Nano-shells have been previously shown to have tunable absorption frequencies, dependent on the ratio of their inner and outer radii. Inspired by this, we ask: can a nano-shell increase the absorption of a small core system embedded within it? A theoretical model is constructed to answer this question. A core, composed of a “jellium” ball of the density of gold is embedded within a jellium nano-shell of nanometric diameter. The shell plasmon frequency is tuned to the core absorption line. A calculation based the time-dependent density functional theory was performed showing a ten fold increase in core excitation yield.
Single-molecule Förster resonance energy transfer (smFRET) is utilized to study the structure and dynamics of many biomolecules, such as proteins, DNA, and their various complexes. The structural assessment is based on the well-known Förster relationship between the measured efficiency of energy transfer between a donor (D) and an acceptor (A) dye and the distance between them. Classical smFRET analysis methods called photon distribution analysis (PDA) take into account photon shot-noise, D–A distance distribution, and, more recently, interconversion between states in order to extract accurate distance information. It is known that rapid D–A distance fluctuations on the order of the D lifetime (or shorter) can increase the measured mean FRET efficiency and thus decrease the estimated D–A distance. Nonetheless, this effect has been so far neglected in smFRET experiments, potentially leading to biases in estimated distances. Here we introduce a PDA approach dubbed Monte Carlo diffusion-enhanced photon inference (MC-DEPI). MC-DEPI recolor detected photons of smFRET experiments taking into account dynamics of D–A distance fluctuations, multiple interconverting states, and photoblinking. Using this approach, we show how different underlying conditions may yield identical FRET histograms and how the additional information from fluorescence decays helps in distinguishing between the different conditions. We also introduce a machine learning fitting approach for retrieving the D–A distance distribution, decoupled from the above-mentioned effects. We show that distance interpretation of smFRET experiments of even the simplest dsDNA is nontrivial and requires decoupling the effects of rapid D–A distance fluctuations on FRET in order to avoid systematic biases in the estimation of the D–A distance distribution.
Protein folding is a fundamental process in biology, key to understanding many human diseases. Experimentally, proteins often appear to fold via simple two- or three-state mechanisms involving mainly native-state interactions, yet recent network models built from atomistic simulations of small proteins suggest the existence of many possible metastable states and folding pathways. We reconcile these two pictures in a combined experimental and simulation study of acyl-coenzyme A binding protein (ACBP), a two-state folder (folding time ~10 ms) exhibiting residual unfolded-state structure, and a putative early folding intermediate. Using single-molecule FRET in conjunction with side-chain mutagenesis, we first demonstrate that the denatured state of ACBP at near-zero denaturant is unusually compact and enriched in long-range structure that can be perturbed by discrete hydrophobic core mutations. We then employ ultrafast laminar-flow mixing experiments to study the folding kinetics of ACBP on the microsecond time scale. These studies, along with Trp-Cys quenching measurements of unfolded-state dynamics, suggest that unfolded-state structure forms on a surprisingly slow (~100 μs) time scale, and that sequence mutations strikingly perturb both time-resolved and equilibrium smFRET measurements in a similar way. A Markov state model (MSM) of the ACBP folding reaction, constructed from over 30 ms of molecular dynamics trajectory data, predicts a complex network of metastable stables, residual unfolded-state structure, and kinetics consistent with experiment but no well-defined intermediate preceding the main folding barrier. Taken together, these experimental and simulation results suggest that the previously characterized fast kinetic phase is not due to formation of a barrier-limited intermediate but rather to a more heterogeneous and slow acquisition of unfolded-state structure.
This paper details the making, characterization, and use of a simple and versatile capillary-based co-axial single-molecule mixing device which has a response time of 5-10 milliseconds and which can be used to monitor bioconformational reactions and/or transient conformational states under non-equilibrium reactions conditions with single molecule resolution. The device's co-axial geometry allows three-dimensional hydrodynamic focusing of sample fluids to diffraction-limited dimensions where diffusional mixing is rapid and efficient. Its capillary-based design enables rapid in-lab construction of mixers without the need for expensive lithography-based microfabrication facilities. In-line filtering of sample fluids using granulated silica particles virtually eliminates clogging and extends the lifetime of each device to many months. A major technical challenge dealt with here is the translation of spatial distances from the mixing region into time-points for kinetic analyses. In order to obtain the required distance-to-time transfer and instrument response functions for the device we characterize its fluid flow and mixing properties using both Fluorescence Cross-Correlation Spectroscopy (FCCS) velocimetry and computational fluid dynamics (CFD) simulations. We then apply the mixer to single molecule FRET protein folding studies of Chymotrypsin Inhibitor protein 2. By transiently populating the unfolded state of CI2 under non-equilibrium in-vitro re-folding conditions, we spatially and temporally resolve the denaturant-dependent non-specific collapse of the unfolded state from the barrier-limited folding transition of CI2.
In their Perspective,
Kelley
et al .
report from a recent symposium on single-molecule spectroscopy. The symposium demonstrates that the field has matured and is now providing unprecedented insights in biology and materials science.
Type-II ZnSe/CdS voltage-sensing seeded nanorods (vsNRs) were functionalized with α-helical peptides and zwitterionic-decorated lipoic acids (zw-LAs). Specific membrane targeting with high loading efficiency and minimal nonspecific binding was achieved. These vsNRs display quantum yield (QY) modulation as a function of membrane potential (MP) changes, as demonstrated at the ensemble level for (i) vesicles treated with valinomycin and (ii) wild-type HEK cells under alternating buffers with different [K+]. ΔF/F of ∼ 1% was achieved.
The measurement data files in this repository have been used in the following publications: 48-spot single-molecule FRET setup with periodic acceptor excitation. Ingargiola et al., bioRxiv (2017), doi: https://doi.org/10.1101/156182 Optical crosstalk in SPAD arrays for high-throughput single-molecule fluorescence spectroscopy. Ingargiola et al., bioRxiv (2017), doi: https://doi.org/10.1101/207118
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