Structural Changes of Yellow Cameleon Domains Observed by Quantitative FRET Analysis and Polarized Fluorescence Correlation Spectroscopy

Förster resonance energy transfer (FRET) is a widely used method for monitoring interactions between or within biological macromolecules conjugated with suitable donor-acceptor pairs. Donor fluorescence lifetimes in absence and presence of acceptor molecules are often measured for the observation of FRET. However, these lifetimes may originate from interacting and noninteracting molecules, which hampers quantitative interpretation of FRET data. We describe a methodology for the detection of FRET that monitors the rise time of acceptor fluorescence on donor excitation thereby detecting only those molecules undergoing FRET. The large advantage of this method, as compared to donor fluorescence quenching method used more commonly, is that the transfer rate of FRET can be determined accurately even in cases where the FRET efficiencies approach 100% yielding highly quenched donor fluorescence. Subsequently, the relative orientation between donor and acceptor chromophores is obtained from time-dependent fluorescence anisotropy measurements carried out under identical conditions of donor excitation and acceptor detection. The FRET based calcium sensor Yellow Cameleon 3.60 (YC3.60) was used because it changes its conformation on calcium binding, thereby increasing the FRET efficiency. After mapping distances and orientation angles between the FRET moieties in YC3.60, cartoon models of this FRET sensor with and without calcium could be created. Independent support for these representations came from experiments where the hydrodynamic properties of YC3.60 under ensemble and single-molecule conditions on selective excitation of the acceptor were determined. From rotational diffusion times as found by fluorescence correlation spectroscopy and consistently by fluorescence anisotropy decay analysis it could be concluded that the open structure (without calcium) is flexible as opposed to the rather rigid closed conformation. The combination of two independent methods gives consistent results and presents a rapid and specific methodology to analyze structural and dynamical changes in a protein on ligand bindin

Nanosecond Dynamics of Single-Molecule Fluorescence Resonance Energy Transfer

Motivated by recent experiments on photon statistics from individual dye pairs planted on biomolecules and coupled by fluorescence resonance energy transfer (FRET), we show here that the FRET dynamics can be modelled by Gaussian random processes with colored noise. Using Monte-Carlo numerical simulations, the photon intensity correlations from the FRET pairs are calculated, and are turned out to be very close to those observed in experiment. The proposed stochastic description of FRET is consistent with existing theories for microscopic dynamics of the biomolecule that carries the FRET coupled dye pairs.Comment: 8 pages, 1 figure. accepted to J.Phys.Chem.

Fluorescence energy transfer enhancement in aluminum nanoapertures

Zero-mode waveguides (ZMWs) are confining light into attoliter volumes, enabling single molecule fluorescence experiments at physiological micromolar concentrations. Among the fluorescence spectroscopy techniques that can be enhanced by ZMWs, F\"{o}rster resonance energy transfer (FRET) is one of the most widely used in life sciences. Combining zero-mode waveguides with FRET provides new opportunities to investigate biochemical structures or follow interaction dynamics at micromolar concentration with single molecule resolution. However, prior to any quantitative FRET analysis on biological samples, it is crucial to establish first the influence of the ZMW on the FRET process. Here, we quantify the FRET rates and efficiencies between individual donor-acceptor fluorophore pairs diffusing in aluminum zero-mode waveguides. Aluminum ZMWs are important structures thanks to their commercial availability and the large literature describing their use for single molecule fluorescence spectroscopy. We also compare the results between ZMWs milled in gold and aluminum, and find that while gold has a stronger influence on the decay rates, the lower losses of aluminum in the green spectral region provide larger fluorescence brightness enhancement factors. For both aluminum and gold ZMWs, we observe that the FRET rate scales linearly with the isolated donor decay rate and the local density of optical states (LDOS). Detailed information about FRET in ZMWs unlocks their application as new devices for enhanced single molecule FRET at physiological concentrations

Linear approaches to intramolecular Förster Resonance Energy Transfer probe measurements for quantitative modeling

Numerous unimolecular, genetically-encoded Forster Resonance Energy Transfer (FRET) probes for monitoring biochemical activities in live cells have been developed over the past decade. As these probes allow for collection of high frequency, spatially resolved data on signaling events in live cells and tissues, they are an attractive technology for obtaining data to develop quantitative, mathematical models of spatiotemporal signaling dynamics. However, to be useful for such purposes the observed FRET from such probes should be related to a biological quantity of interest through a defined mathematical relationship, which is straightforward when this relationship is linear, and can be difficult otherwise. First, we show that only in rare circumstances is the observed FRET linearly proportional to a biochemical activity. Therefore in most cases FRET measurements should only be compared either to explicitly modeled probes or to concentrations of products of the biochemical activity, but not to activities themselves. Importantly, we find that FRET measured by standard intensity-based, ratiometric methods is inherently non-linear with respect to the fraction of probes undergoing FRET. Alternatively, we find that quantifying FRET either via (1) fluorescence lifetime imaging (FLIM) or (2) ratiometric methods where the donor emission intensity is divided by the directly-excited acceptor emission intensity (denoted R<sub>alt</sub>) is linear with respect to the fraction of probes undergoing FRET. This linearity property allows one to calculate the fraction of active probes based on the FRET measurement. Thus, our results suggest that either FLIM or ratiometric methods based on R<sub>alt</sub> are the preferred techniques for obtaining quantitative data from FRET probe experiments for mathematical modeling purpose

Nanophotonic enhancement of the F\"orster resonance energy transfer rate on single DNA molecules

Nanophotonics achieves accurate control over the luminescence properties of a single quantum emitter by tailoring the light-matter interaction at the nanoscale and modifying the local density of optical states (LDOS). This paradigm could also benefit to F\"orster resonance energy transfer (FRET) by enhancing the near-field electromagnetic interaction between two fluorescent emitters. Despite the wide applications of FRET in nanosciences, using nanophotonics to enhance FRET remains a debated and complex challenge. Here, we demonstrate enhanced energy transfer within single donor-acceptor fluorophore pairs confined in gold nanoapertures. Experiments monitoring both the donor and the acceptor emission photodynamics at the single molecule level clearly establish a linear dependence of the FRET rate on the LDOS in nanoapertures. These findings are applied to enhance the FRET rate in nanoapertures up to six times, demonstrating that nanophotonics can be used to intensify the near-field energy transfer and improve the biophotonic applications of FRET

Ligand regulation of the quaternary organization of cell surface M3 muscarinic acetylcholine receptors analyzed by fluorescence resonance energy transfer (FRET) imaging and homogenous time-resolved FRET

Flp-In T-REx 293 cells expressing a wild type human M muscarinic acetylcholine receptor construct constitutively and able to express a Receptor Activated Solely by Synthetic Ligand (RASSL) form of this receptor on demand maintained response to the muscarinic agonist carbachol but developed response to clozapine-N-oxide only upon induction of the RASSL. The two constructs co-localized at the plasma membrane and generated strong ratiometric fluorescence resonance energy transfer (FRET) signals consistent with direct physical interactions. Increasing levels of induction of the FRET-donor RASSL did not alter wild type receptor FRET-acceptor levels substantially. However, ratiometric FRET was modulated in a bell-shaped fashion with maximal levels of the donor resulting in decreased FRET. Carbachol, but not the antagonist atropine, significantly reduced the FRET signal. Cell surface homogenous time-resolved FRET, based on SNAP-tag technology and employing wild type and RASSL forms of the human M receptor expressed stably in Flp-In TREx 293 cells, also identified cell surface dimeric/oligomeric complexes. Now, however, signals were enhanced by appropriate selective agonists. At the wild type receptor large increases in FRET signal to carbachol and acetylcholine were concentration-dependent with EC values consistent with the relative affinities of the two ligands. These studies confirm the capacity of the human M muscarinic acetylcholine receptor to exist as dimeric/oligomeric complexes at the surface of cells and demonstrate that the organization of such complexes can be modified by ligand binding. However, conclusions as to the effect of ligands on such complexes may depend on the approach used