Single-Molecule Characterization of Photophysical and Colloidal Properties of Biocompatible Quantum Dots

Colloidal semiconductor nanocrystals (NCs) have recently been introduced as novel fluorescent labels for various biological applications. Their unique optical properties — tunable narrow emission spectrum, broad excitation spectrum, high photostability and long fluorescent lifetimes (on the order of tens of nanoseconds) — make them attractive probes in experiments involving long observation times, multicolor and time-gated detection. Photophysical properties were investigated at the ensemble and single-molecule level for CdSe or CdTe core, CdSe/ZnS core-shell and surface-modified NCs. The use of NCs as fluorescent probes in biological applications requires various synthesis routes and surface modifications to enable solubility in aqueous solution and to allow labeling of biological macromolecules. Due to NC’s sensitivity to surface-defects chemical treatments have a significant influence on photophysical properties and need to be thoroughly monitored. Single-molecule fluorescence detection was used to characterize NC fluorescence, observe intermittency in the emission (blinking), and unravel a non-fluorescent subpopulation of NCs whose fluorescence can be restored through photo-induced activation. Stage-scanning confocal, epifluorescence and objective-type total-internal-reflection microscopy, were applied to observe surface-immobilized NCs. Fluorescence lifetimes were determined to be around 20 ns for single particles showing deviations from a mono-exponential decay. This observation was proven to be characteristic of single particles by monitoring photon antibunching. Fluorescence correlation spectroscopy (FCS) was used to characterize photophysical and colloidal properties in solution. It was shown to be a powerful technique to rapidly evaluate information on synthesis and surface modifications. These are essential to achieve water-solubility and bio-conjugation and dramatically influence the optical performance. FCS allows measuring concentration of fluorescent particles, an average brightness and particle size. From these observables, the brightness per particle can be estimated, something not possible in ensemble measurements due to the presence of absorbing but dark particles. The ratio of dark to fluorescent NCs was estimated and concentration changes due to photo-induced activation were observed. Particle sizes measured by FCS were compared to transmission electron microscopy and found in good agreement down to 7 nm. The correlation amplitude was observed to be excitation power dependent which was attributed to saturation and optical trapping effects. An electronic polarizability was evaluated and found to be two orders of magnitude larger than reported for measurements at non-resonant wavelength. Monte-Carlo simulations (MCS) were performed to compute autocorrelation functions under the influence of power-law blinking. Diffusion through a confocal volume, excitation-emission cycles with defined rate constants and on/off blinking were incorporated into MCS and used to investigate influences of saturation, size-distributions, photobleaching and blinking. The results were compared to experimental data of various NCs. Simulations account for both types of experimentally observed effects of blinking in FCS curves: significant deviation from a diffusion-model observed at high excitation powers; and no deviation from a diffusion-model despite the existence of blinking, observed at low excitation powers. Simulations showed that blinking does not influence FCS data for certain power law parameters

Sensing ligand binding to a clinically relevant lectin by tryptophan fluorescence anisotropy

Increasing insights into the involvement of endogenous lectins in disease processes fuel the interest to develop potent inhibitors. As a consequence, robust assay procedures are required. Due to their activity as adhesion/growth-regulatory effectors this study focussed on galectins. The human proto-type galectin-1 was selected as representative of this family with conserved presence of a tryptophan moiety in the binding site. This structural feature was taken advantage of to establish its use as reporter for ligand contact measuring polarized fluorescence emission. The experimentally determined anisotropy r0 was about 0.2, altered by about 5% in the presence of the cognate disaccharide lactose. This parameter change enabled calculating the equilibrium binding constant and kinetic rate constants. The detailed analysis of the depolarization process further indicated fast conformational dynamics within the binding site. Since an inherent property of the protein was exploited, no labeling is needed. Owing to tryptophan’s presence in carbohydrate-binding sites, also in other classes of lectins as well as in carbohydrate-binding modules and glycoenzymes (glycosyltransferases, glycosidases), this assay procedure can have relevance beyond galectins

Upregulation of CD38 expression on multiple myeloma cells by novel HDAC6 inhibitors is a class effect and augments the efficacy of daratumumab

Multiple myeloma (MM) is incurable, so there is a significant unmet need for effective therapy for patients with relapsed or refractory disease. This situation has not changed despite the recent approval of the anti-CD38 antibody daratumumab, one of the most potent agents in MM treatment. The efficiency of daratumumab might be improved by combining it with synergistic anti-MM agents. We therefore investigated the potential of the histone deacetylase (HDAC) inhibitor ricolinostat to up-regulate CD38 on MM cells, thereby enhancing the performance of CD38-specific therapies. Using quantitative reverse transcription polymerase chain reaction and flow cytometry, we observed that ricolinostat significantly increases CD38 RNA levels and CD38 surface expression on MM cells. Super-resolution microscopy imaging of MM cells by direct stochastic optical reconstruction microscopy confirmed this rise with molecular resolution and revealed homogeneous distribution of CD38 molecules on the cell membrane. Particularly important is that combining ricolinostat with daratumumab induced enhanced lysis of MM cells. We also evaluated next-generation HDAC6 inhibitors (ACY-241, WT- 161) and observed similar increase of CD38 levels suggesting that the upregulation of CD38 expression on MM cells by HDAC6 inhibitors is a class effect. This proof-of-concept illustrates the potential benefit of combining HDAC6 inhibitors and CD38-directed immunotherapy for MM treatme

Hydrogen-Bond Driven Loop-Closure Kinetics in Unfolded Polypeptide Chains

Characterization of the length dependence of end-to-end loop-closure kinetics in unfolded polypeptide chains provides an understanding of early steps in protein folding. Here, loop-closure in poly-glycine-serine peptides is investigated by combining single-molecule fluorescence spectroscopy with molecular dynamics simulation. For chains containing more than 10 peptide bonds loop-closing rate constants on the 20–100 nanosecond time range exhibit a power-law length dependence. However, this scaling breaks down for shorter peptides, which exhibit slower kinetics arising from a perturbation induced by the dye reporter system used in the experimental setup. The loop-closure kinetics in the longer peptides is found to be determined by the formation of intra-peptide hydrogen bonds and transient β-sheet structure, that accelerate the search for contacts among residues distant in sequence relative to the case of a polypeptide chain in which hydrogen bonds cannot form. Hydrogen-bond-driven polypeptide-chain collapse in unfolded peptides under physiological conditions found here is not only consistent with hierarchical models of protein folding, that highlights the importance of secondary structure formation early in the folding process, but is also shown to speed up the search for productive folding events

Locan

<p>locan 0.17.0</p>If you use this software, please cite it as below

Super-resolution microscopy reveals local accumulation of plasma membrane gangliosides at Neisseria meningitidis Invasion Sites

Neisseria meningitidis (meningococcus) is a Gram-negative bacterium responsible for epidemic meningitis and sepsis worldwide. A critical step in the development of meningitis is the interaction of bacteria with cells forming the blood-cerebrospinal fluid barrier, which requires tight adhesion of the pathogen to highly specialized brain endothelial cells. Two endothelial receptors, CD147 and the β2-adrenergic receptor, have been found to be sequentially recruited by meningococci involving the interaction with type IV pilus. Despite the identification of cellular key players in bacterial adhesion the detailed mechanism of invasion is still poorly understood. Here, we investigated cellular dynamics and mobility of the type IV pilus receptor CD147 upon treatment with pili enriched fractions and specific antibodies directed against two extracellular Ig-like domains in living human brain microvascular endothelial cells. Modulation of CD147 mobility after ligand binding revealed by single-molecule tracking experiments demonstrates receptor activation and indicates plasma membrane rearrangements. Exploiting the binding of Shiga (STxB) and Cholera toxin B (CTxB) subunits to the two native plasma membrane sphingolipids globotriaosylceramide (Gb3) and raft-associated monosialotetrahexosylganglioside GM1, respectively, we investigated their involvement in bacterial invasion by super-resolution microscopy. Structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM) unraveled accumulation and coating of meningococci with GM1 upon cellular uptake. Blocking of CTxB binding sites did not impair bacterial adhesion but dramatically reduced bacterial invasion efficiency. In addition, cell cycle arrest in G1 phase induced by serum starvation led to an overall increase of GM1 molecules in the plasma membrane and consequently also in bacterial invasion efficiency. Our results will help to understand downstream signaling events after initial type IV pilus-host cell interactions and thus have general impact on the development of new therapeutics targeting key molecules involved in infection

Dynamics of unfolded polypeptide chains in crowded environment studied by fluorescence correlation spectroscopy

Neuweiler H, Löllmann M, Doose S, Sauer M. Dynamics of unfolded polypeptide chains in crowded environment studied by fluorescence correlation spectroscopy. JOURNAL OF MOLECULAR BIOLOGY. 2007;365(3):856-869.Proteins have evolved to fold and function within a cellular environment that is characterized by high macromolecular content. The earliest step of protein folding represents intrachain contact formation of amino acid residues within an unfolded polypeptide chain. It has been proposed that macromolecular crowding can have significant effects on rates and equilibria of biomolecular processes. However, the kinetic consequences on intrachain diffusion of polypeptides, have not been tested experimentally, yet. Here, we demonstrate that selective fluorescence quenching of the oxazine fluorophore MR121 by the amino acid tryptophan (Trp) in combination with fast fluorescence correlation spectroscopy (FCS) can be used to monitor end-to-end contact formation rates of unfolded polypeptide chains. MR121 and Trp were incorporated at the terminal ends of polypeptides. consisting of repetitive units of glycine (G) and serine (S) residues. End-to-end contact formation and dissociation result in "off" and "on" switching of MR121 fluorescence and underlying kinetics can be revealed in FCS experiments with nanosecond time resolution. We revisit previous experimental studies concerning the dependence of end-to-end contact formation rates on polypeptide chain length, showing that kinetics can be described by Gaussian chain theory. We further investigate effects of solvent viscosity and temperature on contact formation rates demonstrating that intrachain diffusion represents a purely diffusive, entropy-controlled process. Finally, we study the influence of macromolecular crowding on polypepticle chain dynamics. The data presented demonstrate that intrachain diffusion is fast in spite of hindered diffusion caused by repulsive interactions with macromolecules. Findings can be explained by effects of excluded volume reducing chain entropy and therefore accelerating the loop search process. Our results suggest that within a cellular environment the early formation of structural elements in k unfolded proteins can still proceed quite efficiently in spite of hindered L diffusion caused by high macromolecular content. (c) 2006 Elsevier Ltd. All rights reserved