Fluorescence energy transfer as a tool to locate functional sites in membrane proteins

Gutiérrez‐Merino, Carlos; Centeno, Francisco; García‐Martín, Elena; Merino, Jaime M.

doi:10.1042/bst0220784

Cited by 14 publications

(13 citation statements)

References 8 publications

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“…To use FRET conditions to characterize discrete sites for lipids at the proteinlipid interface, it is necessary to know the relative position of the donor and acceptor molecules. In a previous work, taking advantage of an analytical approach developed by Gutierrez-Merino et al (31,32), a minimum donor-acceptor distance of 14 ( 1 Å could be calculated for the Laurdan-AChR pair in the T. marmorata native membrane (13), in agreement with the postulated location of Laurdan in other membranes (11,27,28) and with the overall dimensions of the AChR and its transmembrane region in particular (30).…”

Section: Discussionsupporting

confidence: 61%

Disclosure of Discrete Sites for Phospholipid and Sterols at the Protein−Lipid Interface in Native Acetylcholine Receptor-Rich Membrane

Antollini

Barrantes

1998

Biochemistry

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There is an increasing body of evidence to support the notion that the function of the nicotinic acetylcholine receptor (AChR) is influenced by its lipid microenvironment [see Barrantes, F. J. (1993) FASEB J. 7, 1460-1467]. We have recently made use of the so-called generalized polarization (GP) of the fluorescent probe Laurdan (6-dodecanoyl-2-(dimethylamino)naphthalene) to learn about the physical state of the lipids in Torpedo marmorata AChR native membrane [Antollini, S. S., Soto, M. A., Bonini de Romanelli, I., Gutiérrez Merino, C., Sotomayor, P., and Barrantes, F. J. (1996) Biophys. J. 70, 1275-1284] and cells expressing endogenous or heterologous AChR [Zanello, L. P., Aztiria, E., Antollini, S., and Barrantes, F. J. (1996) Biophys. J. 70, 2155-2164]. In the present work, Laurdan GP was measured in T. marmorata native AChR membrane by direct excitation or under energy transfer conditions in the presence of exogenous lipids. GP was found to diminish in these two regions upon addition of oleic acid and dioleoylphosphatidylcholine and not to vary significantly upon addition of cholesterol hemisuccinate, indicating an increase in the polarity of the single, ordered-liquid lipid phase in the two former cases. Complementary information about the bulk lipid order was obtained from measurements of fluorescence anisotropy of DPH and two of its derivatives. The membrane order diminished in the presence of oleic acid and dioleoylphosphatidylcholine. The location of Laurdan was determined using the parallax method. Laurdan lies at approximately 10 A from the center of the bilayer, i.e., at depth of approximately 5 A from the lipid-water interface. Exogenous lipids modified the energy transfer efficiency from the intrinsic fluorescence to Laurdan. This strategy is introduced as a new analytic tool that discloses for the first time the occurrence of discrete and independent sites for phospholipids and sterols, respectively, both accessible to fatty acids, and presumably located at a shallow depth close to the phospholipid polar head region in the native AChR membrane.

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Section: Discussionsupporting

confidence: 61%

Disclosure of Discrete Sites for Phospholipid and Sterols at the Protein−Lipid Interface in Native Acetylcholine Receptor-Rich Membrane

Antollini

Barrantes

1998

Biochemistry

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“…However, as every Cy3 fluorescent secondary antibody is labelled with more than 1 mol of Cy3 per mol of antibody and also due to the presence of more acceptor molecules in the vicinity of the donor in protein-rich microdomains, FRET between Alexa488-IgG and Cy3-IgG secondary antibodies is a case of FRET from one donor to multiple acceptors. The major advantages of this FRET case are (1) there is a minimum error in the assumption of random orientation between donor and acceptors, and (2) the separation distance for measurable FRET efficiency increases by nearly two-fold [49,51].…”

Section: Fluorescence Resonance Energy Transfer (Fret)mentioning

confidence: 99%

“…Having in mind the volume and geometry of IgG [68], the value of the distance for 50% FRET efficiency of the donor/acceptor pairs used herein (R 0 values), between 5 and 6 nm [50], and the fact that FRET efficiency is lower than 10% for a separation distance of 2·R 0 in the case of a single donor/acceptor pair [48,49] and also in the case of a highly packed and close to hexagonalcentered geometry of multiple acceptors per donor [49,51], we can conclude that the absence of statistically significant FRET implies a separation distance higher than 80 nm when using double staining with antibodies to generate the donor/acceptor pair and higher than 50 nm when only one of the proteins of interest is stained using the primary and labelled secondary antibody complex. Taking into account the molecular sizes of the plasma membrane calcium transport systems studied herein, e.g.…”

Section: Page 21 Of 47mentioning

confidence: 99%

“…For a single donor/acceptor pair with random orientation, the FRET efficiency (E) depends upon their separation (r): E = r -6 / (R 0 -6 + r -6 ), where R 0 is the distance at which the efficiency of FRET is 50% [48,49]. Alexa488 and Cy3 dyes form a good donor/acceptor pair for FRET, having a R 0 value between 5 and 6 nm [50].…”

Section: Fluorescence Resonance Energy Transfer (Fret)mentioning

confidence: 99%

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Caveolin-rich lipid rafts of the plasma membrane of mature cerebellar granule neurons are microcompartments for calcium/reactive oxygen and nitrogen species cross-talk signaling

Marques‐da‐Silva

Gutiérrez‐Merino

2014

Cell Calcium

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“…The analysis of the fluorescence energy transfer data, obtained by assessing the degree of donor (protein intrinsic fluorescence) quenching (see above), was carried out as indicated in Gutierrez-Merino et al (1994, 1995. The method was calibrated using experimental data for the fluorescein isothiocyanate-phosphatidylethanolamine/rhodamine-phosphatidylcholine and fluorescein isothiocyanate-phosphatidylethanolamine/XRITC-phosphatidylethanolamine donor/emission pairs and simulated using the data of Fung and Stryer (1978) for various other pairs.…”

Section: Analysis Of Forster Energy Transfer Datamentioning

confidence: 99%

Physical state of bulk and protein-associated lipid in nicotinic acetylcholine receptor-rich membrane studied by laurdan generalized polarization and fluorescence energy transfer

Antollini¹,

Soto²,

Romanelli³

et al. 1996

Biophysical Journal

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The spectral properties of the fluorescent probe laurdan (6-dodecanoyl-2-dimethylaminonaphthalene) were exploited to learn about the physical state of the lipids in the nicotinic acetylcholine receptor (AChR)-rich membrane and compare them with those in reconstituted liposomes prepared from lipids extracted from the native membrane and those formed with synthetic phosphatidylcholines. In all cases redshifts of 50 to 60 nm were observed as a function of temperature in the spectral emission maximum of laurdan embedded in these membranes. The so-called generalized polarization of laurdan exhibited high values (0.6 at 5 degrees C) in AChR-rich membranes, diminishing by approximately 85% as temperature increased, but no phase transitions with a clear Tm were observed. A still unexploited property of laurdan, namely its ability to act as a fluorescence energy transfer acceptor from tryptophan emission, has been used to measure properties of the protein-vicinal lipid. Energy transfer from the protein in the AChR-rich membrane to laurdan molecules could be observed upon excitation at 290 nm. The efficiency of this process was approximately 55% for 1 microM laurdan. A minimum donor-acceptor distance r of 14 +/- 1 A could be calculated considering a distance 0 < H < 10 A for the separation of the planes containing donor and acceptor molecules, respectively. This value of r corresponds closely to the diameter of the first-shell protein-associated lipid. A value of approximately 1 was calculated for Kr, the apparent dissociation constant of laurdan, indicating no preferential affinity for the protein-associated probe, i.e., random distribution in the membrane. From the spectral characteristics of laurdan in the native AChR-rich membrane, differences in the structural and dynamic properties of water penetration in the protein-vicinal and bulk bilayer lipid regions can be deduced. We conclude that 1) the physical state of the bulk lipid in the native AChR-rich membrane is similar to that of the total lipids reconstituted in liposomes, exhibiting a decreasing polarity and an increased solvent dipolar relaxation at the hydrophilic/hydrophobic interface upon increasing the temperature; 2) the wavelength dependence of laurdan generalized polarization spectra indicates the presence of a single, ordered (from the point of view of molecular axis rotation)-liquid (from the point of view of lateral diffusion) lipid phase in the native AChR membrane; 3) laurdan molecules within energy transfer distance of the protein sense protein-associated lipid, which differs structurally and dynamically from the bulk bilayer lipid in terms of polarity and molecular motion and is associated with a lower degree of water penetration.

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Fluorescence energy transfer as a tool to locate functional sites in membrane proteins

Cited by 14 publications

References 8 publications

Disclosure of Discrete Sites for Phospholipid and Sterols at the Protein−Lipid Interface in Native Acetylcholine Receptor-Rich Membrane

Disclosure of Discrete Sites for Phospholipid and Sterols at the Protein−Lipid Interface in Native Acetylcholine Receptor-Rich Membrane

Caveolin-rich lipid rafts of the plasma membrane of mature cerebellar granule neurons are microcompartments for calcium/reactive oxygen and nitrogen species cross-talk signaling

Physical state of bulk and protein-associated lipid in nicotinic acetylcholine receptor-rich membrane studied by laurdan generalized polarization and fluorescence energy transfer

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