A Four-Amino Acid Linker between Repeats in the α‑Synuclein Sequence Is Important for Fibril Formation

α-Synuclein is a 140-amino acid protein that can switch conformation among intrinsically disordered in solution, helical on a membrane, and β-sheet in amyloid fibrils. Using the fluorescence of single-tryptophan mutants, we determined the immersion of different regions of the protein into lipid membranes. Our results suggest the presence of a flexible break close to residues 52–55 between two helical domains. The four-amino acid linker is not necessary for membrane binding but is important for fibril formation. A deletion mutant lacking this linker aggregates extremely slowly and slightly inhibits wild-type aggregation, possibly by blocking the growing ends of fibrils

Self-Assembly of Protein Fibrils into Suprafibrillar Aggregates: Bridging the Nano- and Mesoscale

We report on <i>in vitro</i> self-assembly of nanometer-sized α-synuclein amyloid fibrils into well-defined micrometer-sized suprafibrillar aggregates with sheet-like or cylindrical morphology depending on the ionic strength of the solution. The cylindrical suprafibrillar structures are heavily hydrated, suggesting swollen gel-like particles. In contrast to higher order structures formed by other negatively charged biopolymers, multivalent ions are not required for the suprafibrillar aggregates to form. Their formation is induced by both mono- and divalent counterions. The self-assembly process is not mediated by protein-specific interactions but rather by the cooperative action of long-range electrostatic repulsion and short-range attraction. Understanding the mechanism driving the self-assembly might give us valuable insight into the pathological formation of fibrillar superstructures such as Lewy bodies and neuritesdistinct signatures of Parkinson’s diseaseand will open the possibility to utilize the self-assembly process for the design of novel fibril-based smart nanostructured materials

Oriented Protein Immobilization using Covalent and Noncovalent Chemistry on a Thiol-Reactive Self-Reporting Surface

We report the fabrication of a patterned protein array using three orthogonal methods of immobilization that are detected exploiting a fluorogenic surface. Upon reaction of thiols, the fluorogenic tether reports the bond formation by an instantaneous rise in (blue) fluorescence intensity providing a means to visualize the immobilization even of nonfluorescent biomolecules. First, the covalent, oriented immobilization of a visible fluorescent protein (TFP) modified to display a single cysteine residue was detected. Colocalization of the fluorescence of the immobilized TFP and the fluorogenic group provided a direct tool to distinguish covalent bond formation from physisorption of proteins. Subsequent orthogonal immobilization of thiol-functionalized biomolecules could be conveniently detected by fluorescence microscopy using the fluorogenic surface. A thiol-modified nitrilotriacetate ligand was immobilized for binding of hexahistidine-tagged red-fluorescing TagRFP, while an appropriately modified biotin was immobilized for binding of Cy5-labeled streptavidin

Super-resolution images of internalized α-syn aggregates in endosomal vesicles in time.

<p>(a) dSTORM image of a cell treated for half an hour with α-syn -Alexa532 aggregates. A detailed view of the aggregates in the cell membrane is shown below a). (b) After 2 hours of incubation, α-syn aggregates are internalized in vesicles. Detailed view of the aggregates in a vesicle shown in the image below b). (c) Internalized α-syn aggregates after 24 hours of incubation, with two different sized clusters highlighted bellow image c).</p

Size distribution of α-syn aggregates in endosomal vesicles in time.

<p>(a)-(c) Histogram of FWHM of intracellular α-syn clusters in time. (ANOVA significance levels: (a)-(b): 10⁻⁴; (b)-(c):5×10⁻³; ((a)-(c):10⁻⁷). (d) A decrease in α-syn cluster size is observed in the mean average FWHM of α-syn clusters in time (median and 50% interval).</p

Internalization of α-syn sonicated fibrils in human neuroblastoma cells.

<p>Images show co-localization of Alexa 532 labeled α-syn aggregates (green) with LysoTracker Deep Red (red). SH-SY5Y cells were treated with 50 nM LysoTracker Deep Red, then washed, incubated further with Alexa532-labeled α-syn sonicated fibrils and imaged live on a confocal microscope.</p

Super-resolution imaging of the <i>in vitro</i> prepared α-syn fibrils.

<p>(a) AFM and (b) dSTORM images of intact wild-type α-syn fibrils covalently labeled with the NHS derivate of Alexa 532 fluorophore. (c) AFM and (d) dSTORM images of sonicated labeled α-syn fibrils.</p

Silver Nanoparticle Aggregates as Highly Efficient Plasmonic Antennas for Fluorescence Enhancement

The enhanced local fields around plasmonic structures can lead to enhancement of the excitation and modification of the emission quantum yield of fluorophores. So far, high enhancement of fluorescence intensity from dye molecules was demonstrated using bow-tie gap antenna made by e-beam lithography. However, the high manufacturing cost and the fact that currently there are no effective ways to place fluorophores only at the gap prevent the use of these structures for enhancing fluorescence-based biochemical assays. We report on the simultaneous modification of fluorescence intensity and lifetime of dye-labeled DNA in the presence of aggregated silver nanoparticles. The nanoparticle aggregates act as efficient plasmonic antennas, leading to more than 2 orders of magnitude enhancement of the <i>average</i> fluorescence. This is comparable to the best-reported fluorescence enhancement for a single molecule but here applies to the average signal detected from all fluorophores in the system. This highlights the remarkable efficiency of this system for surface-enhanced fluorescence. Moreover, we show that the fluorescence intensity enhancement varies with the plasmon resonance position and measure a significant reduction (300×) of the fluorescence lifetime. Both observations are shown to be in agreement with the electromagnetic model of surface-enhanced fluorescence