[show abstract][hide abstract] ABSTRACT: Integration of the HIV-1 cDNA into the human genome is catalyzed by the viral integrase (IN) protein. Several studies have shown the importance of cellular cofactors that interact with integrase and affect viral integration and infectivity. In this study, we produced a stable complex between HIV-1 integrase, viral U5 DNA, the cellular cofactor LEDGF/p75 and the integrase binding domain of INI1 (INI1-IBD), a subunit of the SWI/SNF chromatin remodeling factor. The stoichiometry of the IN/LEDGF/INI1-IBD/DNA complex components was found to be 4/2/2/2 by mass spectrometry and Fluorescence Correlation Spectroscopy. Functional assays showed that INI1-IBD inhibits the 3' processing reaction but does not interfere with specific viral DNA binding. Integration assays demonstrate that INI1-IBD decreases the amount of integration events but inhibits by-product formation such as donor/donor or linear full site integration molecules. Cryo-electron microscopy locates INI1-IBD within the cellular DNA binding site of the IN/LEDGF complex, constraining the highly flexible integrase in a stable conformation. Taken together, our results suggest that INI1 could stabilize the PIC in the host cell, by maintaining integrase in a stable constrained conformation which prevents non-specific interactions and auto integration on the route to its integration site within nucleosomes, while LEDGF organizes and stabilizes an active integrase tetramer suitable for specific vDNA integration. Moreover, our results provide the basis for a novel type of integrase inhibitor (conformational inhibitor) representing a potential new strategy for use in human therapy.
[show abstract][hide abstract] ABSTRACT: Biomembranes are ordered and dynamic nanoscale structures critical for
cell functions. The biological functions of the membranes strongly
depend on their physicochemical properties, such as electrostatics,
phase state, viscosity, polarity and hydration. These properties are
essential for the membrane structure and the proper folding and function
of membrane proteins. To monitor these properties, fluorescence
techniques and notably, two-photon microscopy appear highly suited due
to their exquisite sensitivity and their capability to operate in
complex biological systems, such as living cells and tissues. In this
context, we have developed multiparametric environment-sensitive
fluorescent probes tailored for precise location in the membrane
bilayer. We notably developed probes of the 3-hydroxychromone family,
characterized by an excited state intramolecular proton transfer
reaction, which generates two tautomeric emissive species with
well-separated emission bands. As a consequence, the response of these
probes to changes in their environment could be monitored through
changes in the ratios of the two bands, as well as through changes in
the fluorescence lifetimes. Using two-photon ratiometric imaging and
FLIM, these probes were used to monitor the surface membrane potential,
and were applied to detect apoptotic cells and image membrane domains.
[show abstract][hide abstract] ABSTRACT: During reverse transcription, the HIV-1 RNA is converted by the reverse transcriptase (RT) into proviral DNA. RT is
assisted by the HIV-1 nucleocapsid (NCp7) protein that notably increases the ability of RT to synthesize DNA through
pause sites. Using single molecule FRET, we monitored the NCp7 effect on the binding of RT to nucleic acid sequences
corresponding to two different pause sites. NCp7 was found to modify the distribution of RT orientations on the
oligonucleotides and decrease the residence time of RT on one of the pause sites. These results give direct insight into
the NCp7 molecular mechanism in reverse transcription.
Single Molecule Spectroscopy and Superresolution Imaging VI, edited by Jörg Enderlein, Ingo Gregor, Zygmunt Karol Gryczynski, Rainer Erdmann, Felix Koberling, Proc. of SPIE, USA; 02/2013
[show abstract][hide abstract] ABSTRACT: Expression of antibody fragments in mammalian cells (intrabodies) is used to probe the target protein or interfere with its biological function. We previously described the in vitro characterisation of a single-chain Fv (scFv) antibody fragment (F5) isolated from an intrabody library that binds to the oncoprotein gankyrin (GK) in solution. Here, we have isolated several other scFvs that interact with GK in the presence of F5 and tested whether they allow, when fused to fluorescent proteins, to detect by FRET endogenous GK in living cells. The binding of pairs of scFvs to GK was analysed by gel filtration and the ability of each scFv to mediate nuclear import/export of GK was determined. Binding between scFv-EGFP and RFP-labelled GK in living cells was detected by fluorescence lifetime imaging microscopy (FLIM). After co-transfection of two scFvs fused to EGFP and RFP respectively, that form a tri-molecular complex with GK in vitro, FRET signal was measured. This system allowed us to observe that GK is monomeric and distributed throughout the cytoplasm and nucleus of several cancer cell lines. Our results show that pairs of fluorescently labelled intrabodies can be monitored by FLIM-FRET microscopy and that this technique allows the detection of lowly expressed endogenous proteins in single living cells.
Experimental Cell Research 01/2013; · 3.56 Impact Factor
[show abstract][hide abstract] ABSTRACT: Single molecule experiments were used to investigate the photobleaching mechanism of single Cy3 molecules encapsulated in large unilamellar vesicles. Analysis of thousands of individual fluorescence traces evidenced that the survival probability distribution is monoexponential with an average survival time depending on the laser intensity, the concentration of molecular oxygen, the temperature and the rigidity of the medium. Taken together, our data suggest that the photobleaching of Cy3 molecules is governed by two parallel pathways, namely photooxidation and a thermally activated structural rearrangement of the Cy3 molecules in their excited state.
Physical Chemistry Chemical Physics 12/2011; 14(5):1585-8. · 3.83 Impact Factor
[show abstract][hide abstract] ABSTRACT: Addition of Vpr C-terminus to various cell types provokes cell apoptosis. This property was recently shown useful to develop inhibitors of cell proliferation. In that context, we investigated the cellular uptake of rhodamine- and fluorescein-labeled Vpr(52-96) peptides to understand the mechanism of Vpr C-terminus entry into cells. Dynamic light scattering data indicated that this peptide spontaneously formed polydispersed aggregates in cell culture medium. The fluorescently labeled Vpr(52-96) peptide was efficiently internalized, appearing either as large fluorescent patches in the cytoplasm or in a more diffuse form throughout the cell. Using isothermal titration calorimetry, we demonstrated that Vpr(52-96) can tightly associate with heparin, a glycosaminoglycan analog of heparan sulphate, suggesting a central role of the ubiquitous cell surface-associated heparan sulphate proteoglycans for the internalization of Vpr C-terminus. Fluorescently-labeled transferrin and methyl-β-cyclodextrin showed that the Vpr C-terminus was mediated through clathrin- and caveolae/raft-dependent endocytosis. We found that Vpr C-terminus uptake was partly blocked at 4°C suggesting the importance of membrane fluidity for Vpr C-terminus entry. In fact, atomic force microscopy and liposome leakage further indicated that the Vpr peptide can destabilize and disrupt model membrane bilayers, suggesting that this mechanism may contribute to the passive entry of the peptide. Finally, using fluorescence lifetime imaging, we found that the Vpr(52-96) peptide was stable in cells for at least 48h, probably as a consequence of the poor accessibility of the peptide to proteolytic enzymes in aggregates.
[show abstract][hide abstract] ABSTRACT: During formation of HIV particles, the Gag polyproteins are thought to interact with Vpr proteins to promote their encapsidation in the nascent particles. To directly visualize and monitor the formation of the Gag-Vpr complexes and correlate their formation with Vpr oligomerization, we used two photon lifetime imaging microscopy (FLIM) and time laps microscopy on HeLa cells expressing eGFP-labelled Vpr and tetracystein-tagged Gag proteins detected by the biarsenical ReAsH labelling reagent. Using these fluorescent microscopy approaches, we found that Gag proteins interact directly with Vpr proteins, which results in their transfer from the nuclear to the plasma membrane. Vpr oligomerization was found critical for both its interaction with Gag proteins and its transfer to the plasma membrane. Moreover, mutations in the C-terminal domain of Gag abolished the interaction with Vpr and its redistribution to the plasma membrane, indicating that this domain was critical for interaction with Vpr. Taken together, these data show that the Gag polyproteins interact through their C-terminal domain with Vpr oligomers, promoting the redistribution of the latter at the plasma membrane and probably their incorporation into nascent viral particles.
[show abstract][hide abstract] ABSTRACT: The main function of the HIV-1 trans-activator of transcription (Tat protein) is to promote the transcription of the proviral DNA by the host RNA polymerase which leads to the synthesis of large quantities of the full length viral RNA. Tat is also thought to be involved in the reverse transcription (RTion) reaction by a still unknown mechanism. The recently reported nucleic acid annealing activity of Tat might explain, at least in part, its role in RTion. To further investigate this possibility, we carried out a fluorescence study on the mechanism by which the full length Tat protein (Tat(1-86)) and the basic peptide (44-61) direct the annealing of complementary viral DNA sequences representing the HIV-1 transactivation response element TAR, named dTAR and cTAR, essential for the early steps of RTion. Though both Tat(1-86) and the Tat(44-61) peptide were unable to melt the lower half of the cTAR stem, they strongly promoted cTAR/dTAR annealing through non-specific attraction between the peptide-bound oligonucleotides. Using cTAR and dTAR mutants, this Tat promoted-annealing was found to be nucleated through the thermally frayed 3'/5' termini, resulting in an intermediate with 12 intermolecular base pairs, which then converts into the final extended duplex. Moreover, we found that Tat(1-86) was as efficient as the nucleocapsid protein NCp7, a major nucleic acid chaperone of HIV-1, in promoting cTAR/dTAR annealing, and could act cooperatively with NCp7 during the annealing reaction. Taken together, our data are consistent with a role of Tat in the stimulation of the obligatory strand transfers during viral DNA synthesis by reverse transcriptase.
Journal of Molecular Biology 07/2010; 400(3):487-501. · 3.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: Herein, using a recently developed hydration-sensitive ratiometric biomembrane probe based on 3-hydroxyflavone (F2N12S) that binds selectively to the outer leaflet of plasma membranes, we compared plasma membranes of living cells and lipid vesicles as model membranes. Through the spectroscopic analysis of the probe response, we characterized the membranes in terms of hydration and polarity (electrostatics). The hydration parameter value in cell membranes was in between the values obtained with liquid ordered (Lo) and liquid disordered (Ld) phases in model membranes, suggesting that cell plasma membranes exhibit a significant fraction of Lo phase in their outer leaflet. Moreover, two-photon fluorescence microscopy experiments show that cell membranes labeled with this probe exhibit a homogeneous lipid distribution, suggesting that the putative domains in Lo phase are distributed all over the membrane and are highly dynamic. Cholesterol depletion affected dramatically the dual emission of the probe suggesting the disappearance of the Lo phase in cell membranes. These conclusions were corroborated with the viscosity sensitive diphenylhexatriene derivative TMA-DPH, showing membrane fluidity in intact cells intermediate between those for Lo and Ld phases in model membranes, as well as a significant increase in fluidity after cholesterol depletion. Moreover, we observed that cell apoptosis results in a similar loss of Lo phase, which could be attributed to a flip of sphingomyelin from the outer to the inner leaflet of the plasma membrane due to apoptosis-driven lipid scrambling. Our data suggest a new methodology for evaluating the Lo phase in membranes of living cells.
Biochimica et Biophysica Acta 07/2010; 1798(7):1436-43. · 4.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: We describe a simple fluorescent protein-based method to investigate interactions with a viral movement protein in living cells that relies on the in vivo re-localization of proteins in the presence of their interaction partners. We apply this method in combination with fluorescence lifetime imaging microscopy (FLIM) to demonstrate that a domain of the Tobacco mosaic virus (TMV) movement protein (MP) previously predicted to mediate protein:protein interactions is dispensable for these contacts. We suggest that this method can be generalized for analysis of other protein interactions in planta.
The Plant Journal 04/2010; 62(1):171-7. · 6.58 Impact Factor
[show abstract][hide abstract] ABSTRACT: Cholesterol and sphingomyelin form together a highly ordered membrane phase, which is believed to play important biological functions in plasma membranes of mammalian cells. Since sphingomyelin is present mainly at the outer leaflet of cell membranes, monitoring its lipid order requires molecular probes capable to bind specifically at this leaflet and exhibit negligibly slow flip-flop. In the present work, such a probe was developed by modifying the solvatochromic fluorescent dye Nile Red with an amphiphilic anchor group. To evaluate the flip-flop of the obtained probe (NR12S), we developed a methodology of reversible redox switching of its fluorescence at one leaflet using sodium dithionite. This method shows that NR12S, in contrast to parent Nile Red, binds exclusively the outer membrane leaflet of model lipid vesicles and living cells with negligible flip-flop in the time scale of hours. Moreover, the emission maximum of NR12S in model vesicles exhibits a significant blue shift in liquid ordered phase (sphingomyelin-cholesterol) as compared to liquid disordered phase (unsaturated phospholipids). As a consequence, these two phases could be clearly distinguished in NR12S-stained giant vesicles by fluorescence microscopy imaging of intensity ratio between the blue and red parts of the probe emission spectrum. Being added to living cells, NR12S binds predominantly, if not exclusively, their plasma membranes and shows an emission spectrum intermediate between those in liquid ordered and disordered phases of model membranes. Importantly, the emission color of NR12S correlates well with the cholesterol content in cell membranes, which allows monitoring the cholesterol depletion process with methyl-beta-cyclodextrin by fluorescence spectroscopy and microscopy. The attractive photophysical and switching properties of NR12S, together with its selective outer leaflet staining and sensitivity to cholesterol and lipid order, make it a new powerful tool for studying model and cell membranes.
Journal of the American Chemical Society 03/2010; 132(13):4907-16. · 10.68 Impact Factor
[show abstract][hide abstract] ABSTRACT: The multifunctional HCV core protein consists of a hydrophilic RNA interacting D1 domain and a hydrophobic D2 domain interacting with membranes and lipid droplets. The core D1 domain was found to possess nucleic acid annealing and strand transfer properties. To further understand these chaperone properties, we investigated how the D1 domain and two peptides encompassing the D1 basic clusters chaperoned the annealing of complementary canonical nucleic acids that correspond to the DNA sequences of the HIV-1 transactivation response element TAR and its complementary cTAR. The core peptides were found to augment cTAR-dTAR annealing kinetics by at least three orders of magnitude. The annealing rate was not affected by modifications of the dTAR loop but was strongly reduced by stabilization of the cTAR stem ends, suggesting that the core-directed annealing reaction is initiated through the terminal bases of cTAR and dTAR. Two kinetic pathways were identified with a fast pre-equilibrium intermediate that then slowly converts into the final extended duplex. The fast and slow pathways differed by the number of base pairs, which should be melted to nucleate the intermediates. The three peptides operate similarly, confirming that the core chaperone properties are mostly supported by its basic clusters.
Nucleic Acids Research 02/2010; 38(11):3632-42. · 8.28 Impact Factor
[show abstract][hide abstract] ABSTRACT: In a search for environmentally sensitive (solvatochromic) dyes with superior properties, we extended the electronic conjugation of one of the best solvatochromic dyes, Prodan, by substituting its naphthalene core with fluorene. The newly synthesized fluorene derivatives bearing strong electron-donor (dialkylamino) and -acceptor (carbonyl) groups at the 2 and 7 positions showed red-shifted absorption (close to 400 nm), twice as large of a absorption coefficient (43 000 M -1 cm -1), and a manifold larger two-photon absorption cross section (∼400 GM) compared to Prodan. Studies in solvents revealed much stronger fluorescence solvatochromism of the new dyes, which is connected with their twice as large transition dipole moment (14.0 D). Similarly to Prodan, they exhibit high fluorescence quantum yields, while their photostability is largely improved. Thus, substitution of the naphthalene core in Prodan with fluorene resulted in new fluorophores with superior spectroscopic and solvatochromic properties. We expect them to find a variety of applications as environmentally sensitive probes and labels in biology. SECTION Kinetics, Spectroscopy F luorescent dyes found a variety of applications as probes and labels in biology. 1 Of special interest are the environmentally sensitive (solvatochromic) fluores-cent dyes 2-13 that exhibit strong changes in their dipole moment upon electronic excitation 14 and thus show red shifts in their emission maximum upon an increase in their solvent polarity. 15,16 Typical examples are dansyl deriva-tives, 17 Prodan, 18 Nile Red, 19,20 Fluoroprobe, 21 dimethylamino-phthalimide (DMAP), 11-13 3-hydroxychromones, 22,23 and so forth. Due to their sensitivity to the local environment, these dyes found a number of applications for monitoring protein 2-5,11-13 and DNA 6,7 interactions and probing biophy-sical properties of biomembranes. 8-10 However, none of these dyes meet simultaneously all of the desired spectro-scopic requirements for biological applications, absorption in the visible (>400 nm), high absorption coefficient (>30 000 M -1 cm -1), high fluorescence quantum yield (>50%) and photostability, as well as strong solvatochro-mism. For instance, dansyl derivatives, 17 DMAP, and its analogues 11-13 show a low absorption coefficient, while Nile Red exhibits relatively weak solvatochromism. 19,20 The most solvatochromic dye described to date, Fluoroprobe, exhibits poor absorption properties and low fluorescence quantum yield in polar solvents. 21 3-Hydroxyflavone derivatives, show-ing strong two-color solvatochromism, 22,23 suffer from poor photostability. 24 Among the best solvatochromic dyes re-ported so far are Prodan 18 and dapoxyl 25 derivatives, for which most of the mentioned criteria are fulfilled. Prodan, which is composed of a naphthalene aromatic core substi-tuted at the 2 and 6 positions with donor and acceptor groups (Chart 1), and its derivatives are probably among the most popular environmentally sensitive dyes for biological applica-tions, particularly for probing the local environment of lipid membranes, proteins, and DNA. 2-10 However, Prodan fluoro-phore absorbs in the UV range, which limits its applications in biology. A recently developed extended analogue of Prodan, Anthradan, showed significantly red-shifted absorption (around 460 nm) and emission, but its low absorption coefficient (12,000 M -1 cm -1) is a severe drawback. 26 All of these examples clearly show a strong need for improved solvent-sensitive fluorescent dyes. In the present work, we substituted the naphthalene aromatic core of Prodan with fluorene, which presents longer electronic conjugation together with rigid conjugated struc-ture. Though fluorene was extensively used as a building block for the development of dyes with good two-photon absorption properties, 27-32 the structural analogues of Prodan, bearing electron-donor dialkylamino and -acceptor carbonyl groups at the 2 and 7 positions, were not described. Therefore, we have synthesized two fluorene derivatives, FR0 (7-diethylamino-9,9-dimethyl-9H-fluorene-2-carbaldehyde) and FR8 (1-(7-diethylamino-9,9-dimethyl-9H-fluoren-2-yl)-nonan-1-one). Our spectroscopic studies show that compared to Prodan, the new dyes exhibit a 2-fold larger solvatochromism and absorp-tion coefficient, a manifold larger two-photon absorption
[show abstract][hide abstract] ABSTRACT: The functions of cell plasma membranes strongly rely on their physicochemical properties, which determine the membrane structure, control the transport of molecular species, and determine the folding and function of membrane proteins. Among these properties, the membrane electrostatics, phase state, hydration, and dynamics are of particular importance and their monitoring appears critical to further understand the structure and functions of biomembranes. Among the different techniques used to reach this aim, fluorescence-based techniques are of special interest, due notably, to their single-molecule sensitivity and their ability to operate from the molecular to the animal level. However, several intrinsic properties of the membranes render their analysis by fluorescence techniques particularly challenging. Indeed, the membrane is highly anisotropic with steep gradients of its physico-chemical properties within lipid membrane width, which does not exceed 5 nm. In addition, the membrane interior is viscous and constrained, which strongly limits the probe motion and relaxation processes. As a result, approximating the probe environment as an isotropic medium is no more valid and thus, a switch to a molecular-scale interpretation of the fluorescence data is needed. To reach this aim, probes with controlled and precise depth and orientation in the bilayer should be designed. In addition, since multiple parameters are to be measured, multiparametric probes able to simultaneously monitor several parameters through different channels should be developed.
[show abstract][hide abstract] ABSTRACT: Materials and methods All the solvents and chemicals were purchased from Aldrich. The solvents were of spectroscopic grade. Absorption and fluorescence spectra were recorded on Cary 4 spectrophotometer (Varian) and FluoroLog spectrofluorometer (Jobin Yvon, Horiba), respectively. Fluorescence quantum yields were determined by taking Prodan in ethanol (quantum yield, QY = 71 %) 1,2 as a reference. The quantum yield values were corrected for the solvent refractive index. For spectroscopic measurements, 1 μM solutions of dyes were used. All the spectra are corrected from the background (spectrum of neat solvent). In photodegradation assays, a 0.33 μM solution of a given dye in a quartz micro-cuvette (50 μM volume) was illuminated by a 360 nm light of Xenon lamp of a FluoroLog spectrofluorometer (slits were open to 8 nm). During the time of illumination (5000 seconds), the fluorescence at the maximum was recorded as a function of time. The fluorescence photodegradation curves were fitted to a single exponential decay function: I = y 0 +exp(-t/τ d), where y 0 = 0 (final intensity after total photobleaching), τ d is the degradation constant, I is the measured fluorescence intensity, and t is the time of experiment. Two-photon absorption cross section measurements were performed using Rhodamine B calibration standard according to the method of Webb et al. 3,4 Two-photon excitation was provided by a mode-locked titanium-sapphire laser (Tsunami, Spectra Physics) with a pulse duration of 200 fs. The laser was focused by an achromatic lens (f = 2 cm) in a cuvette containing the dye (30-100 μM in an appropriate solvent) and the spectra were recorded with a fibered spectrometer (Avantes) by collecting the fluorescence emission at 90° with a 20X Olympus objective. Rhodamine B in methanol was used as
[show abstract][hide abstract] ABSTRACT: Plasmodesmata (PD) are essential but poorly understood structures in plant cell walls that provide symplastic continuity and intercellular communication pathways between adjacent cells and thus play fundamental roles in development and pathogenesis. Viruses encode movement proteins (MPs) that modify these tightly regulated pores to facilitate their spread from cell to cell. The most striking of these modifications is observed for groups of viruses whose MPs form tubules that assemble in PDs and through which virions are transported to neighbouring cells. The nature of the molecular interactions between viral MPs and PD components and their role in viral movement has remained essentially unknown. Here, we show that the family of PD-located proteins (PDLPs) promotes the movement of viruses that use tubule-guided movement by interacting redundantly with tubule-forming MPs within PDs. Genetic disruption of this interaction leads to reduced tubule formation, delayed infection and attenuated symptoms. Our results implicate PDLPs as PD proteins with receptor-like properties involved the assembly of viral MPs into tubules to promote viral movement.
[show abstract][hide abstract] ABSTRACT: During HIV-1 assembly, the viral protein R (Vpr) is incorporated into newly made viral particles via an interaction with the C-terminal domain of the Gag polyprotein precursor Pr55(Gag). Vpr has been implicated in the nuclear import of newly made viral DNA and subsequently in its transcription. In addition, Vpr can affect the cell physiology by causing G(2)/M cell cycle arrest and apoptosis. Vpr can form oligomers, but their roles have not yet been investigated. We have developed fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer-based assays to monitor the interaction between Pr55(Gag) and Vpr in HeLa cells. To that end, we used enhanced green fluorescent protein-Vpr that can be incorporated into the virus and tetracysteine (TC)-tagged Pr55(Gag)-TC. This TC motif is tethered to the C terminus of Pr55(Gag) and does not interfere with Pr55(Gag) trafficking and the assembly of virus-like particles (VLPs). Results show that the Pr55(Gag)-Vpr complexes accumulated mainly at the plasma membrane. In addition, results with Pr55(Gag)-TC mutants confirm that the (41)LXXLF domain of Gag-p6 is essential for Pr55(Gag)-Vpr interaction. We also report that Vpr oligomerization is crucial for Pr55(Gag) recognition and its accumulation at the plasma membrane. On the other hand, Pr55(Gag)-Vpr complexes are still formed when Pr55(Gag) carries mutations impairing its multimerization. These findings suggest that Pr55(Gag)-Vpr recognition and complex formation occur early during Pr55(Gag) assembly.
Journal of Virology 11/2009; 84(3):1585-96. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: Quadrupolar Bodipy dyes exhibiting TPA activity and high brightness with an emission at 660 nm were synthesized and internalized in HeLa cells, whereupon FLIM experiments were conducted.
[show abstract][hide abstract] ABSTRACT: New boron containing two-photon absorbing fluorophores have been prepared. Centered on a pyrazabole central core, various conjugated systems and end groups were investigated to modulate their physicochemical properties (alkoxy, diphenylamino, and boron dipyromethene groups). One and two-photon photophysical characterizations were performed, showing efficient fluorescence in organic solvents. High two-photon absorption cross sections were determined in the 500-800 nm range. Two-photon excited microscopy images were also obtained with these new boron containing fluorescent bioprobes with laser intensities in the milliwatt range.
[show abstract][hide abstract] ABSTRACT: Ligand binding to G protein-coupled receptors is a complex process that involves sequential receptor conformational changes, ligand translocation, and possibly ligand-induced receptor oligomerization. Binding events at muscarinic acetylcholine receptors are usually interpreted from radioligand binding studies in terms of two-step ligand-induced receptor isomerization. We report here, using a combination of fluorescence approaches, on the molecular mechanisms for Bodipy-pirenzepine binding to enhanced green fluorescent protein (EGFP)-fused muscarinic M1 receptors in living cells. Real time monitoring, under steady-state conditions, of the strong fluorescence energy transfer signal elicited by this interaction permitted a fine kinetic description of the binding process. Time-resolved fluorescence measurements allowed us to identify discrete EGFP lifetime species and to follow their redistribution upon ligand binding. Fluorescence correlation spectroscopy, with EGFP brightness analysis, showed that EGFP-fused muscarinic M1 receptors predominate as monomers in the absence of ligand and dimerize upon pirenzepine binding. Finally, all these experimental data could be quantitatively reconciled into a three-step mechanism, with four identified receptor conformational states. Fast ligand binding to a peripheral receptor site initiates a sequence of conformational changes that allows the ligand to access to inner regions of the protein and drives ligand-receptor complexes toward a high affinity dimeric state.
Journal of Biological Chemistry 06/2009; 284(29):19533-43. · 4.65 Impact Factor