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

Locan

<p>locan 0.19.0</p>If you use this software, please cite it as below

Locan

<p>locan 0.18.0</p>If you use this software, please cite it as below

Locan

<p>locan 0.18.0</p>If you use this software, please cite it as below