Single-Molecule Orientation Imaging Reveals Two Distinct Binding Configurations on Amyloid Fibrils.

Fluorescence readouts for an amyloid fibril sensor critically depend on its molecular interaction and local environment offered by the available structural motifs. Here we employ polarized points accumulation for imaging in nanoscale topography with intramolecular charge transfer probes transiently bound to amyloid fibrils to investigate the organization of fibril nanostructures and probe binding configurations. Besides the in-plane (θ ≈ 90°) mode for binding on the fibril surface parallel to the long fibril axis, we also observed a sizable population of over 60% out-of-plane (θ < 60°) dipoles for rotor probes experiencing a varying degree of orientational mobility. Highly confined dipoles exhibiting an out-of-plane configuration probably reflect tightly bound dipoles in the inner channel grooves, while the weakly bound ones on amyloid enjoy rotational flexibility. Our observation of an out-of-plane binding mode emphasizes the pivotal role played by the electron donor amino group toward fluorescence detection and hence the emergence of anchored probes alongside conventional groove binders.

Inter-molecular interaction kinetics: tale of photon anti-bunching and bunching in fluorescence correlation spectroscopy (FCS).

Molecular interactions are fundamental to any chemical or biological processes, and their rates define the operational sequence and control for any desirable product. Here, we deliberate on a recently developed novel fluorescence spectroscopic method, which combines fluorescence photon anti-bunching, photon bunching, time-correlated single-photon counting (TCSPC), and steady-state fluorescence spectroscopy, to study composite chemical reactions with single molecule sensitivity. The proposed method captures the full picture of the multifaceted quenching kinetics, which involves static quenching by ground state complexation and collisional quenching in the excited state under dynamic exchange of fluorophore in a heterogeneous media, and which cannot be seen by steady-state or lifetime measurements alone. Photon correlation in fluorescence correlation spectroscopy (FCS) provides access to interrogate interaction dynamics from picosecond to seconds, stitching all possible stages of dye-quencher interaction in a micellar media. This is not possible with the limited time window available to conventional ensemble techniques like TCSPC, flash photolysis, transient absorption, stop-flow, etc. The basic premises of such unified global analysis and sanctity of extracted parameters critically depends on the minimum but precise description of reaction scheme, for which careful inspection of ensemble spectroscopy data for photo-physical behaviour is very important. Though in this contribution we discussed and demonstrated the merits of photon antibunching and bunching spectroscopy for dye-quencher interaction in cationic cetyltrimethylammonium bromide (CTAB) micellar solution by photo-induced electron transfer mechanism and the influence of micellar charge and microenvironment on the interaction kinetics, but in principal similar arguments are equally applicable to any other interaction mechanisms which alter fluorescence photon correlations, like Förster resonance energy transfer (FRET), proton transfer, isomerisation, etc.

Picosecond to Second Fluorescence Correlation Spectroscopy for Studying Solute Exchange and Quenching Dynamics in Micellar Media.

Numerous studies have been devoted to understand the reaction kinetics in micelles, where the accessible kinetic time window is often limited by the dynamic range of the employed spectroscopic technique. This is usually accompanied by a selection of probes that comfortably explore time scales where slow solute exchange kinetics is negligible, as compared to the fast excited state reactions. This has led to an undervaluation of the role played by dynamic partitioning of hydrophilic solutes in microheterogeneous media. Here, we employ fluorescence correlation spectroscopy (FCS) and the zwitterionic dye Rhodamine 110 to quantitatively explore the impact of solute exchange on the photoinduced electron transfer between this dye and N,N-dimethylaniline in micellar media. Our study elucidates the coupling and interplay between the kinetics of photophysics, quenching, and solute exchange through a quantitative unified molecular-state quenching-kinetic model that describes the steady-state ensemble and FCS data from subnanosecond photon antibunching to millisecond diffusions.

Binding Constant Determined from the Angstrom-Scale Change in Hydrodynamic Radius of Transferrin upon Binding with Europium Using Dual-Focus Fluorescence Correlation Spectroscopy.

Monitoring the binding of a large fluorescently tagged molecule to a small solute by fluorescence correlation spectroscopy (FCS) is rather uncommon because the binding-related change in diffusion coefficient is very small. Here, we use a high-precision variant of FCS, namely, dual-focus FCS (2fFCS), for measuring the angstrom-scale change of the hydrodynamic radius of the bilobal metal transport protein transferrin (Tf) upon binding europium ions. Applying a sequential 1:2 complexation model, we use these measurements for determining the binding constants (K). Our results show a 0.7 Å change of the protein's hydrodynamic radius upon 1:1 Tf-Eu complex formation and a second change of 1.8 Å upon subsequent binding of a second europium ion. More than one unit variation in logK indicates an intrinsic dissimilarity in metal affinity of the C- and N-lobes of Tf, which agrees well with earlier reported ensemble spectroscopy results.

Determining Metal Ion Complexation Kinetics with Fluorescent Ligands by Using Fluorescence Correlation Spectroscopy.

Fluorescence correlation spectroscopy (FCS) has been extensively used to measure equilibrium binding constants (K) or association and dissociation rates in many reversible chemical reactions across chemistry and biology. For the majority of investigated reactions, the binding constant was on the order of ∼100 M-1 , with dissociation constants faster or equal to 103  s-1 , which ensured that enough association/dissociation events occur during the typical diffusion-determined transition time of molecules through the FCS detection volume. However, complexation reactions involving metal ions and chelating ligands exhibit equilibrium constants exceeding 104  M-1 . In the present paper, we explore the applicability of FCS for measuring reaction rates of such complexation reactions, and apply it to binding of iron, europium and uranyl ions to a fluorescent chelating ligand, calcein. For this purpose, we exploit the fact that the ligand fluorescence becomes strongly quenched after binding a metal ion, which results in strong intensity fluctuations that lead to a partial correlation decay in FCS. We also present measurements for the strongly radioactive ions of 241 Am3+ , where the extreme sensitivity of FCS allows us to work with sample concentrations and volumes that exhibit close to negligible radioactivity levels. A general discussion of the applicability of FCS to the investigation of metal-ligand binding reactions concludes our paper.