bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2023.04.03.535482

Fluorescence lifetime analysis of smFRET with contribution of PIFE on donor and acceptor

Jazani, S.·Ha, T.
View original source ↗Export metadata ↓

Abstract

Single-molecule fluorescence resonance energy transfer (FRET) is a powerful technique based on dipole-dipole interaction between donor and acceptor fluorophores to observe inter- and intra-molecular dynamics in realtime with sensitivity to macro-molecular distances ([~] 2.5-10 nm). That said, some fluorophores have an inherent characteristic known as protein induced fluorescence enhancement (PIFE). PIFE is a photo-physical feature of dyes undergoing cis-trans transitions and occurs for protein-dye interactions closer than 3 nm. Here, the challenge is uncoupling the PIFE effect in the FRET data. Ignoring the PIFE effect in the analysis of the FRET data may lead to misinterpretation of the system under investigation. As a solution to this problem, we develop a computational framework based on Bayesian statistics to analyze the fluorescence lifetime signals of the donor and acceptor channels which allows us to uncouple the PIFE effects from the FRET. Our framework can extract any changes in the FRET efficiency simultaneously with any changes in the fluorescence lifetimes of the donor and acceptor due to the PIFE effect. In addition, our framework can provide other parameters, such as the donor and acceptor excitation rates, background photon rates, and detectors cross-talk ratios. Our framework extracts all these parameters by analyzing a single photon arrival time trace with only a few thousand photons.

Explore related subjects

biophysicsPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Jazani, S., Ha, T.. 2023-04-06. Fluorescence lifetime analysis of smFRET with contribution of PIFE on donor and acceptor. https://doi.org/10.1101/2023.04.03.535482

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Mechanism of molecular recognition revealed through dynamic drug binding pathways to SARS-CoV-2 main protease

Characterization of drug-binding pathways remains experimentally limited by transient intermediates and computationally challenging due to long timescales intractable for conventional molecular dynamics. To address these challenges, we combined solution NMR titrations with weighted ensemble (WE) enhanced sampling simulations to resolve atomistic pathways of nirmatrelvir binding to the SARS-CoV-2 main protease. NMR titration revealed residue-dependent heterogeneity spanning fast, intermediate, and slow exchange regimes. WE simulations complement the NMR by providing insights into unassigned residues and adding time-resolved and three-dimensional structural context. We map key interactions along two distinct binding pathways, provide dynamic explanations for residues involved in resistance, and capture unique backbone conformations compared to those sampled in unbound or bound states. Our comprehensive binding model is consistent with a combined conformational selection and induced fit mechanism in which early transient contacts are made with residues E47 and L50 and allosteric motions are centered around residue V204 of the distal domain. This synergistic application of WE and titration NMR enables a more comprehensive characterization of drug binding than either method alone, providing an integrated framework that may have broader applicability to defining structure-kinetic relationships and guiding design of next-generation inhibitors.

biophysics↗
bioRxiv2026

Fibers and Glasses are Competing Material States in FUS Protein Condensation

Dense, well-ordered material states of proteins form the amyloid fibers that are a hallmark of neurodegenerative disease in the brain. Beyond forming amyloid fibers, some of these proteins can also adopt other material states termed condensates which are initially liquid-like but evolve to a soft, glassy phase. Fiber growth requires a large supply of monomers and, thus, it is often speculated that fibers emerge from within a dense condensate as it ages and its microscopic dynamics slow into a glassy state. Here, we use the well-established model system Fused in Sarcoma (FUS) to directly observe, quantify and theoretically describe fiber growth and its interplay with condensates. We report the discovery that fibers grow overwhelmingly in the dilute phase surrounding the condensates while the condensates concurrently evolve to a glassy arrested solid. The resulting protein fibers and glassy condensates are both distinct solid-like phases that coexist but do not directly interconvert. Taken together, these findings reveal that there are two competitive aging pathways in FUS condensation that are linked through phase separation kinetics.

biophysics↗
bioRxiv2026

A Minimally Perturbative DARPin Probe for Quantitative Fluorescence Imaging of the Human TCR-CD3 Complex

Fluorescence microscopy is a powerful tool for dissecting the molecular mechanisms of T-cell antigen recognition in living cells, but its quantitative insight critically depends on non-perturbative, high-quality probes. Here, we repurpose a small (~15 kDa) CD3epsilon-binding DARPin (designed ankyrin repeat proteins) to a fluorescent label for T-cell receptor (TCR)/CD3 complexes on primary human CD8+ T-cells, with the aim of generating a powerful tool for quantitative analysis, single-molecule tracking, and advanced imaging of TCR dynamics. We show that the DARPin binds CD3{varepsilon} with high affinity and selectivity and using single molecule tracking and brightness analysis, we characterize the TCR-CD3 diffusion behavior and show that the DARPin binds to both CD3epsilon; subunits. Importantly, labeling preserves antigen sensitivity: on supported lipid bilayers presenting cognate pMHC, T-cells remain responsive, assemble synapses, form TCR microclusters, and initiate signaling similar to unlabeled controls. We further demonstrate compatibility with lattice light-sheet microscopy for volumetric imaging of T-cell - APC interactions in living cells. Together, these results establish DARPins as versatile, minimally perturbative probes for high resolution, quantitative studies of T cell synapse organization and signaling.

biophysics↗