The self-assembly of peptides is ak ey direction for fabrication of advanced materials.N ovel approaches for fine tuning of macroscopic and microscopic properties of peptide self-assemblies are of ah igh demand for constructing biomaterials with desired properties.Inthis work, while studying the kinetics of the Fmoc-Diphenylalanine (Fmoc-FF) dipeptide self-assembly using the Thioflavin T( ThT) dye,w eo bserved that the presence of ThT strongly modifies structural and mechanical properties of the Fmoc-FF hydrogel. Notably,t he presence of ThT resulted in at enfold increase of the gelation time and in the formation of short and dense fibers in the hydrogel. As ar esult of these morphological alteration higher thermal stability,a nd most important, tenfold increase of the hydrogel rigidity was achieved. Hence,T hT not only slowed the kinetics of the Fmoc-FF hydrogel formation, but also strongly enhanced its mechanical properties.I nt his study,w e provideadetailed description of the ThT effect on the hydrogel properties and suggest the mechanisms for this phenomenon, paving the wayf or the novel approach to the control of the peptide hydrogels micro-and macroscale properties.
The aggregation of red blood cells (RBC) is of importance for hemorheology, while its mechanism remains debatable. The key question is the role of the adsorption of macromolecules on RBC membranes, which may act as "bridges" between cells. It is especially important that dextran is considered to induce "bridge"-less aggregation due to the depletion forces. We revisit the dextran-RBC interaction on the single cell level using the laser tweezers combined with microfluidic technology and fluorescence microscopy. An immediate sorption of ~10 molecules of 70 kDa dextran per cell was observed. During the incubation of RBC with dextran, a gradual tenfold increase of adsorption was found, accompanied by a moderate change in the RBC deformability. The obtained data demonstrate that dextran sorption and incubation-induced changes of the membrane properties must be considered when studying RBC aggregation .
Fluorescent dye Thioflavin T (ThT) is a widely used probe for the detection of amyloid fibrils, which are protein aggregates involved in the pathogenesis of neurodegenerative disorders. Upon the formation of a complex with amyloids, the fluorescence quantum yield of ThT increases 1000-fold due to a dramatic reduction of the nonradiative decay rate. This is accompanied by a remarkable change of ThT fluorescence lifetime τ from ~1 to ~1000 ps, thus making it possible to assess ThT binding to different systems using τ as an indicator. However, when measuring ThT interaction with proteins, one can observe that the binding affinity determined from the ThT fluorescence intensity's dependence on protein concentration may be orders of magnitude lower than that determined using τ. Here we show that this discrepancy at least partly originates from a limited temporal resolution when determining the fluorescence lifetime of ThT in the ThT-protein system using the time-correlated single photon counting technique (TCSPC), which is usually characterized by a ~100 ps instrument response function. This results in the situation when a small fraction (~1%) of ThT molecules with a relatively slow decay (τ ~ 1000 ps) completely disguises the impact of ThT molecules with an ultrafast decay (τ ~ 1 ps) to the overall measured fluorescence decay curve. Moreover, using the femtosecond-resolved fluorescence up-conversion technique, we demonstrate that not only free ThT molecules but also a subpopulation of protein-bound ThT molecules exhibits fluorescence decay on a 1 ps timescale. The obtained results are of critical importance for a reliable interpretation of protein binding and aggregation experiments when using a ThT assay Astro Ltd
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