurements are restricted by the short (~1ms) diffusion limited observation time. Surface immobilized measurements can extend the observation window, but at the expense of the molecule’s translational and rotational degrees of freedom. Moreover, there is always a concern that immobilization may perturb the biomolecule’s function. We overcome these limitations by combining smFRET optics with the capability to isolate individual molecules in solution using an Anti-Brownian ELectrokinetic (ABEL) trap. Our new platform, ABEL-FRET, enables photon-by-photon recording of smFRET trajectories over tens of seconds in solution, without tethering the molecule to a surface. We first demonstrate ABELFRET using short (~10bp) DNA rulers and achieve near shot-noise limited precision of ΔE~0.01 for 5,000 photons, which enables resolution of single base pair differences in a mixture of FRET-labeled dsDNA molecules. We also demonstrate the capability to make simultaneous measurements of donor fluorescence lifetime and smFRET.

Resolving Mixtures in Solution by Single-Molecule Rotational Diffusivity.

Sensing the size of individual molecules in an ensemble has proven to be a powerful tool to investigate biomolecular interactions and association-dissociation processes. In biologically relevant solution environments, molecular size is often sensed by translational or rotational diffusivity. The rotational diffusivity is more sensitive to the size and conformation of the molecules as it is inversely proportional to the cube of the hydrodynamic radius, as opposed to the inverse linear dependence of the translational diffusion coefficient. Single-molecule rotational diffusivity has been measured with time-resolved fluorescence anisotropy decay, but the ability to sense different sizes has been restricted by the limited number of photons available or has required surface attachment to observe each molecule longer, and the attachment may be perturbative. To address these limitations, we show how to measure and monitor single-molecule rotational diffusivity by combining the solution-phase Anti-Brownian ELectrokinetic (ABEL) trap and maximum likelihood analysis of time-resolved fluorescence anisotropy based on the information inherent in each detected photon. We demonstrate this approach by resolving a mixture of single- and double-stranded fluorescently labeled DNA molecules at equilibrium, freely rotating in a native solution environment. The rotational diffusivity, fluorescence brightness and lifetime, and initial and steady-state anisotropy are simultaneously determined for each trapped single DNA molecule. The time resolution and precision of this method are analyzed using statistical signal analysis and simulations. We present key parameters that define the usefulness of a particular fluorescent label for extracting molecular size information from single-molecule rotational diffusivity measurements.

Single-Molecule Measurements of Quenching and Photophysical Heterogeneity in Phycobiliproteins

Phycobilisomes, the membrane-associated light-harvesting antenna system of cyanobacteria, dynamically adjust to changing irradiation by modulating energy capture and transfer over a few seconds or longer, for example by genetic regulation, structural reorganization, or non-photochemical quenching by Orange Carotenoid Protein (OCP). Recent observation of excitation-dependent photodynamics in single intact surface-immobilized phycobilisomes, including “blinking” to dark or dim states, suggests that the phycobilisome itself may also possess intrinsic molecular mechanisms to regulate energy transfer. Here, we confine single phycobiliproteins in free solution to a small measurement region for several seconds at a time using an Anti-Brownian ELectrokinetic (ABEL) trap, and simultaneously monitor changes in multiple spectroscopic properties (brightness, fluorescence anisotropy, fluorescence lifetime, and emission spectrum). We are able to trap single intact phycobilisomes and isolated substructures thereof, including phycobilisomes quenched by OCP. We find that even the smallest constituent phycobilisome subunits exhibit surprisingly complex photodynamics. For example, in monomers of C-phycocyanin (C-PC), a phycobiliprotein that contains three covalently bound and chemically identical chromophores, we observe reversible switching among at least seven distinct photophysical states (Squires and Moerner, PNAS 2017). The spectroscopic properties of these states and the transitions among them allow us to identify each state as a unique combination of chromophores participating in energy transfer. A simple computational FRET model closely predicts the observed states, and additionally reveals that the primary acceptor chromophore in C-PC may sometimes act as a quencher. The complex photodynamics and quenching that we observe for individual subunits of the phycobilisome are likely one source of the dynamics observed for the full phycobilisome complex, and may provide an intrinsic mechanism for short-timescale modulation of energy transfer to the reaction center.

Revealing Conformational Variants of Solution-Phase Intrinsically Disordered Tau Protein at the Single-Molecule Level.

Intrinsically disordered proteins, such as tau protein, adopt a variety of conformations in solution, complicating solution-phase structural studies. We employed an anti-Brownian electrokinetic (ABEL) trap to prolong measurements of single tau proteins in solution. Once trapped, we recorded the fluorescence anisotropy to investigate the diversity of conformations sampled by the single molecules. A distribution of anisotropy values obtained from trapped tau protein is conspicuously bimodal while those obtained by trapping a globular protein or individual fluorophores are not. Time-resolved fluorescence anisotropy measurements were used to provide an explanation of the bimodal distribution as originating from a shift in the compaction of the two different families of conformations.

Revealing Conformational Variants of Solution‐Phase Intrinsically Disordered Tau Protein at the Single‐Molecule Level

AbstractIntrinsically disordered proteins, such as tau protein, adopt a variety of conformations in solution, complicating solution‐phase structural studies. We employed an anti‐Brownian electrokinetic (ABEL) trap to prolong measurements of single tau proteins in solution. Once trapped, we recorded the fluorescence anisotropy to investigate the diversity of conformations sampled by the single molecules. A distribution of anisotropy values obtained from trapped tau protein is conspicuously bimodal while those obtained by trapping a globular protein or individual fluorophores are not. Time‐resolved fluorescence anisotropy measurements were used to provide an explanation of the bimodal distribution as originating from a shift in the compaction of the two different families of conformations.

Direct single-molecule measurements of phycocyanobilin photophysics in monomeric C-phycocyanin

Phycobilisomes are highly organized pigment-protein antenna complexes found in the photosynthetic apparatus of cyanobacteria and rhodophyta that harvest solar energy and transport it to the reaction center. A detailed bottom-up model of pigment organization and energy transfer in phycobilisomes is essential to understanding photosynthesis in these organisms and informing rational design of artificial light-harvesting systems. In particular, heterogeneous photophysical behaviors of these proteins, which cannot be predicted de novo, may play an essential role in rapid light adaptation and photoprotection. Furthermore, the delicate architecture of these pigment-protein scaffolds sensitizes them to external perturbations, for example, surface attachment, which can be avoided by study in free solution or in vivo. Here, we present single-molecule characterization of C-phycocyanin (C-PC), a three-pigment biliprotein that self-assembles to form the midantenna rods of cyanobacterial phycobilisomes. Using the Anti-Brownian Electrokinetic (ABEL) trap to counteract Brownian motion of single particles in real time, we directly monitor the changing photophysical states of individual C-PC monomers from Spirulina platensis in free solution by simultaneous readout of their brightness, fluorescence anisotropy, fluorescence lifetime, and emission spectra. These include single-chromophore emission states for each of the three covalently bound phycocyanobilins, providing direct measurements of the spectra and photophysics of these chemically identical molecules in their native protein environment. We further show that a simple Förster resonant energy transfer (FRET) network model accurately predicts the observed photophysical states of C-PC and suggests highly variable quenching behavior of one of the chromophores, which should inform future studies of higher-order complexes.

Single Molecule Studies of Tau Protein in the Abel Trap

Tau protein is an intrinsically disordered protein that stabilizes microtubules of neurons mainly in the central nervous system. Abnormalities in Tau are thought to be involved in Alzheimer's disease. The aggregation process of monomeric Tau into an oligomeric species and the intermediate conformational states that may facilitate aggregation are of particular interest. To investigate the disordered nature and aggregation of Tau protein, single, solution-phase Tau proteins and oligomers were isolated in a microfluidic trap, called the Anti-Brownian Electrokinetic (ABEL) Trap. The ABEL trap cancels Brownian motion of a molecule of interest via electrokinetic and/or electroosmotic flow, allowing prolonged examination of a single, fluorescently-labeled analyte in solution. Combining this technique with fluorescence anisotropy enabled studies into how monomer structure and aggregation changes with cellular conditions known to accelerate aggregation, including concentration effects, solution viscosity, heparin, and cellular crowding. Characteristics of disordered Tau were compared with those of a globular protein of similar size, microbial transglutaminase (MTG). The ultimate goal is to provide the missing link between non-aggregated, monomer Tau protein and the toxic species that result in devastating disease, offering insight into how this pathway may be upset.

ChemInform Abstract: Single‐Molecule Spectroscopy of Photosynthetic Proteins in Solution: Exploration of Structure—Function Relationships

AbstractReview: 43 refs.

Elucidation of the Photodynamics of Single Photosynthetic LH2 Complexes in Solution

Photosynthetic organisms function under low light by converting photoenergy to chemical energy with near-unity quantum efficiency and under high light by dissipating unused photoenergy to prevent formation of deleterious photoproducts. There is widespread interest in how this dual functionality is achieved, because this balance enables efficient light harvesting under the fluctuating intensities of sunlight at the earth's surface. One obstacle to characterizing the molecular mechanisms responsible for this balance is that the excited-state properties of photosynthetic proteins vary drastically between individual proteins (due to static heterogeneity) and even within a single protein over time (due to dynamic heterogeneity). In ensemble measurements, these excited-state properties appear as a static, average value. To overcome this averaging, we investigate light-harvesting complex 2 (LH2), the primary antenna in purple bacteria, at the single-molecule level. Using a novel technique, the Anti-Brownian ELectrokinetic (ABEL) trap, we study individual LH2 complexes in a solution-phase environment to eliminate perturbations due to immobilization schemes, which can alter the protein structure and function. Furthermore, we perform the first simultaneous measurements of fluorescence intensity, lifetime, and emission spectra from individual proteins. We identify three distinct functional conformations of LH2, two of which correspond to a quenched and an unquenched form, and observe transitions occurring between these forms on a timescale of seconds. Our results reveal that individual LH2 complexes undergo photoactivated switching to the quenched state, and thermally revert to the ground state. This is a previously unknown, reversible mechanism for dissipation of excitation energy activated by high light conditions, and may be one component by which photosynthetic organisms flourish under varying light intensities.

Single-molecule spectroscopy of photosynthetic proteins in solution: exploration of structure–function relationships

In photosynthetic light harvesting, absorbed photoenergy transfers through networks of pigment–protein complexes to a central location, known as the reaction center, for conversion to chemical energy. This process occurs with remarkable near-unity quantum efficiency. Pigment–protein complexes exhibit emergent properties very different from those of their component molecules, resulting from interactions among the pigments and between the pigments and the surrounding protein environment. Thus, the precise molecular mechanisms of the energy transport process are complex and coupled, obscuring their molecular origin. Furthermore, because these interactions are sensitive to the molecular structure, they vary from complex to complex and vary in time for each individual complex due to fluctuations of the protein's conformation. To explore the effect of complex-to-complex variation, we study individual photosynthetic proteins, one at a time. We use a solution-phase, single-molecule technique, known as the Anti-Brownian Electrokinetic (ABEL) trap, to elucidate the conformational dynamics of single photosynthetic pigment–protein complexes without introducing additional perturbations from immobilization strategies. After reviewing the principles of the ABEL trap, we demonstrate its application to the study of several photosynthetic pigment–protein complexes. We demonstrate that the ABEL trap approach can lead to an increased understanding of photosynthetic complexes by presenting three examples: (1) analysis of photodegradation pathways, (2) characterization of complex-to-complex heterogeneity, and (3) identification of distinct functional forms.