Here, we use single-molecule techniques to study the aggregation of α-synuclein, protein whose misfolding and deposition is associated with Parkinson's disease. We identify a conformational change from initially formed oligomers stable, more compact proteinase-K-resistant as key step that leads ultimately fibril formation. The result structural conversion generate much higher levels oxidative stress in rat primary neurons than do initially, showing they are damaging cells. remarkably slow,...

A key issue in understanding the pathogenic conditions associated with aberrant aggregation of misfolded proteins is identification and characterization species formed during process. Probing nature such has, however, proved to be extremely challenging conventional techniques because their transient heterogeneous character. We describe here application a two-color single-molecule fluorescence technique examine assembly oligomeric SH3 domain PI3 kinase. The experiments show that at stage...

Caught in a trap and on camera: A nanopipette is used for electrodeless dielectrophoresis clear evidence shown, by using wide-field fluorescence imaging (see image), the reversible trapping of Alexa-488-labeled proteins (protein G immunoglobulin G) also fluorophore alone. The dielectrophoretic concentration enhanced at least factor 300 these fluorophore-labeled proteins. Supporting Information this article available WWW under http://www.wiley-vch.de/contents/jc_2002/2005/z500196_s.html or...

The T cell receptor (TCR) expressed on most cells is a protein complex consisting of TCRalphabeta heterodimers that bind antigen and cluster differentiation (CD) 3epsilondelta, epsilongamma, zetazeta dimers initiate signaling. A long-standing controversy concerns whether there one, or more than alphabeta heterodimer per complex. We used form single-molecule spectroscopy to investigate this question live hybridomas. method relies detecting coincident fluorescence from single molecules labeled...

We report the measurement of surface conductivity biological macromolecules by dielectrophoretic trapping at tip a glass nanopipet. find that threshold voltage for is function salt concentration and can be directly linked to effective biomolecule its solvation shell. The conductivities obtained 20-mer single-stranded DNA, 40-mer double-stranded yellow fluorescent protein are $7.9\ifmmode\pm\else\textpm\fi{}1.9\text{ }\text{ }\mathrm{nS}$, $5.3\ifmmode\pm\else\textpm\fi{}0.7\text{...

The results of some recent observations radio sources have been used to investigate the "luminosity function" for sources. Without making any assumptions about their nature, it can be shown from alone that most lying in given range flux density are extra-galactic with an emission at 178 Mc/s which exceeds 10 24 watts (c/s) −1 ster . If is assumed either physical dimensions or optical luminosities comparable those Galaxy, then must lie 3 × 25 –10 27

We have extended the method of single-molecule fluorescence, two-color coincidence detection (TCCD) to detect coincident events due a low fraction complex against background chance events, monomers. developed two complementary methods determine number using experimental data and without need for additional experiments. show that subtraction level is essential accurate quantification relative complexes their stoichiometry. By performing experiments on model samples made from...

There is an intrinsic repulsion between glass and cell surfaces that allows noninvasive scanning ion conductance microscopy (SICM) of cells which must be overcome in order to form the gigaseals used for patch clamping investigations channels. However, interactions physiological solutions electrolytes, including presence this repulsion, example, do not obviously agree with standard Derjaguin-Landau-Verwey-Overbeek (DLVO) colloid theory accurate at much lower salt concentrations. In paper we...

Directly examining subcellular mechanics whilst avoiding excessive strain of a live cell requires the precise control light stress on very small areas, which is fundamentally difficult. Here we use glass nanopipet out contact with plasma membrane to both exert and also accurately monitor cellular compression. This allows mapping stiffness at lateral resolution finer than 100 nm. We calculate exerts as sum intrinsic pressure between tip face plus its direct any glycocalyx, evaluated from gap...

A fluorescent nanosensor based on reporter dye molecules trapped in the tip of a nanopipette has been developed. This 100 nm sized shown to be capable measuring local pH and mapping sodium concentration with temporal resolution few milliseconds.

We have experimentally determined the optimal flow velocities to characterize or count single molecules by using a simple microfluidic device perform two-color coincidence detection (TCCD) and pair Förster resonance energy transfer (spFRET) confocal fluorescence spectroscopy on traveling at speeds of up 10 cm s–1. show that flowing fluorophores ≥0.5 s–1 reduces photophysical processes competing with fluorescence, enabling use high excitation irradiances partially compensate for short...

In this work we show how hydrodynamic forces can be used to locally trap molecules in a supported lipid bilayer (SLB). The method uses the drag arising from flow through conical pipette with tip radius of 1–1.5 μm, placed approximately 1 μm above investigated SLB. This results localized forcefield that acts on protruding SLB, yielding size given by tip. We demonstrate concept trapping protein streptavidin, bound biotin receptors It is also shown static and kinetic information about...

Telomerase is a nonclassical DNA polymerase that uses its integral RNA as template to synthesize telomeric repeats onto chromosome ends. The molecular mechanism of telomerase unique and involves translocation step after the synthesis each repeat. To directly measure enzymatic turnover substrate efficiency we have extended our two-color single molecule fluorescence coincidence method (Anal.Chem. 2003, 75, 1664−1670). employs Cy5-dATP incorporation into primer has been prelabeled with...

We discuss how the ideal formalism of computational mechanics can be adapted to apply a noninfinite series corrupted and correlated data, that is typical most observed natural time series. Specifically, simple filter removes corruption creates rare unphysical causal states demonstrated, concept effective soficity introduced. believe cannot applied noisy finite data without invoking an argument based upon soficity. A related distinction between noise unresolved structure also defined: Noise...

In der Falle und mit Kamera eingefangen: Mit einer Nanopipette für die elektrodenlose Dielektrophorese ließen sich Alexa-488-markierte Proteine (Protein G Immunglobulin G) das freie Fluorophor reversibel einfangen, wie Weitfeld-Fluoreszenz-Imaging (siehe Bild) eindeutig belegt. Die dielektrophoretische Konzentration steigt bei diesen Fluorophor-markierten Proteinen um wenigstens den Faktor 300 an. Supporting Information for this article is available on the WWW under...

Two-color coincidence detection is a single-molecule fluorescence technique that capable of resolving subpopulations biomolecular complexes at very low concentrations. In this paper, we have developed method automatically determines appropriate thresholds for the analysis sets two-color data. This has distinct advantage allowing rapid determination optimized in reproducible fashion. The trade-offs involved such selections are should be high enough both to exceed background photon count rates...

Optogenetic actuators have revolutionized the resolution at which biological processes can be controlled. In plants, deployment of optogenetics is challenging due to need for these light-responsive systems function in context horticultural light environments. Furthermore, many available optogenetic are based on plant photoreceptors that might crosstalk with endogenous signaling processes, while others depend exogenously supplied cofactors. To overcome such challenges, we developed...

Cells naturally operate on the nanoscale level, with molecules combining together to form complex molecular machines, which can work enable normal cell function or go wrong as in case of many diseases. Visualizing these key processes has been difficult and two main approaches have used date; nanometer resolution imaging fixed cells using electron microscopy, live optical fluorescence a few hundred nanometers. Scanning probe microscopy potential allow be imaged at noncontact method based use...

Single-molecule fluorescence resonance energy transfer (FRET) is commonly used to probe different conformations and conformational dynamics of single biomolecules. However, the analysis raw burst traces not always straightforward. The presence a "zero peak" skewness peaks at high low FRET efficiencies in proximity ratio histograms make accurate evaluation histogram challenging task. This further compounded by difficulty associated with siting two fluorophores optimal range each other. Here...

This paper discusses physical and structural aspects of the mechanisms herpes simplex virus (HSV) uses for membrane fusion. Calculations show that glycoprotein D has such avidity its receptors it can hold virion against plasma a neuron strongly enough B (gB) to disrupt both leaflets bilayer. The strong electric field generated by cell potential across perforations at this disruption would break hydrogen bonds securing gB fusion loops, leading viral membranes. mechanism agrees with high...