DNA double-strand breaks (DSBs) are repaired at DSB sites. How sites assemble and how broken is prevented from separating not understood. Here we uncover that the synapsis of mediated by sensor protein poly(ADP-ribose) (PAR) polymerase 1 (PARP1). Using bottom-up biochemistry, reconstitute functional show form through co-condensation PARP1 multimers with DNA. The co-condensates exert mechanical forces to keep ends together become enzymatically active for PAR synthesis. PARylation promotes...

Protein conformational transitions form the molecular basis of many cellular processes, such as signal transduction and membrane traffic. However, in cases, little is known about their structural dynamics. Here we have used dynamic single-molecule fluorescence to study at high time resolution, syntaxin 1, a soluble N -ethylmaleimide-sensitive factor attachment protein receptors essential for exocytotic fusion. Sets double mutants were randomly labeled with mix donor acceptor dye resonance...

We establish a probability distribution analysis (PDA) method for the of fluorescence resonance energy transfer (FRET) signals to determine with high precision originating value shot-noise-limited signal distribution. PDA theoretical distributions are calculated explicitly including crosstalk, stochastic variations, and background represent minimum width that FRET must have. In this way an unambiguous distinction is made between shot-noise broadened by heterogeneities. This simultaneously...

By using single-molecule multiparameter fluorescence detection, resonance energy transfer experiments, and newly developed data analysis methods, this study demonstrates directly the existence of three structurally distinct forms reverse transcriptase (RT):nucleic acid complexes in solution. Single-molecule detection also provides first information on structure a complex not observed by x-ray crystallography. This species did incorporate nucleotides is from other two species. We determined...

Two complementary methods in confocal single-molecule fluorescence spectroscopy are presented to analyze conformational dynamics by Förster resonance energy transfer (FRET) measurements considering simulated and experimental data. First, an extension of photon distribution analysis (PDA) is applied characterize exchange between two or more states via global the shape FRET peaks for different time bins. PDA accurately predicts efficiency histograms presence fluctuations, taking into account...

The nucleosome has a central role in the compaction of genomic DNA and control accessibility for transcription replication. To help understanding mechanism opening closing these processes, we studied disassembly mononucleosomes by quantitative single-molecule FRET with high spatial resolution, using SELEX-generated “Widom 601” positioning sequence labeled donor acceptor fluorophores. Reversible dissociation was induced increasing NaCl concentration. At least 3 species different were...

Abstract The dynamic architecture of chromatin fibers, a key determinant genome regulation, is poorly understood. Here, we employ multimodal single-molecule Förster resonance energy transfer studies to reveal structural states and their interconversion kinetics in fibers. We show that nucleosomes engage short-lived (micro- milliseconds) stacking interactions with one neighbors. This results discrete tetranucleosome units distinct interaction registers interconvert within hundreds...

GBPs are essential for immunity against intracellular pathogens, especially Toxoplasma gondii control. Here, the molecular interactions of murine (mGBP1/2/3/5/6), homo- and hetero-multimerization properties mGBP2 its function in parasite killing were investigated by mutational, Multiparameter Fluorescence Image Spectroscopy, live cell microscopy methodologies. Control T. replication requires GTP hydrolysis isoprenylation thus, enabling reversible oligomerization vesicle-like structures....

Abstract An analysis method of lifetime, polarization and spectrally filtered fluorescence correlation spectroscopy, referred to as FCS (fFCS), is introduced. It uses, but not limited to, multiparameter detection differentiate between molecular species with respect their spectral information. Like the recently introduced lifetime spectroscopy (FLCS) [ Chem. Phys. Lett. 2002 , 353 439–445], fFCS based on pulsed laser excitation. However, it uses species‐specific resolved decays generate...

In plants, the flagellin and CLAVATA3 signaling pathways act through induced preassembled receptor complexes, respectively.

Abstract We use a hybrid fluorescence spectroscopic toolkit to monitor T4 Lysozyme (T4L) in action by unraveling the kinetic and dynamic interplay of conformational states. In particular, combining single-molecule ensemble multiparameter detection, EPR spectroscopy, mutagenesis, FRET-positioning screening, other biochemical biophysical tools, we characterize three short-lived states over ns-ms timescale. The 33 FRET-derived distance sets, screen available T4L structures, reveal that solution...

Conformational dynamics of biomolecules are fundamental importance for their function. Single-molecule studies Förster Resonance Energy Transfer (smFRET) between a tethered donor and acceptor dye pair powerful tool to investigate the structure labeled molecules. However, capturing quantifying conformational in intensity-based smFRET experiments remains challenging when occur on sub-millisecond timescale. The method multiparameter fluorescence detection addresses this challenge by...

Abstract Phase separation and percolation contribute to phase transitions of multivalent macromolecules. Contributions are evident through the viscoelasticity condensates formation heterogeneous distributions nano- mesoscale pre-percolation clusters in sub-saturated solutions. Here, we show that formed solutions FET (FUS-EWSR1-TAF15) proteins affected differently by glutamate versus chloride. These differences on nanoscale, gleaned using a suite methods deployed across wide range protein...

We present an advanced time-correlated single photon counting (TCSPC) technique that delivers traditional fluorescence correlation (FCS) or cross (FCCS) and lifetime data simultaneously. Newly developed electronics allow for detection registration of events over time periods hours with picoseconds accuracy. Subsequent software-correlation yields curves covering more than 12 orders magnitude in time. At the same time, original data, containing all information accessible by techniques, can be...

Dye photobleaching is a major constraint of fluorescence readout within range applications. In this study, we investigated the influence in experiments applying multicolor laser as well Förster resonance energy transfer (FRET) mediated excitation using several red-emitting dyes frequently used or FRET acceptors. The chosen (cyanine 5 (Cy5), MR121, Alexa660, Alexa680, Atto647N, Atto655) have chemically distinct chromophore systems and can be excited at 650 nm. Several analysis techniques been...

The control of supramolecular systems requires a thorough understanding their dynamics on molecular level. We present fluorescence correlation spectroscopy (FCS) as powerful spectroscopic tool to study with single molecule sensitivity. formation complex between β-cyclodextrin (β-CD) host and pyronines Y (PY) B (PB) guests is studied by FCS. Global target analysis full curves newly derived theoretical model yields in experiment the lifetimes diffusion coefficients free complexed rate...

Single-molecule FRET experiments on freely diffusing rigid molecules frequently show efficiency (E) distributions broader than those defined by photon statistics. It is often unclear whether the observed extra broadening can be attributed to a physical donor−acceptor distance (RDA) distribution. Using double-stranded DNA (dsDNA) labeled with Alexa488 and Cy5 (or Alexa647) as test system, we investigate various possible contributions E distribution width. On basis of simultaneous analysis...

Characterization of polyacrylamide hydrogels with dextran host molecules using four complementary methods.

Nucleosomes play a dual role in compacting the genome and regulating access to DNA. To unravel underlying mechanism, we study fluorescently labeled mononucleosomes by multi-parameter FRET measurements characterize their structural dynamic heterogeneity upon NaCl-induced destabilization. Species-selective fluorescence lifetime analysis photon distribution reveal intermediates during nucleosome opening lead coherent kinetic model. In octasomes hexasomes interface between H2A-H2B dimers...

p27$^{Kip1}$ (p27) is an intrinsically disordered protein (IDP) that folds upon binding to cyclin-dependent kinase (Cdk)$/$cyclin complexes (e.g., Cdk2$/$cyclin A), inhibiting their catalytic activity and causing cell cycle arrest. However, division progresses when stably A-bound p27 phosphorylated on one or two structurally occluded tyrosine residues $[$tyrosines 88 (Y88) 74 (Y74)$]$ a distal threonine residue $[$threonine 187 (T187)$]$. These events trigger ubiquitination degradation of...

Analysis of anisotropy in single-molecule fluorescence experiments using the probability distribution analysis (PDA) method is presented. The theory anisotropy-PDA an extension PDA recently developed for Förster resonance energy transfer (FRET) signals [Antonik, M.; et al. J. Phys. Chem. B 2006, 110, 6970]. predicts shape histograms any given expected ensemble anisotropy, signal intensity distribution, and background. Further improvements allow one to work with very low photon numbers, i.e.,...

Probability distribution analysis (PDA) [M. Antonik et al., J. Phys. Chem. B 2006, 110, 6970] allows one to quantitatively analyze single-molecule (SM) data obtained in Forster resonance energy transfer (FRET) or fluorescence polarization experiments. By taking explicitly background and shot noise contributions into account, PDA accurately predicts the shape of one-dimensional histograms various parameters, such as FRET efficiency anisotropy. In order describe complex experimental SM-FRET...