Single-molecule Spectroscopy Studies Research Articles

Recent advances in single-molecule spectroscopy studies on light-harvesting processes in oxygenic photosynthesis

Photosynthetic light-harvesting complexes (LHCs) play a crucial role in concentrating the photon energy from the sun that otherwise excites a typical pigment molecule, such as chlorophyll-a, only several times a second. Densely packed pigments in the complexes ensure efficient energy transfer to the reaction center. At the same time, LHCs have the ability to switch to an energy-quenching state and thus play a photoprotective role under excessive light conditions. Photoprotection is especially important for oxygenic photosynthetic organisms because toxic reactive oxygen species can be generated through photochemistry under aerobic conditions. Because of the extreme complexity of the systems in which various types of pigment molecules strongly interact with each other and with the surrounding protein matrixes, there has been long-standing difficulty in understanding the molecular mechanisms underlying the flexible switching between the light-harvesting and quenching states. Single-molecule spectroscopy studies are suitable to reveal the conformational dynamics of LHCs reflected in the fluorescence properties that are obscured in ordinary ensemble measurements. Recent advanced single-molecule spectroscopy studies have revealed the dynamical fluctuations of LHCs in their fluorescence peak position, intensity, and lifetime. The observed dynamics seem relevant to the conformational plasticity required for the flexible activations of photoprotective energy quenching. In this review, we survey recent advances in the single-molecule spectroscopy study of the light-harvesting systems of oxygenic photosynthesis.

Single Molecule Spectroscopy Studies of Acid-Base Chemical Gradients Using Nile Red as a Probe of Local Surface Acidity.

Single molecule spectroscopy studies of local acidity along bifunctional acid-base gradients are reported. Gradients are prepared by directional vapor phase diffusion and subsequent reaction of 3-aminopropyl-trimethoxysilane with a uniform silica film. Gradient formation is confirmed by spectroscopic ellipsometry and by static water contact angle measurements. X-ray photoelectron spectroscopy is used to characterize the nitrogen content and degree of nitrogen protonation along the gradient. Nile Red is employed as the probe dye in single molecule spectroscopy studies of these gradients. While Nile Red is well-known for its solvent sensitivity, it is used here, for the first time, to sense the acid/base properties of the film in two-color wide-field fluorescence imaging experiments. The data reveal broad bimodal distributions of Nile Red emission spectra that vary along the gradient direction. The single molecule results are consistent with solution phase ensemble acid/base studies of the dye. The former reveal a gradual transition from a surface dominated by basic aminosilane sites at the high-amine end of the gradient to one dominated by acidic silanol sites at the low-amine end. The sub-diffraction-limited spatial resolution afforded by superlocalization of the single molecules reveals spatial correlations in the acid/base properties of the gradient over ∼200 nm distances. These studies provide data relevant to the use of aminosilane-modified silica in bifunctional, cooperative chemical catalysis.

The role of far-red spectral states in the energy regulation of phycobilisomes

The main light-harvesting pigment-protein complex of cyanobacteria and certain algae is the phycobilisome, which harvests sunlight and regulates the flow of absorbed energy to provide the photochemical reaction centres with a constant energy throughput. At least two light-driven mechanisms of excited energy quenching in phycobilisomes have been identified: the dominant mechanism in many strains of cyanobacteria depends on the orange carotenoid protein (OCP), while the second mechanism is intrinsically available to a phycobilisome and is possibly activated faster than the former. Recent single molecule spectroscopy studies have shown that far-red (FR) emission states are related to the OCP-dependent mechanism and it was proposed that the second mechanism may involve similar states. In this study, we examined the dynamics of simultaneously measured emission spectra and intensities from a large set of individual phycobilisome complexes from Synechocystis PCC 6803. Our results suggest a direct relationship between FR spectral states and thermal energy dissipating states and can be explained by a single phycobilin pigment in the phycobilisome core acting as the site of both quenching and FR emission likely due to the presence of a charge-transfer state. Our experimental method provides a means to accurately resolve the fluorescence lifetimes and spectra of the FR states, which enabled us to quantify a kinetic model that reproduces most of the experimentally determined properties of the FR states.

Single-Molecule Spectroscopy Study of Crowding-Induced Protein Spontaneous Denature and Crowding-Perturbed Unfolding-Folding Conformational Fluctuation Dynamics.

The effects of molecular crowding on protein folding-unfolding processes are of importance for understanding protein function and structure dynamics in living cells. The enhancement of protein stability as a result of reduced entropic effect in the presence of molecular crowding is well understood both experimentally and theoretically. However, because of the complexity and interplay between various interactions existing in an equally favored environment of protein folding and unfolding conformational dynamics, such a simple reduced entropic enhancement model does not suffice to describe protein folding conformational dynamics under a protein crowding condition. In this paper, we report our observation on that single protein molecules spontaneously denature into unfolded proteins and folding-unfolding fluctuations in solution of crowding reagent Ficoll 70. We have identified that such protein dynamics involves a combined mechanism of polymer-polymer interaction, entropic effects, and protein solvation dynamics. We characterize the protein folding-unfolding dynamics by using single-molecule spectroscopy to obtain detailed molecular dynamic scale information on the protein folding-unfolding conformational fluctuation dynamics. Our findings suggest that the complex unfolding dynamic processes are spontaneous denature of single protein molecules induced by molecular crowding effect which has been elusive for analysis in ensemble-averaged measurements. Furthermore, the energy needed for the spontaneous unfolding is at the biological accessible force fluctuation level, which suggests a strong implication of significant human health relevance and importance. The new knowledge of the inhomogeneous protein unfolding processes can serve as a step forward to a mechanistic understanding of human diseases associated with molecular crowding, protein aggregates, fibril formation, as well as gene translational regulation processes typically under a molecular crowded local environment.

Design principles of natural light-harvesting as revealed by single molecule spectroscopy

Biology offers a boundless source of adaptation, innovation, and inspiration. A wide range of photosynthetic organisms exist that are capable of harvesting solar light in an exceptionally efficient way, using abundant and low-cost materials. These natural light-harvesting complexes consist of proteins that strongly bind a high density of chromophores to capture solar photons and rapidly transfer the excitation energy to the photochemical reaction centre. The amount of harvested light is also delicately tuned to the level of solar radiation to maintain a constant energy throughput at the reaction centre and avoid the accumulation of the products of charge separation. In this Review, recent developments in the understanding of light-harvesting by plants will be discussed, based on results obtained from single molecule spectroscopy studies. Three design principles of the main light-harvesting antenna of plants will be highlighted: (a) fine, photoactive control over the intrinsic protein disorder to efficiently use intrinsically available thermal energy dissipation mechanisms; (b) the design of the protein microenvironment of a low-energy chromophore dimer to control the amount of shade absorption; (c) the design of the exciton manifold to ensure efficient funneling of the harvested light to the terminal emitter cluster.

Sizing up single-molecule enzymatic conformational dynamics

Enzymatic reactions and related protein conformational dynamics are complex and inhomogeneous, playing crucial roles in biological functions. The relationship between protein conformational dynamics and enzymatic reactions has been a fundamental focus in modern enzymology. It is extremely difficult to characterize and analyze such complex dynamics in an ensemble-averaged measurement, especially when the enzymes are associated with multiple-step, multiple-conformation complex chemical interactions and transformations. Beyond the conventional ensemble-averaged studies, real-time single-molecule approaches have been demonstrated to be powerful in dissecting the complex enzymatic reaction dynamics and related conformational dynamics. Single-molecule enzymology has come a long way since the early demonstrations of the single-molecule spectroscopy studies of enzymatic dynamics about two decades ago. The rapid development of this fundamental protein dynamics field is hand-in-hand with the new development of single-molecule imaging and spectroscopic technology and methodology, theoretical model analyses, and correlations with biological preparation and characterization of the enzyme protein systems. The complex enzymatic reactions can now be studied one molecule at a time under physiological conditions. Most exciting developments include active manipulation of enzymatic conformational changes and energy landscape to regulate and manipulate the enzymatic reactivity and associated conformational dynamics, and the new advancements have established a new stage for studying complex protein dynamics beyond by simply observing but by actively manipulating and observing the enzymatic dynamics at the single-molecule sensitivity temporally and spatially.

Excitations get random

The fate of newly created excited states in conjugated materials is not fully understood, with unanswered questions regarding where exactly excitons form and their subsequent behaviour. Now, single-molecule spectroscopy studies of large conjugated molecular wheels reveal that excitons localize at random positions around the wheel rims.

The Specificity of Controlled Protein Disorder in the Photoprotection of Plants

Light-harvesting pigment-protein complexes of photosystem II of plants have a dual function: they efficiently use absorbed energy for photosynthesis at limiting sunlight intensity and dissipate the excess energy at saturating intensity for photoprotection. Recent single-molecule spectroscopy studies on the trimeric LHCII complex showed that environmental control of the intrinsic protein disorder could in principle explain the switch between their light-harvesting and photoprotective conformations in vivo. However, the validity of this proposal depends strongly on the specificity of the protein dynamics. Here, a similar study has been performed on the minor monomeric antenna complexes of photosystem II (CP29, CP26, and CP24). Despite their high structural homology, similar pigment content and organization compared to LHCII trimers, the environmental response of these proteins was found to be rather distinct. A much larger proportion of the minor antenna complexes were present in permanently weakly fluorescent states under most conditions used; however, unlike LHCII trimers the distribution of the single-molecule population between the strongly and weakly fluorescent states showed no significant sensitivity to low pH, zeaxanthin, or low detergent conditions. The results support a unique role for LHCII trimers in the regulation of light harvesting by controlled fluorescence blinking and suggest that any contribution of the minor antenna complexes to photoprotection would probably involve a distinct mechanism.

Blinking Dynamics: Photoluminenscence Blinking Dynamics of Colloidal Quantum Dots in the Presence of Controlled External Electron Traps (Small 2/2012)

A single-molecule spectroscopy study on colloidal quantum dots reveals the effect of the external charge trap on the photoluminescence blinking dynamics. The study uses a series of colloidal quantum-dot-bridge-fullerene dimers with varying bridge lengths where the fullerene moiety acts as a well-defined, well-positioned external electron trap. A single external electron trap is shown to be able to switch a quantum dot from having “telegraph-like”, two-state on/off blinking behavior into exhibiting quasi- continuous, multistate blinking. Additionally, charge transfer followed by charge recombination is found to be an important mechanism in determining the blinking behavior of isolated quantum dots.

Heterogeneous Diffusion in Thin Polymer Films As Observed by High-Temperature Single-Molecule Fluorescence Microscopy

Single-molecule fluorescence microscopy was used to investigate the dynamics of perylene diimide (PDI) molecules in thin supported polystyrene (PS) films at temperatures up to 135 °C. Such high temperatures, so far unreached in single-molecule spectroscopy studies, were achieved using a custom-built setup which allows for restricting the heated mass to a minimum. This enables temperature-dependent single-molecule fluorescence studies of structural dynamics in the temperature range most relevant to the processing and to applications of thermoplastic materials. In order to ensure that polymer chains were relaxed, a molecular weight of 3000 g/mol, clearly below the entanglement length of PS, was chosen. We found significant heterogeneities in the motion of single PDI probe molecules near T(g). An analysis of the track radius of the recorded single-probe molecule tracks allowed for a distinction between mobile and immobile molecules. Up to the glass transition temperature in bulk, T(g,bulk), probe molecules were immobile; at temperatures higher than T(g,bulk) + 40 K, all probe molecules were mobile. In the range between 0 and 40 K above T(g,bulk) the fraction of mobile probe molecules strongly depends on film thickness. In 30-nm thin films mobility is observed at lower temperatures than in thick films. The fractions of mobile probe molecules were compared and rationalized using Monte Carlo random walk simulations. Results of these simulations indicate that the observed heterogeneities can be explained by a model which assumes a T(g) profile and an increased probability of probe molecules remaining at the surface, both effects caused by a density profile with decreasing polymer density at the polymer-air interface.

Unravelling Electronic Energy Transfer in Single Conjugated Polyelectrolytes Encapsulated in Lipid Vesicles

A method for the study of conjugated polyelectrolyte (CPE) photophysics in solution at the single molecule level is described. Extended observation times of single polymer molecules are enabled by the encapsulation of the CPEs within 200 nm lipid vesicles, which are in turn immobilized on a surface. When combined with a molecular-level visualization of vesicles and CPE via cryo-TEM, these single molecule spectroscopy studies on CPEs enable us to directly correlate the polymer conformation with its spectroscopic features. These studies are conducted with poly[5-methoxy-2-(3-sulfopropoxy)-1,4-phenylene-vinylene] (MPS-PPV, a negatively charged CPE), when encapsulated in neutral and in negatively-charged lipid vesicles. MPS-PPV exists as a freely diffusing polymer when confined in negatively-charged vesicles. Individual MPS-PPV molecules adopt a collapsed-chain conformation leading to efficient energy migration over multiple chromophores. Both the presence of stepwise photobleaching in fluorescence intensity/time trajectories and emission from low energy chromophores along the chain are observed. These results correlate with the amplified sensing potential reported for MPS-PPV in aqueous solution. When confined within neutral vesicles, single MPS-PPV molecules adopt an extended conformation upon insertion in the lipid bilayer. In this case emission arises from multiple chromophores within the isolated polymer chains, leading to an exponential decay of the intensity over time and a broad blue-shifted emission spectrum.

Unraveling electronic energy transfer in single conjugated polyelectrolytes encapsulated in lipid vesicles.

A method for the study of conjugated polyelectrolyte (CPE) photophysics in solution at the single-molecule level is described. Extended observation times of single polymer molecules are enabled by the encapsulation of the CPEs within 200-nm lipid vesicles, which are in turn immobilized on a surface. When combined with a molecular-level visualization of vesicles and CPE via cryo-transmission electron microscopy, these single-molecule spectroscopy studies on CPEs enable us to directly correlate the polymer conformation with its spectroscopic features. These studies are conducted with poly[5-methoxy-2-(3-sulfopropoxy)-1,4-phenylene-vinylene] (MPS-PPV, a negatively charged CPE), when encapsulated in neutral and in negatively charged lipid vesicles. MPS-PPV exists as a freely diffusing polymer when confined in negatively charged vesicles. Individual MPS-PPV molecules adopt a collapsed-chain conformation leading to efficient energy migration over multiple chromophores. Both the presence of stepwise photobleaching in fluorescence intensity-time trajectories and emission from low-energy chromophores along the chain are observed. These results correlate with the amplified sensing potential reported for MPS-PPV in aqueous solution. When confined within neutral vesicles, single MPS-PPV molecules adopt an extended conformation upon insertion in the lipid bilayer. In this case emission arises from multiple chromophores within the isolated polymer chains, leading to an exponential decay of the intensity over time and a broad blue-shifted emission spectrum.

A Dynamic t-SNARE Complex

Syntaxin-1/SNAP-25 heterodimers with 1:1 stoichiometry likely play a key role in neurotransmitter release but they have been difficult to characterize. In this issue of Structure, Weninger et al. present a beautiful single molecule spectroscopy study showing the dynamic nature of these heterodimers and how they are influenced by other factors.

Liposome Encapsulation of Conjugated Polyelectrolytes: Toward a Liposome Beacon

Herein we report the encapsulation of fluorescent conjugated polyelectrolytes (CPEs) within 100 nm diameter liposomes, their subsequent specific immobilization onto polyethylene-glycol-coated glass coverslips and their observation via single molecule spectroscopy. We believe that this novel architecture, a surface tethered liposome beacon, will prove instrumental in developing membrane sensing schemes. It will further facilitate single molecule spectroscopy studies on the spectroscopic properties of CPEs.

Probing polyproline structure and dynamics by photoinduced electron transfer provides evidence for deviations from a regular polyproline type II helix.

Polyprolines are well known for adopting a regular polyproline type II helix in aqueous solution, rendering them a popular standard as molecular ruler in structural molecular biology. However, single-molecule spectroscopy studies based on Förster resonance energy transfer (FRET) have revealed deviations of experimentally observed end-to-end distances of polyprolines from theoretical predictions, and it was proposed that the discrepancy resulted from dynamic flexibility of the polyproline helix. Here, we probe end-to-end distances and conformational dynamics of poly-l-prolines with 1-10 residues using fluorescence quenching by photoinduced-electron transfer (PET). A single fluorophore and a tryptophan residue, introduced at the termini of polyproline peptides, serve as sensitive probes for distance changes on the subnanometer length scale. Using a combination of ensemble fluorescence and fluorescence correlation spectroscopy, we demonstrate that polyproline samples exhibit static structural heterogeneity with subpopulations of distinct end-to-end distances that do not interconvert on time scales from nano- to milliseconds. By observing prolyl isomerization through changes in PET quenching interactions, we provide experimental evidence that the observed heterogeneity can be explained by interspersed cis isomers. Computer simulations elucidate the influence of trans/cis isomerization on polyproline structures in terms of end-to-end distance and provide a structural justification for the experimentally observed effects. Our results demonstrate that structural heterogeneity inherent in polyprolines, which to date are commonly applied as a molecular ruler, disqualifies them as appropriate tool for an accurate determination of absolute distances at a molecular scale.

A detailed single molecule spectroscopy study of the vibronic states and energy transfer pathways of the conjugated polymer MEH-PPV

The low energy onset of the optically accessible density of electronic states (OADOS) of the conjugated polymer MEH-PPV has been investigated by low temperature fluorescence single molecule spectroscopy (SMS). The first extensive set of high signal-to-noise SMS data have been acquired for MEH-PPV samples as a function of molecular weight confirming the previously reported bimodal energy gap distribution. These data have been acquired under sample and radiation conditions that inhibit photophysical dynamics that have distorted previous SMS studies. A molecule by molecule vibronic analysis has yielded an experimentally determined spectral density of the S 0–S 1 energy gaps for the individual chromophores for MEH-PPV. The SMS data has also led to new insights on the energy transfer pathways for conjugated polymers, and on the molecular nature of the “blue” and “red” sites of conjugated polymers. Finally, a combined analysis of the new SMS data with the previously obtained bulk absorption and emission data for MEH-PPV has offered clear evidence that the low energy onset of the OADOS for MEH-PPV, in analogy to the emission spectral density, confirms the existence of a bimodal energy gap distribution.

High sensitivity optical microscope for single molecule spectroscopy studies

We describe the setup and apply two algorithms for fast imaging in a sample raster scanning two photon microscope. Imaging can be performed at a rate of 1–100 lines per second with a closed loop piezo actuator, and the detection is performed via avalanche photodiodes. This allows to investigate single molecule emission with 50 ms time resolution. In a slow scanning algorithm we have implemented fluorescence fluctuation analysis by computing the photon counting histogram (PCH) on each pixel of the image. In a fast-scan acquistion method the image acquistion rate is 5 lines per second on a large field of view and high resolution(50 nm scanning step, 100×100 μm2 field of view) and ≅100 lines per second on smaller field of views with optically limited resolution (200 nm scanning step, 20×20 μm2 field of view). This figure, which is lower than the typical value for normal confocal scanning imaging (≅500 lines per second), allows nevertheless to perform imaging studies of extended samples in reasonable times for intracellular kinetics and interactions. With this setup and by means of the PCH analysis we are able to discriminate between local concentration and molecular brightness on extended samples also at the level of the single molecule.

Single-molecule spectroscopy studies of conformational change dynamics in enzymatic reactions.

Protein conformational dynamics, often associated with static and dynamic inhomogeneities, plays a crucial role in biomolecular functions. It is extremely difficult to characterize such inhomogeneous dynamics in an ensemble-averaged measurement, especially when the protein involves in a multiple-step complex chemical reaction, such as an enzymatic reaction. Single-molecule spectroscopy is a powerful approach to analyze protein conformational dynamics in real time under physiological conditions, providing dynamic perspectives on a molecular-level understanding of protein structure-function mechanisms. In this minireview, we discuss our recent studies on single-molecule enzymatic reaction dynamics and protein conformational dynamics of the T4 lysozyme hydrolyzation reaction of a polysaccharide by a combined approach of single-molecule spectroscopy, molecular dynamics simulation, and theoretical modeling. Detailed characterization of the complex enzymatic reaction dynamics identified the nature of the inhomogeneity and revealed multiple intermediate conformational states associated with the enzyme-substrate complex formation in the multiple-step enzymatic reaction.

Single-molecule conformational dynamics of fluctuating noncovalent DNA-protein interactions in DNA damage recognition.

Single-molecule spectroscopy is a powerful methodology to characterize inhomogeneous samples 1-4 because information may be hidden or lost in conventional ensemble-averaged experiments due to intrinsic sample heterogeneities 5-7. Here we report a single-molecule spectroscopy study that reveals dynamics of fluctuating molecular noncovalent interactions within single protein-DNA complexes. This data will facilitate a molecule-level understanding for the dynamic mechanisms of the damage-recognition process in DNA repair 8.

Single-Molecule Spectroscopy Studies of Molecular Dynamics in Chemical and Biological Systems*

Article Single-Molecule Spectroscopy Studies of Molecular Dynamics in Chemical and Biological Systems* was published on January 1, 1998 in the journal Zeitschrift für Physikalische Chemie (volume 1, issue 1).