共查询到20条相似文献,搜索用时 31 毫秒
1.
Fluorescence resonance energy transfer (FRET) from a donor-labelled molecule to an acceptor-labelled molecule is a useful, proximity-based fluorescence tool to discriminate molecular states on the surface and in the interior of cells. Most microscope-based determinations of FRET yield only a single value, the interpretation of which is necessarily model-dependent. In this paper we demonstrate two new measurements of FRET heterogeneity using selective donor photobleaching in combination with synchronous donor/acceptor detection based on either (1) full kinetic analysis of donor-detected and acceptor-detected donor photobleaching or (2) a simple time-based ratiometric approach. We apply the new methods to study the cell surface distribution of concanavalin A yielding estimates of FRET and non-FRET population distributions, as well as FRET efficiencies within the FRET populations. 相似文献
2.
Reliable and global measurement of fluorescence resonance energy transfer using fluorescence microscopes 总被引:15,自引:0,他引:15
下载免费PDF全文
![点击此处可从《Biophysical journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Green fluorescence protein (GFP)-based fluorescence resonance energy transfer (FRET) is increasingly used in investigation of inter- and intramolecular interactions in living cells. In this report, we present a modified method for FRET quantification in cultured cells using conventional fluorescence microscopy. To reliably measure FRET, three positive control constructs in which a cyan fluorescence protein and a yellow fluorescence protein were linked by peptides of 15, 24, or 37 amino acid residues were prepared. FRET was detected using a spectrofluorometer, a laser scanning confocal microscope, and an inverted fluorescence microscope. Three calculation methods for FRET quantification using fluorescence microscopes were compared. By normalization against expression levels of GFP fusion proteins, the modified method gave consistent FRET values that could be compared among different cells with varying protein expression levels. Whole-cell global analysis using this method allowed FRET measurement with high spatial resolutions. Using such a procedure, the interaction of synaptic proteins syntaxin and the synaptosomal associated protein of 25 kDa (SNAP-25) was examined in PC12 cells, which showed strong FRET on plasma membranes. These results demonstrate the effectiveness of the modified method for FRET measurement in live cell systems. 相似文献
3.
The assembly of multiprotein complexes at the membrane interface governs many signaling processes in cells. However, very few methods exist for obtaining biophysical information about protein complex formation at the membrane. We used single molecule fluorescence resonance energy transfer to study complexin and synaptotagmin interactions with the SNARE complex in deposited lipid bilayers. Using total internal reflectance microscopy, individual binding events at the membrane could be resolved despite an excess of unbound protein in solution. Fluorescence resonance energy transfer (FRET)-efficiency derived distances for the complexin-SNARE interaction were consistent with the crystal structure of the complexin-SNARE complex. The unstructured N-terminal region of complexin showed broad distributions of FRET efficiencies to the SNARE complex, suggesting that information on conformational variability can be obtained from FRET efficiency distributions. The low-affinity interaction of synaptotagmin with the SNARE complex changed dramatically upon addition of Ca2+ with high FRET efficiency interactions appearing between the C2B domain and linker domains of synaptotagmin and the membrane proximal portion of the SNARE complex. These results demonstrate that single molecule FRET can be used as a "spectroscopic ruler" to simultaneously gain structural and kinetic information about transient multiprotein complexes at the membrane interface. 相似文献
4.
Förster resonance energy transfer (FRET) is an exquisitely sensitive method for detection of molecular interactions and conformational changes in living cells. The recent advent of fluorescence imaging technology with single-molecule (or molecular-complex) sensitivity, together with refinements in the kinetic theory of FRET, provide the necessary tool kits for determining the stoichiometry and relative disposition of the protomers within protein complexes (i.e., quaternary structure) of membrane receptors and transporters in living cells. In contrast to standard average-based methods, this method relies on the analysis of distributions of apparent FRET efficiencies, Eapp, across the image pixels of individual cells expressing proteins of interest. The most probable quaternary structure of the complex is identified from the number of peaks in the Eapp distribution and their dependence on a single parameter, termed pairwise FRET efficiency. Such peaks collectively create a unique FRET spectrum corresponding to each oligomeric configuration of the protein. Therefore, FRET could quite literally become a spectrometric method—akin to that of mass spectrometry—for sorting protein complexes according to their size and shape. 相似文献
5.
Studies of protein interactions have increased our understanding and knowledge of biological processes. Assays that utilize fluorescent proteins, such as fluorescence resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC), have enabled direct visualization of protein interactions in living cells. However, these assays are primarily suitable for a pair of interacting proteins, and methods to visualize and identify multiple protein complexes in vivo are very limited. This protocol describes the recently developed BiFC-FRET assay, which allows visualization of ternary complexes in living cells. We discuss how to design the BiFC-FRET assay on the basis of the validation of BiFC and FRET assays and how to perform transfection experiments for acquisition of fluorescent images for net FRET calculation. We also provide three methods for normalization of the FRET efficiency. The assay employs a two-chromophore and three-filter FRET setup and is applicable to epifluorescence microscopes. The entire protocol takes about 2-3 weeks to complete. 相似文献
6.
The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial applications as well as increases basic fundamental biological knowledge. Förster (Fluorescence) Resonance Energy Transfer (FRET) between a donor molecule in an electronically excited state and a nearby acceptor molecule has been frequently utilized for studies of protein-protein interactions in living cells. The proteins of interest are tagged with two different types of fluorescent probes and expressed in biological cells. The fluorescent probes are then excited, typically using laser light, and the spectral properties of the fluorescence emission emanating from the fluorescent probes is collected and analyzed. Information regarding the degree of the protein interactions is embedded in the spectral emission data. Typically, the cell must be scanned a number of times in order to accumulate enough spectral information to accurately quantify the extent of the protein interactions for each region of interest within the cell. However, the molecular composition of these regions may change during the course of the acquisition process, limiting the spatial determination of the quantitative values of the apparent FRET efficiencies to an average over entire cells. By means of a spectrally resolved two-photon microscope, we are able to obtain a full set of spectrally resolved images after only one complete excitation scan of the sample of interest. From this pixel-level spectral data, a map of FRET efficiencies throughout the cell is calculated. By applying a simple theory of FRET in oligomeric complexes to the experimentally obtained distribution of FRET efficiencies throughout the cell, a single spectrally resolved scan reveals stoichiometric and structural information about the oligomer complex under study. Here we describe the procedure of preparing biological cells (the yeast Saccharomyces cerevisiae) expressing membrane receptors (sterile 2 α-factor receptors) tagged with two different types of fluorescent probes. Furthermore, we illustrate critical factors involved in collecting fluorescence data using the spectrally resolved two-photon microscopy imaging system. The use of this protocol may be extended to study any type of protein which can be expressed in a living cell with a fluorescent marker attached to it.Download video file.(90M, mov) 相似文献
7.
F?rster's resonance energy transfer (FRET) can be used to study protein-protein interactions in living cells. Numerous methods to measure FRET have been devised and implemented; however, the accuracy of these methods is unknown, which makes interpretation of FRET efficiency values difficult if not impossible. This problem exists due to the lack of standards with known FRET efficiencies that can be used to validate FRET measurements. The advent of spectral variants of green fluorescent protein and easy access to cell transfection technology suggests a simple solution to this problem: the development of genetic constructs with known FRET efficiencies that can be replicated with high fidelity and freely distributed. In this study, fluorescent protein constructs with progressively larger separation distances between donors and acceptors were generated and FRET efficiencies were measured using fluorescence lifetime spectroscopy, sensitized acceptor emission, and spectral imaging. Since the results from each method were in good agreement, the FRET efficiency value of each construct could be determined with high accuracy and precision, thereby justifying their use as standards. 相似文献
8.
Millington M Grindlay GJ Altenbach K Neely RK Kolch W Bencina M Read ND Jones AC Dryden DT Magennis SW 《Biophysical chemistry》2007,127(3):155-164
We have used widefield photon-counting FLIM to study FRET in fixed and living cells using control FRET pairs. We have studied fixed mammalian cells expressing either cyan fluorescent protein (CFP) or a fusion of CFP and yellow fluorescent protein (YFP), and living fungal cells expressing either Cerulean or a Cerulean-Venus fusion protein. We have found the fluorescence behaviour to be essentially identical in the mammalian and fungal cells. Importantly, the high-precision FLIM data is able to reproducibly resolve multiple fluorescence decays, thereby revealing new information about the fraction of the protein population that undergoes FRET and reducing error in the measurement of donor-acceptor distances. Our results for this simple control system indicate that the in vivo FLIM-FRET studies of more complex protein-protein interactions would benefit greatly from such quantitative measurements. 相似文献
9.
Sean C. Warren Anca Margineanu Dominic Alibhai Douglas J. Kelly Clifford Talbot Yuriy Alexandrov Ian Munro Matilda Katan Chris Dunsby Paul M. W. French 《PloS one》2013,8(8)
Fluorescence lifetime imaging (FLIM) is widely applied to obtain quantitative information from fluorescence signals, particularly using Förster Resonant Energy Transfer (FRET) measurements to map, for example, protein-protein interactions. Extracting FRET efficiencies or population fractions typically entails fitting data to complex fluorescence decay models but such experiments are frequently photon constrained, particularly for live cell or in vivo imaging, and this leads to unacceptable errors when analysing data on a pixel-wise basis. Lifetimes and population fractions may, however, be more robustly extracted using global analysis to simultaneously fit the fluorescence decay data of all pixels in an image or dataset to a multi-exponential model under the assumption that the lifetime components are invariant across the image (dataset). This approach is often considered to be prohibitively slow and/or computationally expensive but we present here a computationally efficient global analysis algorithm for the analysis of time-correlated single photon counting (TCSPC) or time-gated FLIM data based on variable projection. It makes efficient use of both computer processor and memory resources, requiring less than a minute to analyse time series and multiwell plate datasets with hundreds of FLIM images on standard personal computers. This lifetime analysis takes account of repetitive excitation, including fluorescence photons excited by earlier pulses contributing to the fit, and is able to accommodate time-varying backgrounds and instrument response functions. We demonstrate that this global approach allows us to readily fit time-resolved fluorescence data to complex models including a four-exponential model of a FRET system, for which the FRET efficiencies of the two species of a bi-exponential donor are linked, and polarisation-resolved lifetime data, where a fluorescence intensity and bi-exponential anisotropy decay model is applied to the analysis of live cell homo-FRET data. A software package implementing this algorithm, FLIMfit, is available under an open source licence through the Open Microscopy Environment. 相似文献
10.
Zeynep Dereli‐Korkut Harmeet Gandhok Ling Ge Zeng Sidra Waqas Xuejun Jiang Sihong Wang 《Biotechnology and bioengineering》2013,110(5):1396-1404
Targeted therapy involving the activation of death receptors DR4 and/or DR5 by its ligand, TRAIL, can selectively induce apoptosis in certain tumor cells. In order to profile the dynamic activation or trimerization of TRAIL–DR4 in live cells in real‐time, the development of an apoptosis reporter cell line is essential. Fluorescence resonance energy transfer (FRET) technology via a FRET pair, cyan fluorescence protein (CFP) and yellow fluorescence protein (YFP), was used in this study. DR4‐CFP and DR4‐YFP were stably expressed in human lung cancer PC9 cells. Flow cytometer sorting and limited dilution coupled with fluorescence microscopy were used to select a monoclonal reporter cell line with high and compatible expression levels of DR4‐CFP and DR4‐YFP. FRET experiments were conducted and FRET efficiencies were monitored according to the Siegel's YFP photobleaching FRET protocol. Upon TRAIL induction a significant increase in FRET efficiencies from 5% to 9% demonstrated the ability of the DR4‐CFP/YFP reporter cell line in monitoring the dynamic activation of TRAIL pathways. 3D reconstructed confocal images of DR4‐CFP/YFP reporter cells exhibited a colocalized expression of DR4‐CFP and DR4‐YFP mainly on cell membranes. FRET results obtained during this study complements the use of epi‐fluorescence microscopy for FRET analysis. The real‐time FRET analysis allows the dynamic profiling of the activation of TRAIL pathways by using the time‐lapse fluorescence microscopy. Therefore, DR4‐CFP/YFP PC9 reporter cells along with FRET technology can be used as a tool for anti‐cancer drug screening to identify compounds that are capable of activating TRAIL pathways. Biotechnol. Bioeng. 2013; 110: 1396–1404. © 2012 Wiley Periodicals, Inc. 相似文献
11.
Jelle Hendrix Waldemar Schrimpf Matthias H?ller Don?C. Lamb 《Biophysical journal》2013,105(4):848-861
Fluorescence fluctuation imaging is a powerful means to investigate dynamics, interactions, and stoichiometry of proteins inside living cells. Pulsed interleaved excitation (PIE) is the method of nanosecond alternating excitation with time-resolved detection and allows accurate, independent, and quasi-simultaneous determination of fluorescence intensities and lifetimes of different fluorophores. In this work, we combine pulsed interleaved excitation with fluctuation imaging methods (PIE-FI) such as raster image correlation spectroscopy (RICS) or number and brightness analysis (N&B). More specifically, we show that quantitative measurements of diffusion and molecular brightness of Venus fluorescent protein (FP) can be performed in solution with PIE-RICS and compare PIE-RICS with single-point PIE-FCS measurements. We discuss the advantages of cross-talk free dual-color PIE-RICS and illustrate its proficiency by quantitatively comparing two commonly used FP pairs for dual-color microscopy, eGFP/mCherry and mVenus/mCherry. For N&B analysis, we implement dead-time correction to the PIE-FI data analysis to allow accurate molecular brightness determination with PIE-NB. We then use PIE-NB to investigate the effect of eGFP tandem oligomerization on the intracellular maturation efficiency of the fluorophore. Finally, we explore the possibilities of using the available fluorescence lifetime information in PIE-FI experiments. We perform lifetime-based weighting of confocal images, allowing us to quantitatively determine molecular concentrations from 100 nM down to <30 pM with PIE-raster lifetime image correlation spectroscopy (RLICS). We use the fluorescence lifetime information to perform a robust dual-color lifetime-based FRET analysis of tandem fluorescent protein dimers. Lastly, we investigate the use of dual-color RLICS to resolve codiffusing FRET species from non-FRET species in cells. The enhanced capabilities and quantitative results provided by PIE-FI make it a powerful method that is broadly applicable to a large number of interesting biophysical studies. 相似文献
12.
Fluorescence fluctuation imaging is a powerful means to investigate dynamics, interactions, and stoichiometry of proteins inside living cells. Pulsed interleaved excitation (PIE) is the method of nanosecond alternating excitation with time-resolved detection and allows accurate, independent, and quasi-simultaneous determination of fluorescence intensities and lifetimes of different fluorophores. In this work, we combine pulsed interleaved excitation with fluctuation imaging methods (PIE-FI) such as raster image correlation spectroscopy (RICS) or number and brightness analysis (N&B). More specifically, we show that quantitative measurements of diffusion and molecular brightness of Venus fluorescent protein (FP) can be performed in solution with PIE-RICS and compare PIE-RICS with single-point PIE-FCS measurements. We discuss the advantages of cross-talk free dual-color PIE-RICS and illustrate its proficiency by quantitatively comparing two commonly used FP pairs for dual-color microscopy, eGFP/mCherry and mVenus/mCherry. For N&B analysis, we implement dead-time correction to the PIE-FI data analysis to allow accurate molecular brightness determination with PIE-NB. We then use PIE-NB to investigate the effect of eGFP tandem oligomerization on the intracellular maturation efficiency of the fluorophore. Finally, we explore the possibilities of using the available fluorescence lifetime information in PIE-FI experiments. We perform lifetime-based weighting of confocal images, allowing us to quantitatively determine molecular concentrations from 100 nM down to <30 pM with PIE-raster lifetime image correlation spectroscopy (RLICS). We use the fluorescence lifetime information to perform a robust dual-color lifetime-based FRET analysis of tandem fluorescent protein dimers. Lastly, we investigate the use of dual-color RLICS to resolve codiffusing FRET species from non-FRET species in cells. The enhanced capabilities and quantitative results provided by PIE-FI make it a powerful method that is broadly applicable to a large number of interesting biophysical studies. 相似文献
13.
Borst JW Laptenok SP Westphal AH Kühnemuth R Hornen H Visser NV Kalinin S Aker J van Hoek A Seidel CA Visser AJ 《Biophysical journal》2008,95(11):5399-5411
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 binding. 相似文献
14.
A. J. W. G. Visser S. P. Laptenok N. V. Visser A. van Hoek D. J. S. Birch J.-C. Brochon J. W. Borst 《European biophysics journal : EBJ》2010,39(2):241-253
Förster resonance energy transfer (FRET) is a powerful method for obtaining information about small-scale lengths between biomacromolecules. Visible fluorescent proteins (VFPs) are widely used as spectrally different FRET pairs, where one VFP acts as a donor and another VFP as an acceptor. The VFPs are usually fused to the proteins of interest, and this fusion product is genetically encoded in cells. FRET between VFPs can be determined by analysis of either the fluorescence decay properties of the donor molecule or the rise time of acceptor fluorescence. Time-resolved fluorescence spectroscopy is the technique of choice to perform these measurements. FRET can be measured not only in solution, but also in living cells by the technique of fluorescence lifetime imaging microscopy (FLIM), where fluorescence lifetimes are determined with the spatial resolution of an optical microscope. Here we focus attention on time-resolved fluorescence spectroscopy of purified, selected VFPs (both single VFPs and FRET pairs of VFPs) in cuvette-type experiments. For quantitative interpretation of FRET–FLIM experiments in cellular systems, details of the molecular fluorescence are needed that can be obtained from experiments with isolated VFPs. For analysis of the time-resolved fluorescence experiments of VFPs, we have utilised the maximum entropy method procedure to obtain a distribution of fluorescence lifetimes. Distributed lifetime patterns turn out to have diagnostic value, for instance, in observing populations of VFP pairs that are FRET-inactive. 相似文献
15.
Fanjie Meng Mathias M.J. Bellaiche Jae-Yeol Kim Gül H. Zerze Robert B. Best Hoi Sung Chung 《Biophysical journal》2018,114(4):870-884
Monomers of amyloid-β (Aβ) protein are known to be disordered, but there is considerable controversy over the existence of residual or transient conformations that can potentially promote oligomerization and fibril formation. We employed single-molecule Förster resonance energy transfer (FRET) spectroscopy with site-specific dye labeling using an unnatural amino acid and molecular dynamics simulations to investigate conformations and dynamics of Aβ isoforms with 40 (Aβ40) and 42 residues (Aβ42). The FRET efficiency distributions of both proteins measured in phosphate-buffered saline at room temperature show a single peak with very similar FRET efficiencies, indicating there is apparently only one state. 2D FRET efficiency-donor lifetime analysis reveals, however, that there is a broad distribution of rapidly interconverting conformations. Using nanosecond fluorescence correlation spectroscopy, we measured the timescale of the fluctuations between these conformations to be ~35 ns, similar to that of disordered proteins. These results suggest that both Aβ40 and Aβ42 populate an ensemble of rapidly reconfiguring unfolded states, with no long-lived conformational state distinguishable from that of the disordered ensemble. To gain molecular-level insights into these observations, we performed molecular dynamics simulations with a force field optimized to describe disordered proteins. We find, as in experiments, that both peptides populate configurations consistent with random polymer chains, with the vast majority of conformations lacking significant secondary structure, giving rise to very similar ensemble-averaged FRET efficiencies. 相似文献
16.
Chen Y Mills JD Periasamy A 《Differentiation; research in biological diversity》2003,71(9-10):528-541
Interacting proteins assemble into molecular machines that control cellular homeostasis in living cells. While the in vitro screening methods have the advantage of providing direct access to the genetic information encoding unknown protein partners, they do not allow direct access to interactions of these protein partners in their natural environment inside the living cell. Using wide-field, confocal, or two-photon (2p) fluorescence resonance energy transfer (FRET) microscopy, this information can be obtained from living cells and tissues with nanometer resolution. One of the important conditions for FRET to occur is the overlap of the emission spectrum of the donor with the absorption spectrum of the acceptor. As a result of spectral overlap, the FRET signal is always contaminated by donor emission into the acceptor channel and by the excitation of acceptor molecules by the donor excitation wavelength. Mathematical algorithms are required to correct the spectral bleed-through signal in wide-field, confocal, and two-photon FRET microscopy. In contrast, spectral bleed-through is not an issue in FRET/FLIM imaging because only the donor fluorophore lifetime is measured; also, fluorescence lifetime imaging microscopy (FLIM) measurements are independent of excitation intensity or fluorophore concentration. The combination of FRET and FLIM provides high spatial (nanometer) and temporal (nanosecond) resolution when compared to intensity-based FRET imaging. In this paper, we describe various FRET microscopy techniques and its application to protein-protein interactions. 相似文献
17.
Quantitative time domain analysis of lifetime‐based Förster resonant energy transfer measurements with fluorescent proteins: Static random isotropic fluorophore orientation distributions
下载免费PDF全文
![点击此处可从《Journal of biophotonics》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Yuriy Alexandrov Dino S. Nikolic Christopher Dunsby Paul M. W. French 《Journal of biophotonics》2018,11(7)
Förster resonant energy transfer (FRET) measurements are widely used to obtain information about molecular interactions and conformations through the dependence of FRET efficiency on the proximity of donor and acceptor fluorophores. Fluorescence lifetime measurements can provide quantitative analysis of FRET efficiency and interacting population fraction. Many FRET experiments exploit the highly specific labelling of genetically expressed fluorescent proteins, applicable in live cells and organisms. Unfortunately, the typical assumption of fast randomization of fluorophore orientations in the analysis of fluorescence lifetime‐based FRET readouts is not valid for fluorescent proteins due to their slow rotational mobility compared to their upper state lifetime. Here, previous analysis of effectively static isotropic distributions of fluorophore dipoles on FRET measurements is incorporated into new software for fitting donor emission decay profiles. Calculated FRET parameters, including molar population fractions, are compared for the analysis of simulated and experimental FRET data under the assumption of static and dynamic fluorophores and the intermediate regimes between fully dynamic and static fluorophores, and mixtures within FRET pairs, is explored. Finally, a method to correct the artefact resulting from fitting the emission from static FRET pairs with isotropic angular distributions to the (incorrect) typically assumed dynamic FRET decay model is presented. 相似文献
18.
Imaging protein-protein interactions using fluorescence resonance energy transfer microscopy 总被引:21,自引:0,他引:21
Kenworthy AK 《Methods (San Diego, Calif.)》2001,24(3):289-296
Fluorescence resonance energy transfer (FRET) detects the proximity of fluorescently labeled molecules over distances >100 A. When performed in a fluorescence microscope, FRET can be used to map protein-protein interactions in vivo. We here describe a FRET microscopy method that can be used to determine whether proteins that are colocalized at the level of light microscopy interact with one another. This method can be implemented using digital microscopy systems such as a confocal microscope or a wide-field fluorescence microscope coupled to a charge-coupled device (CCD) camera. It is readily applied to samples prepared with standard immunofluorescence techniques using antibodies labeled with fluorescent dyes that act as a donor and acceptor pair for FRET. Energy transfer efficiencies are quantified based on the release of quenching of donor fluorescence due to FRET, measured by comparing the intensity of donor fluorescence before and after complete photobleaching of the acceptor. As described, this method uses Cy3 and Cy5 as the donor and acceptor fluorophores, but can be adapted for other FRET pairs including cyan fluorescent protein and yellow fluorescent protein. 相似文献
19.
André Zeug Andrew Woehler Erwin Neher Evgeni?G. Ponimaskin 《Biophysical journal》2012,103(9):1821-1827
Förster resonance energy transfer (FRET) has become an important tool for analyzing different aspects of interactions among biological macromolecules in their native environments. FRET analysis has also been successfully applied to study the spatiotemporal regulation of various cellular processes using genetically encoded FRET-based biosensors. A variety of procedures have been described for measuring FRET efficiency or the relative abundance of donor-acceptor complexes, based on analysis of the donor fluorescence lifetime or the spectrally resolved fluorescence intensity. The latter methods are preferable if one wants to not only quantify the apparent FRET efficiencies but also calculate donor-acceptor stoichiometry and observe fast dynamic changes in the interactions among donor and acceptor molecules in live cells. This review focuses on a comparison of the available intensity-based approaches used to measure FRET. We discuss their strengths and weaknesses in terms of FRET quantification, and provide several examples of biological applications. 相似文献
20.
《Biophysical journal》2020,118(8):1861-1875
Many membrane proteins are thought to function as dimers or higher oligomers, but measuring membrane protein oligomerization in lipid membranes is particularly challenging. Förster resonance energy transfer (FRET) and fluorescence cross-correlation spectroscopy are noninvasive, optical methods of choice that have been applied to the analysis of dimerization of single-spanning membrane proteins. However, the effects inherent to such two-dimensional systems, such as the excluded volume of polytopic transmembrane proteins, proximity FRET, and rotational diffusion of fluorophore dipoles, complicate interpretation of FRET data and have not been typically accounted for. Here, using FRET and fluorescence cross-correlation spectroscopy, we introduce a method to measure surface protein density and to estimate the apparent Förster radius, and we use Monte Carlo simulations of the FRET data to account for the proximity FRET effect occurring in confined two-dimensional environments. We then use FRET to analyze the dimerization of human rhomboid protease RHBDL2 in giant plasma membrane vesicles. We find no evidence for stable oligomers of RHBDL2 in giant plasma membrane vesicles of human cells even at concentrations that highly exceed endogenous expression levels. This indicates that the rhomboid transmembrane core is intrinsically monomeric. Our findings will find use in the application of FRET and fluorescence correlation spectroscopy for the analysis of oligomerization of transmembrane proteins in cell-derived lipid membranes. 相似文献