Improved Resolution of Complex Single-Molecule FRET Systems via Wavelet Shrinkage

The resolution of complex interactions found in single-molecule fluorescence resonance energy transfer (smFRET) experiments is hindered by noise. Wavelet shrinkage is proven to reduce noise, but traditional methods introduce artifacts when acting on discontinuous signals, such as those acquired in smFRET experiments. Modifications to the basic method that are specific to smFRET are developed and tested on simulated systems. Use of the Haar wavelet basis produces the most optimally denoised estimates. We also assess various thresholding methods, develop a time-localized noise estimator, and implement a translation-invariant wavelet transformation to reduce artifacts associated with discontinuities and inadequate distinction of noise. The time-local estimator enhances noise reduction by 5−20%, and translation-invariant transformation nearly eliminates the aforementioned artifacts. Kinetic parameters extracted from denoised estimates are accurate to within 5% of the simulated values. Overall, the improved resolution results in the complete and accurate characterization of both simple and complex smFRET systems

Dye Diffusion at Surfaces: Charge Matters

Fluorescence correlation spectroscopy and single molecule burst analysis were used to measure the effects of glass surface interactions on the diffusion of two common fluorescent dyes, one cationic and one anionic. The effects of dye−surface interactions on measured diffusion rates as a function of distance from the surface were investigated. Use of a three-axis piezo stage, combined with reference calibration measurements, enabled the accurate acquisition of surface-distance-dependent transport data. This analysis reveals attractive interactions between the cationic dye and the surface, which significantly alter the extracted diffusion values and persist in the measurements up to 1.0 μm from the surface. The Coulomb attraction between the cationic dye and the surface also results in rare, long-lived association events that lead to irreproducibility in extracted diffusion values. In addition to an assignment of the association lifetime for these events, this paper demonstrates that, if experiments must be performed with cationic probes near a glass surface, the use of solution electrolytes can eliminate deleterious dye−surface interactions, as the dyes were tested in a variety of environments. Finally, our data demonstrate that a better dye choice is an anionic probe, which exhibits no depth dependence of diffusion characteristics above a glass surface

Fluorescence Correlation Spectroscopy: Criteria for Analysis in Complex Systems

We have evaluated the effect of varying three key parameters for Fluorescence Correlation Spectroscopy analysis, first in the context of a one species/one environment system, and then in a complex system composed of two species, or conversely, two environments. We establish experimentally appropriate settings for the (1) minimum lag time, (2) maximum lag time, and (3) averaging times over which an autocorrelation is carried out, as a function of expected diffusion decay time for a particular solute, and show that use of appropriate settings plays a critical role in recovering accurate and reliable decay times and resulting diffusion constants. Both experimental and simulated data were used to show that for a complex binary system, to extract accurate diffusion constants for both species, decay times must be bounded by adequate minimum and maximum lag times as dictated by the fast and slow diffusing species, respectively. We also demonstrate that even when constraints on experimental conditions do not permit achieving the necessary lag time limits for both of the species in a binary system, the accuracy of the recovered diffusion constant for the one species whose autocorrelation function is fully time-resolved is unaffected by uncertainty in fitting introduced by the presence of the second species

Mechanistic Understanding of the Phosphorylation-Induced Conformational Rigidity at the AMPA Receptor C‑terminal Domain

Phosphorylation at the intracellular C-terminal domain (CTD) of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors induces conformational rigidity. Such intracellular alterations to the AMPA receptor influence its functional responses, which are involved in multiple synaptic processes and neuronal signaling. The structure of the CTD still remains unresolved, which poses challenges toward providing a mechanism for the process of phosphorylation and deciphering the role of each phosphorylation step in causing the resultant conformational behavior. Herein, we utilize smFRET spectroscopy to understand the mechanism of phosphorylation, with the help of strategic point mutations that mimic phosphorylation. Our results reveal that first, phosphorylation at three target sites (S818, S831, and T840) is necessary for the change in the secondary structure of the existing disordered native sequence. Also, the results suggest that the formation of the tertiary structure through electrostatic interaction involving one specific phosphorylation site (S831) stabilizes the structure and renders conformational rigidity

Single Molecule Spectroscopy Reveals Heterogeneous Transport Mechanisms for Molecular Ions in a Polyelectrolyte Polymer Brush

Single molecule polarization and fluorescence correlation spectroscopy were used to evaluate heterogeneous transport mechanisms of molecular ions within supported polyelectrolyte brushes. Modes of diffusive transport include periods of significantly restricted rotational motion, often maintained over tens of milliseconds; periods of fast molecular rotation; and occasional adsorption of fluorescent probe molecules in the brush. The studies reveal rapid switching between orientational states during each observed mode of motion. Through quantitative analysis of state occupation times, the rate constants for transitions from weakly associated to strongly associated states were extracted. Additionally, the pH dependence of the ion transport rates in the brush exhibits an abrupt, rather than continuous, trend. These single molecule studies demonstrate the presence of dynamic anisotropic interactions between the charged molecular probe and the polymer brush and provide experimental evidence of stimuli responsive switchable transport functionality in the polyelectrolyte brush

Heterogeneity and Hysteresis in the Polymer Collapse of Single Core–Shell Stimuli-Responsive Plasmonic Nanohybrids

Broad application of polymeric stimuli-responsive smart nanohybrids requires understanding the mechanisms governing active control. Ensemble techniques have identified inhomogeneous polymer collapse in microgels that potentially arise from heterogeneous interchain interactions and differences in core size. A single-particle examination would establish the influence of core size and internal polymer network heterogeneity on local interactions that contribute to the observed inhomogeneous polymer collapse dynamics of nanohybrids. Using single-particle dark-field spectroscopy, we investigated the complex polymer collapse profiles of core–shell plasmonic nanohybrids comprising thermoresponsive poly­(N-isopropylacrylamide) (pNIPAM)-encapsulated gold nanorods (AuNRs). We report that the polymer collapse behavior was independent of the core size. For thinner polymer shells, we observed hysteresis in the collapse of AuNR@pNIPAMs, likely related to local pNIPAM aggregation due to interchain hydrogen bonding. For thicker polymer shells, we observed a broad polymer collapse distribution that we attributed to a two-step phase transition that arises from a polymer network density gradient. Our single-particle approach relates the internal heterogeneity of the polymer network of nanohybrids to the mechanisms underlying heterogeneous phase transitions that traditional, ensemble-averaged approaches are unable to discern

Naturally Occurring Proteins Direct Chiral Nanorod Aggregation

Serum albumin can template gold nanorods into chiral assemblies, but the aggregation mechanism is not entirely understood. We used circular dichroism spectroscopy and scanning electron microscopy to investigate the role of protein identity/shape, protein/nanorod ratio, and surfactants on chiral protein–nanorod aggregation. Three globular proteinsserum albumin, immunoglobulin, and transferrinproduced similarly sized chiral protein–nanorod aggregates. In solution these aggregates exhibited circular dichroism at the plasmon resonance that switched direction at specific protein/nanorod concentration ratios. Our explanation is that the extent of protein crowding influences protein conformation and therefore protein–protein interactions, which in turn direct nanorod aggregation into preferentially left- or right-handed structures. The fibrous proteins fibrinogen and fibrillar serum albumin also produced chiral nanorod aggregates but did not exhibit a ratio-dependent switch in the circular dichroism direction. In addition, cetyltrimethyl­ammonium bromide micelles prevented all aggregation, providing compelling evidence that protein–protein interactions are crucial for chiral protein–nanorod aggregate formation. The protein-dependent variations in circular dichroism and aggregation reported here present opportunities for future chiral nanostructure engineering and biosensing applications

Transient Three-Dimensional Orientation of Molecular Ions in an Ordered Polyelectrolyte Membrane

Single-molecule fluorescence spectroscopy is employed to reveal 3D details of the mechanisms underpinning ion transport in a polyelectrolyte thin film possessing polymer-brush nanoscale order. The ability to resolve fluorescence emission over three discrete polarization angles reveals that these ordered materials impart 3D orientation to charged, diffusing molecules. The experiments, supported by simulations, report global orientation parameters for molecular transport, track dipole angle progressions over time, and identify a unique transport mechanism: translational diffusion with restricted rotation. In general, realization of this experimental method for translational diffusion in systems exhibiting basic orientation should lend itself to evaluation of transport in a variety of important, ordered, functional materials

Polymer Free Volume Effects on Protein Dynamics in Polystyrene Revealed by Single-Molecule Spectroscopy

Protein–polymer interactions are critical to applications ranging from biomedical devices to chromatographic separations. The mechanistic relationship between the microstructure of polymer chains and protein interactions is challenging to quantify and not well studied. Here, single-molecule microscopy is used to compare the dynamics of two model proteins, α-lactalbumin and lysozyme, at the interface of uncharged polystyrene with varied molecular weights. The two proteins exhibit different surface interaction mechanisms despite having a similar size and structure. α-Lactalbumin exhibits interfacial adsorption–desorption with residence times that depend on polymer molecular weight. Lysozyme undergoes a continuous time random walk at the polystyrene surface with residence times that also depend on the molecular weight of polystyrene. Single-molecule observables suggest that the hindered continuous time random walk dynamics displayed by lysozyme are determined by the polystyrene free volume, a finding supported by thermal annealing and solvent quality studies. Hindered dynamics are dominated by short-range hydrophobic interactions where the contributions of electrostatic forces are negligible. This work establishes a relationship between the microscale structure (i.e., free volume) of polystyrene polymer chains to nanoscale interfacial protein dynamics

Nanoscale Surface-Induced Unfolding of Single Fibronectin Is Restricted by Serum Albumin Crowding

Understanding nanoscale protein conformational changes at solid–liquid interfaces is critical for predicting how proteins will impact the performance of biomaterials in vivo. Crowding is an important contributor to conformational stability. Here we apply single-molecule high resolution imaging with photobleaching to directly measure dye-conjugated fibronectin’s unfolding in varying conditions of crowding with human serum albumin on aminosilanized glass. Using this approach, we identify serum albumin’s crowding mechanism. We find that fibronectin achieves larger degrees of unfolding when not crowded by coadsorbed serum albumin. Serum albumin does not as effectively constrict fibronectin’s conformation if it is sequentially, rather than simultaneously, introduced, suggesting that serum albumin’s crowding mechanism is dependent on its ability to sterically block fibronectin’s unfolding during the process of adsorption. Because fibronectin’s conformation is dependent on interfacial macromolecular crowding under in vitro conditions, it is important to consider the role of in vivo crowding on protein activity