Christy F. Landes

In situ measurement of bovine serum albumin interaction with gold nanospheres

We present in situ observations of adsorption of bovine serum albumin (BSA) on citrate-stabilized gold nanospheres. We implemented scattering correlation spectroscopy as a tool to quantify changes in the nanoparticle brownian motion resulting from BSA adsorption onto the nanoparticle surface. Protein binding was observed as an increase in the nanoparticle hydrodynamic radius. Our results indicate the formation of a protein monolayer at similar albumin concentrations as those found in human blood. Additionally, by monitoring the frequency and intensity of individual scattering events caused by single gold nanoparticles passing the observation volume, we found that BSA did not induce colloidal aggregation, a relevant result from the toxicological viewpoint. Moreover, to elucidate the thermodynamics of the gold nanoparticle-BSA association, we measured an adsorption isotherm which was best described by an anticooperative binding model. The number of binding sites based on this model was consistent with a BSA monolayer in its native state. In contrast, experiments using poly(ethylene glycol)-capped gold nanoparticles revealed no evidence for adsorption of BSA.

Structural landscape of isolated agonist-binding domains from single AMPA receptors

α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors mediate fast excitatory neurotransmission by converting chemical signals into electrical signals. Thus, it is important to understand the relationship between their chemical biology and their function. Single molecule fluorescence resonance energy transfer (smFRET) was used to examine the conformations explored by the agonist binding domain of the AMPA receptor for wild type and T686 mutant proteins. Each form of the agonist binding domain exhibited a dynamic, multi-state sequential equilibrium, which could only be identified using wavelet shrinkage, a signal processing technique that removes experimental shot-noise. These results illustrate that the extent of activation is dependent not on a rigid closed cleft, but instead on the probability that a given subunit will occupy a closed cleft conformation, which in turn is not only determined by the lowest energy state but by the range of states that the protein explores.

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.

Single-Particle Spectroscopy Reveals Heterogeneity in Electrochemical Tuning of the Localized Surface Plasmon

A hyperspectral imaging method was developed that allowed the identification of heterogeneous plasmon response from 50 nm diameter gold colloidal particles on a conducting substrate in a transparent three-electrode spectroelectrochemical cell under non-Faradaic conditions. At cathodic potentials, we identified three distinct behaviors from different nanoparticles within the same sample: irreversible chemical reactions, reversible chemical reactions, and reversible charge density tuning. The irreversible reactions in particular would be difficult to discern in alternate methodologies. Additional heterogeneity was observed when single nanoparticles demonstrating reversible charge density tuning in the cathodic regime were measured dynamically in anodic potential ranges. Some nanoparticles that showed charge density tuning in the cathodic range also showed signs of an additional chemical tuning mechanism in the anodic range. The expected changes in nanoparticle free-electron density were modeled using a charge density-modified Drude dielectric function and Mie theory, a commonly used model in colloidal spectroelectrochemistry. Inconsistencies between experimental results and predictions of this common physical model were identified and highlighted. The broad range of responses on even a simple sample highlights the rich experimental and theoretical playgrounds that hyperspectral single-particle electrochemistry opens.

From tunable core-shell nanoparticles to plasmonic drawbridges: Active control of nanoparticle optical properties

Redox electrochemistry was used to reversibly tune the optical properties of plasmonic core-shell nanoparticles and dimers. The optical properties of metallic nanoparticles are highly sensitive to interparticle distance, giving rise to dramatic but frequently irreversible color changes. By electrochemical modification of individual nanoparticles and nanoparticle pairs, we induced equally dramatic, yet reversible, changes in their optical properties. We achieved plasmon tuning by oxidation-reduction chemistry of Ag-AgCl shells on the surfaces of both individual and strongly coupled Au nanoparticle pairs, resulting in extreme but reversible changes in scattering line shape. We demonstrated reversible formation of the charge transfer plasmon mode by switching between capacitive and conductive electronic coupling mechanisms. Dynamic single-particle spectroelectrochemistry also gave an insight into the reaction kinetics and evolution of the charge transfer plasmon mode in an electrochemically tunable structure. Our study represents a highly useful approach to the precise tuning of the morphology of narrow interparticle gaps and will be of value for controlling and activating a range of properties such as extreme plasmon modulation, nanoscopic plasmon switching, and subnanometer tunable gap applications.

Adsorption and Unfolding of a Single Protein Triggers Nanoparticle Aggregation

The response of living systems to nanoparticles is thought to depend on the protein corona, which forms shortly after exposure to physiological fluids and which is linked to a wide array of pathophysiologies. A mechanistic understanding of the dynamic interaction between proteins and nanoparticles and thus the biological fate of nanoparticles and associated proteins is, however, often missing mainly due to the inadequacies in current ensemble experimental approaches. Through the application of a variety of single molecule and single particle spectroscopic techniques in combination with ensemble level characterization tools, we identified different interaction pathways between gold nanorods and bovine serum albumin depending on the protein concentration. Overall, we found that local changes in protein concentration influence everything from cancer cell uptake to nanoparticle stability and even protein secondary structure. We envision that our findings and methods will lead to strategies to control the associated pathophysiology of nanoparticle exposure in vivo.

Unified superresolution experiments and stochastic theory provide mechanistic insight into protein ion-exchange adsorptive separations

Significance Adsorption of proteins underlies the purification of biopharmaceuticals, as well as therapeutic apheresis, immunoassays, and biosensors. In particular, separation of proteins by interactions with charged ligands on surfaces (ion-exchange chromatography) is an essential tool of the modern pharmaceutical industry. By quantifying the interactions of single proteins with individual charged ligands, we demonstrate that clusters of charges are necessary to create functional adsorption sites and that even chemically identical ligands create sites of varying kinetic properties that depend on steric availability at the interface. Chromatographic protein separations, immunoassays, and biosensing all typically involve the adsorption of proteins to surfaces decorated with charged, hydrophobic, or affinity ligands. Despite increasingly widespread use throughout the pharmaceutical industry, mechanistic detail about the interactions of proteins with individual chromatographic adsorbent sites is available only via inference from ensemble measurements such as binding isotherms, calorimetry, and chromatography. In this work, we present the direct superresolution mapping and kinetic characterization of functional sites on ion-exchange ligands based on agarose, a support matrix routinely used in protein chromatography. By quantifying the interactions of single proteins with individual charged ligands, we demonstrate that clusters of charges are necessary to create detectable adsorption sites and that even chemically identical ligands create adsorption sites of varying kinetic properties that depend on steric availability at the interface. Additionally, we relate experimental results to the stochastic theory of chromatography. Simulated elution profiles calculated from the molecular-scale data suggest that, if it were possible to engineer uniform optimal interactions into ion-exchange systems, separation efficiencies could be improved by as much as a factor of five by deliberately exploiting clustered interactions that currently dominate the ion-exchange process only accidentally.

Denoising Single-Molecule FRET Trajectories with Wavelets and Bayesian Inference

A method to denoise single-molecule fluorescence resonance energy (smFRET) trajectories using wavelet detail thresholding and Bayesian inference is presented. Bayesian methods are developed to identify fluorophore photoblinks in the time trajectories. Simulated data are used to quantify the improvement in static and dynamic data analysis. Application of the method to experimental smFRET data shows that it distinguishes photoblinks from large shifts in smFRET efficiency while maintaining the important advantage of an unbiased approach. Known sources of experimental noise are examined and quantified as a means to remove their contributions via soft thresholding of wavelet coefficients. A wavelet decomposition algorithm is described, and thresholds are produced through the knowledge of noise parameters in the discrete-time photon signals. Reconstruction of the signals from thresholded coefficients produces signals that contain noise arising only from unquantifiable parameters. The method is applied to simulated and observed smFRET data, and it is found that the denoised data retain their underlying dynamic properties, but with increased resolution.

Insights on the role of nucleic acid/protein interactions in chaperoned nucleic acid rearrangements of HIV-1 reverse transcription

HIV-1 reverse transcription requires several nucleic acid rearrangement steps that are “chaperoned” by the nucleocapsid protein (NC), including minus-strand transfer, in which the DNA transactivation response element (TAR) is annealed to the complementary TAR RNA region of the viral genome. These various rearrangement processes occur in NC bound complexes of specific RNA and DNA structures. A major barrier to the investigation of these processes in vitro has been the diversity and heterogeneity of the observed nucleic acid/protein assemblies, ranging from small complexes of only one or two nucleic acid molecules all the way up to large-scale aggregates comprised of thousands of NC and nucleic acid molecules. Herein, we use a flow chamber approach involving rapid NC/nucleic acid mixing to substantially control aggregation for the NC chaperoned irreversible annealing kinetics of a model TAR DNA hairpin sequence to the complementary TAR RNA hairpin, i.e., to form an extended duplex. By combining the flow chamber approach with a broad array of fluorescence single-molecule spectroscopy (SMS) tools (FRET, molecule counting, and correlation spectroscopy), we have unraveled the complex, heterogeneous kinetics that occur during the course of annealing. The SMS results demonstrate that the TAR hairpin reactant is predominantly a single hairpin coated by multiple NCs with a dynamic secondary structure, involving equilibrium between a “Y” shaped conformation and a closed one. The data further indicate that the nucleation of annealing occurs in an encounter complex that is formed by two hairpins with one or both of the hairpins in the “Y” conformation.

Dynamics of an anti-VEGF DNA aptamer: a single-molecule study.

Single-molecule fluorescence resonance energy transfer (SMFRET) was used to study the interaction of a 25-nucleotide (nt) DNA aptamer with its binding target, vascular endothelial growth factor (VEGF). Conformational dynamics of the aptamer were studied in the absence of VEGF in order to characterize fluctuations in the unbound nucleic acid. SMFRET efficiency distributions showed that, while the aptamer favors a base-paired conformation, there are frequent conversions to higher energy conformations. Conversions to higher energy structures were also demonstrated to be dependent on the concentration of Mg2+-counterion by an overall broadening of the SMFRET efficiency distribution at lower Mg2+ concentration. Introduction of VEGF caused a distinct increase in the frequency of lower SMFRET efficiencies, indicating that favorable interaction of the DNA aptamer with its VEGF target directs aptamer structure towards a more open conformation.