Polymer scaling laws of unfolded and intrinsically disordered proteins quantified with single-molecule spectroscopy

Graphene oxide (GO) nanosheets have received a great deal of attention for a wide range of applications from optoelectronic devices to biological sensors. We now report a mechanistic study of the interfacial electron transfer (ET) processes between organic dye molecule, 9-phenyl-2,3,7-trihydroxy-6-fluorone (PF), and nanometre-sized GO sheets using ensemble-averaged and single-molecule spectroscopies. The ET dynamics was characterized by the direct observation of the PF radical cation during the laser flash photolysis, and its reaction rate was determined to be ~10(11) s(-1). The single-molecule fluorescence spectroscopy was utilized to clarify the heterogeneous nature of the interfacial ET within individual composites. Their fluorescence lifetimes and spectra were found to vary from composite to composite, possibly due to the different local structures and molecular interactions. The autocorrelation analysis of fluorescence intensity trajectories also revealed the temporal fluctuation of the ET reactivity.

Single molecule spectroscopy (SMS) has matured to a point where it can be used as a convenient tool to guide organic synthesis and drug discovery, particularly applicable to catalytic systems where questions related to homogeneous vs heterogeneous pathways are important. SMS can look at intimate mechanistic details that can inspire major improvements of the catalyst performance, its recovery, and reuse. Here, we use the click reaction between alkynes and azides as an example where improvements at the bench have been inspired and validated using single-molecule fluorescence spectroscopy.

This review presents an overview of the fluorescence detection and spectroscopy of single molecules (SMS) in liquids and on surfaces under ambient conditions. The various techniques of SMS, such as confocal epifluorescence detection and wide-field imaging are presented and discussed, together with the different methods of data analysis such as fluorescence correlation spectroscopy and burst-by-burst analysis. Selected applications of the various techniques in physics, chemistry, and biology are described.

The properties of solutions of polymers in good and poor solvents and of articles prepared from these solutions

In this perspective, we focus our discussion on how the single-molecule spectroscopy and statistical analysis are able to reveal enzyme hidden properties, taking the study of T4 lysozyme as an example. Protein conformational fluctuations and dynamics play a crucial role in biomolecular functions, such as in enzymatic reactions. Single-molecule spectroscopy is a powerful approach to analyze protein conformational dynamics under physiological conditions, providing dynamic perspectives on a molecular-level understanding of protein structure-function mechanisms. Using single-molecule fluorescence spectroscopy, we have probed T4 lysozyme conformational motions under the hydrolysis reaction of a polysaccharide of E. coli B cell walls by monitoring the fluorescence resonant energy transfer (FRET) between a donor-acceptor probe pair tethered to T4 lysozyme domains involving open-close hinge-bending motions. Based on the single-molecule spectroscopic results, molecular dynamics simulation, a random walk model analysis, and a novel 2D statistical correlation analysis, we have revealed a time bunching effect in protein conformational motion dynamics that is critical to enzymatic functions. Bunching effect implies that conformational motion times tend to bunch in a finite and narrow time window. We show that convoluted multiple Poisson rate processes give rise to the bunching effect in the enzymatic reaction dynamics. Evidently, the bunching effect is likely common in protein conformational dynamics involving in conformation-gated protein functions. In this perspective, we will also discuss a new approach of 2D regional correlation analysis capable of analyzing fluctuation dynamics of complex multiple correlated and anti-correlated fluctuations under a non-correlated noise background. Using this new method, we are able to map out any defined segments along the fluctuation trajectories and determine whether they are correlated, anti-correlated, or non-correlated; after which, a cross correlation analysis can be applied for each specific segment to obtain a detailed fluctuation dynamics analysis.

Integrating single-molecule spectroscopy and simulations for the study of intrinsically disordered proteins.

DNA sliding clamps attach to polymerases and slide along DNA to allow rapid, processive replication of DNA. These clamps contain many positively charged residues that could curtail the sliding due to attractive interactions with the negatively charged DNA. By single-molecule spectroscopy we have observed a fluorescently labeled sliding clamp (polymerase III beta subunit or beta clamp) loaded onto freely diffusing, single-stranded M13 circular DNA annealed with fluorescently labeled DNA oligomers of up to 90 bases. We find that the diffusion constant for the beta clamp diffusing along DNA is on the order of 10(-14) m(2)/s, at least 3 orders of magnitude less than that for diffusion through water alone. We also find evidence that the beta clamp remains at the 3' end in the presence of Escherichia coli single-stranded-binding protein. These results may imply that the clamp not only acts to hold the polymerase on the DNA but also prevents excessive drifting along the DNA.

Internal friction, which reflects the "roughness" of the energy landscape, plays an important role for proteins by modulating the dynamics of their folding and other conformational changes. However, the experimental quantification of internal friction and its contribution to folding dynamics has remained challenging. Here we use the combination of single-molecule Förster resonance energy transfer, nanosecond fluorescence correlation spectroscopy, and microfluidic mixing to determine the reconfiguration times of unfolded proteins and investigate the mechanisms of internal friction contributing to their dynamics. Using concepts from polymer dynamics, we determine internal friction with three complementary, largely independent, and consistent approaches as an additive contribution to the reconfiguration time of the unfolded state. We find that the magnitude of internal friction correlates with the compactness of the unfolded protein: its contribution dominates the reconfiguration time of approximately 100 ns of the compact unfolded state of a small cold shock protein under native conditions, but decreases for more expanded chains, and approaches zero both at high denaturant concentrations and in intrinsically disordered proteins that are expanded due to intramolecular charge repulsion. Our results suggest that internal friction in the unfolded state will be particularly relevant for the kinetics of proteins that fold in the microsecond range or faster. The low internal friction in expanded intrinsically disordered proteins may have implications for the dynamics of their interactions with cellular binding partners.

In the past several years we have demonstrated the metal-enhanced fluorescence (MEF) and the significant changes in the photophysical properties of fluorophores in the presence of metallic nanostructures and nanoparticles using ensemble spectroscopic studies. Here, in the present study, we explored the new insights of these interactions using single-molecule fluorescence spectroscopy. The single molecule study is expected to provide more information, especially on the heterogeneity in the fluorescence enhancement and decrease in lifetimes associated with fluorophore-metal interactions, which is otherwise not possible to observe using ensemble measurements. For the present study, we considered using CdTe nanocrystals (QDots) prepared using modified Weller method as the fluorophores under investigation. QDots having few nanometer sizes, tunable absorption and fluorescence spectral properties, and high photo-stabilities are of important class of fluorescent probes. Because of these unique features Qdots are widely used as probes in various fields, including biological labeling and imaging. These CdTe nanocrystals show characteristic spectral features in solution and on the solid substrate. The CdTe nanocrystals dispersed in PVA and spin-casted on SiFs surface show ∼5-fold increase in fluorescence intensity and ∼3-fold decrease in lifetimes compared to on glass substrate. The data obtained using ensemble and single molecule spectroscopy are complimentary to each other. Additionally as anticipated we have seen increased heterogeneity in the plasmon induced fluorescence modulations. Moreover single molecule spectroscopic study revealed significant reduction in blinking of CdTe nanocrystals on plasmonic nanostructures. Subsequently, we present these important findings on metal-fluorophore interactions of CdTe nanocrystals (QDots) on plasmonic nanostructures.

The structure and function of polymers in confined environments, e.g., biopolymers in the cytoplasm of a cell, are strongly affected by macromolecular crowding. To explore the influence of solvent quality on conformations of crowded polymers, we model polymers as penetrable ellipsoids, whose shape fluctuations are governed by the statistics of self-avoiding walks, appropriate for a polymer in a good solvent. Within this coarse-grained model, we perform Monte Carlo simulations of mixtures of polymers and hard-nanosphere crowders, including trial changes in polymer size and shape. Penetration of polymers by crowders is incorporated via a free energy cost predicted by polymer field theory. To analyze the impact of crowding on polymer conformations in different solvents, we compute the average polymer shape distributions, radius of gyration, volume, and asphericity over ranges of the polymer-to-crowder size ratio and crowder volume fraction. The simulation results are accurately predicted by a free-volume theory of polymer crowding. Comparison of results for polymers in good and theta solvents indicates that excluded-volume interactions between polymer segments significantly affect crowding, especially in the limit of crowders much smaller than polymers. Our approach may help to motivate future experimental studies of polymers in crowded environments, with possible relevance for drug delivery and gene therapy.

A generalized temporal scaling ansatz for the frequency dependence of the loss and storage moduli and for the shear dependence of the viscosity is tested against studies on entangled solutions of star polymers in good and theta solvents. At lower frequencies or shear rates, the ansatz calls for an exponential or stretched-exponential form [e.g., G0 exp(−αων)] for G″(ω)/ω and G′(ω)/ω2, and correspondingly in κ for η(κ). At higher frequencies, the ansatz indicates that each of these quantities has a power-law dependence on its primary variable. The predicted forms are in excellent agreement with literature data on solutions of poly-α-methylstyrene, polybutadiene, polystyrene, and polyisoprene stars. A power-law correlation α∼G02/3 is observed between the zero-frequency, zero-shear modulus G0 and the low-frequency or low-shear decay constant α of the stretched exponential, the same power-law line describing both star and linear polymers in good solvents.

The variation of the diffusion coefficient of polystyrene with molecular weight in bromobenzene, toluene, and methyl ethyl ketone, obtained with a modified Northrop cell, obeyed the relation 1/D = AMε where A and ε are empirical constants. The decrease in the exponent ε with increasing molecular weight agrees qualitatively with the predictions of Debye and Bueche. Both the exponent and the ratio of the diffusion coefficients of a low to high molecular weight sample increased in better solvents, indicating that in such solvents the polystyrene molecules are elongated. Using this ratio as a criterion of solvent power, there is good agreement with the results of swelling and precipitation studies. The decrease in the diffusion coefficient – solvent viscosity product for a low and a high molecular weight polymer in good solvents suggests an increase in the degree of solvation of the polymer. The variation of this product with solvent composition in solvent mixtures indicates the existence of selective solvation by the good solvent. The activation energies of diffusion were of the order of 3 kcal. for both high and low molecular weight polymers in good and poor solvents. This supports Eyring's theory of segment flow for polymer diffusion.

Single-molecule spectroscopy (SMS) is a unique and competent technique to study molecule dynamics and sense biomolecules precisely. The design of an ultrahigh-stability single fluorophore probe with excellent photostability and long-lived dark transient states for single-molecule fluorescence microscopy is challenging. Here, we found that the photostability of bimetallic AuAg28 nanoclusters is better than monometallic Ag29 nanoclusters. The photon antibunching experiments unveiled exceptional brightness and remarkable photostability with high survival times of up to 218 s with minimal blinking. AuAg28 NCs exhibited longer "on" times and shorter "off" times as compared to Ag29 NCs. The statistical analysis was performed on at least 100 molecules that showed single-step photobleaching and almost a 5-fold enhancement in intensity on Au doping in Ag29 NCs. The distinctive and tunable photophysics of metal NCs can offer huge potential in pushing single-molecule dynamic measurements to be carried out biologically.

Enhancing single-molecule fluorescence with nanophotonics

Single-molecule fluorescence spectroscopy is a powerful method to observe real time movements of individual biological molecules while they are functioning without the need for synchronization. Dynamic characteristics of nucleic acids can now be easily and reliably studied, and new applications are emerging in which their recognition and processing by proteins and enzymes are being understood with unprecedented detail. The most recent examples are discussed, including the hairpin ribozyme, Holliday junction, G-quadruplex, Rep helicase, reverse transcriptase, and combination with mechanical manipulation.