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.

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.