A Bayesian method for inferring quantitative information from FRET data

<p>Abstract</p> <p>Background</p> <p>Understanding biological networks requires identifying their elementary protein interactions and establishing the timing and strength of those interactions. Fluorescence microscopy and Förster resonance energy transfer (FRET) have the potential to reveal such information because they allow molecular interactions to be monitored in living cells, but it is unclear how best to analyze FRET data. Existing techniques differ in assumptions, manipulations of data and the quantities they derive. To address this variation, we have developed a versatile Bayesian analysis based on clear assumptions and systematic statistics.</p> <p>Results</p> <p>Our algorithm infers values of the FRET efficiency and dissociation constant, <it>K_d</it>, between a pair of fluorescently tagged proteins. It gives a posterior probability distribution for these parameters, conveying more extensive information than single-value estimates can. The width and shape of the distribution reflects the reliability of the estimate and we used simulated data to determine how measurement noise, data quantity and fluorophore concentrations affect the inference. We are able to show why varying concentrations of donors and acceptors is necessary for estimating <it>K_d</it>. We further demonstrate that the inference improves if additional knowledge is available, for example of the FRET efficiency, which could be obtained from separate fluorescence lifetime measurements.</p> <p>Conclusions</p> <p>We present a general, systematic approach for extracting quantitative information on molecular interactions from FRET data. Our method yields both an estimate of the dissociation constant and the uncertainty associated with that estimate. The information produced by our algorithm can help design optimal experiments and is fundamental for developing mathematical models of biochemical networks.</p

Tau Interaction with Tubulin and Microtubules: From Purified Proteins to Cells

International audienceMicrotubules (MTs) play an important role in many cellular processes and are dynamic structures regulated by an important network of microtubules-associated proteins, MAPs, such as Tau. Tau has been discovered as an essential factor for MTs formation in vitro, and its region implicated in binding to MTs has been identified. By contrast, the affinity, the stoichiometry, and the topology of Tau-MTs interaction remain controversial. Indeed, depending on the experiment conditions a wide range of values have been obtained. In this chapter, we focus on three biophysical methods, turbidimetry, cosedimentation assay, and Förster Resonance Energy Transfer to study Tau-tubulin interaction both in vitro and in cell. We highlight precautions that must be taken in order to avoid pitfalls and we detail the nature of the conclusions that can be drawn from these methods about Tau-tubulin interaction

Revealing the interface in polymer nanocomposites

The morphological characterization of polymer nanocomposites over multiple length scales is a fundamental challenge. Here, we report a technique for high-throughput monitoring of interface and dispersion in polymer nanocomposites based on Förster resonance energy transfer (FRET). Nanofibrillated cellulose (NFC), fluorescently labeled with 5-(4,6-dichlorotriazinyl)- aminofluorescein (FL) and dispersed into polyethylene (PE) doped with Coumarin 30 (C30), is used as a model system to assess the ability of FRET to evaluate the effect of processing on NFC dispersion in PE. The level of energy transfer and its standard deviation, measured by fluorescence spectroscopy and laser scanning confocal microscopy (LSCM), are exploited to monitor the extent of interface formation and composite homogeneity, respectively. FRET algorithms are used to generate color-coded images for a real-space observation of energy transfer efficiency. These images reveal interface formation at a nanoscale while probing a macroscale area that is large enough to be representative of the entire sample. The unique ability of this technique to simultaneously provide orientation/spatial information at a macroscale and nanoscale features, encoded in the FRET signal, provides a new powerful tool for structure-property- processing investigation in polymer nanocomposites.</p

Heme Oxygenase Isoforms Differ in Their Subcellular Trafficking during Hypoxia and Are Differentially Modulated by Cytochrome P450 Reductase

Heme oxygenase (HO) degrades heme in concert with NADPH cytochrome P450 reductase (CPR) which donates electrons to the reaction. Earlier studies reveal the importance of the hydrophobic carboxy-terminus of HO-1 for anchorage to the endoplasmic reticulum (ER) which facilitates the interaction with CPR. In addition, HO-1 has been shown to undergo regulated intramembrane proteolysis of the carboxy-terminus during hypoxia and subsequent translocation to the nucleus. Translocated nuclear HO-1 was demonstrated to alter binding of transcription factors and to alter gene expression. Little is known about the homologous membrane anchor of the HO-2 isoform. The current work is the first systematic analysis in a eukaryotic system that demonstrates the crucial role of the membrane anchor of HO-2 for localization at the endoplasmic reticulum, oligomerization and interaction with CPR. We show that although the carboxy-terminal deletion mutant of HO-2 is found in the nucleus, translocation of HO-2 to the nucleus does not occur under conditions of hypoxia. Thus, we demonstrate that proteolytic regulation and nuclear translocation under hypoxic conditions is specific for HO-1. In addition we show for the first time that CPR prevents this translocation and promotes oligomerization of HO-1. Based on these findings, CPR may modulate gene expression via the amount of nuclear HO-1. This is of particular relevance as CPR is a highly polymorphic gene and deficiency syndromes of CPR have been described in humans

The chlL ( frxC ) gene: Phylogenetic distribution in vascular plants and DNA sequence from Polystichum acrostichoides ( Pteridophyta ) and Synechococcus sp. 7002 ( Cyanobacteria )

We examined chlL ( frxC ) gene evolution using several approaches. Sequences from the chloroplast genome of the fern Polystichum acrostichoides and from the cyanobacterium Synechococcus sp. 7002 were determined and found to be highly conserved. A complete physical map of the fern chloroplast genome and partial maps of other vascular plant taxa show that chlL is located primarily in the small single copy region as in Marchantia polymorpha. A survey of a wide variety of non-angiospermous vascular plant DNAs shows that chlL is widely distributed but has been lost in the pteridophyte Psilotum and (presumably independently) within the Gnetalean gymnosperms.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/41636/1/606_2004_Article_BF00994092.pd