Fábio Fernandes 1, Manuel Prieto 1, Luís M. S. Loura 2,3. Corresponding authors contact:

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1 FRET in membranes Förster resonance energy transfer in membranes: application to lipid domains and protein-lipid selectivity SUMMARY Due to their sensitivity and versatility, the use of fluorescence techniques in membrane biophysics is widespread. Among them, Förster resonance energy transfer (FRET) has risen to great prominence in the last decades. Its unique dependence on interchromophore distances at the 1-10 nm scale is entirely suited to the study of a variety of problems in membrane biophysics. This review is centered on the applications of FRET to the characterization of lipid domains/rafts and protein-lipid selectivity. Formalisms for common situations in this context are presented, and strategies for experimental studies are described and illustrated with significant literature works. Fábio Fernandes 1, Manuel Prieto 1, Luís M. S. Loura 2,3 1 Centro de Química Física Molecular and Institute of Nanosciences and Nanotechnologies, Complexo I, Instituto Superior Técnico, Av. Rovisco Pais, Lisboa, Portugal 2 Faculdade de Farmácia, Universidade de Coimbra, Pólo das Ciências da Saúde, Azinhaga de Santa Comba, Coimbra, Portugal 3 Centro de Química de Coimbra, Largo D. Dinis, Rua Larga, Coimbra, Portugal Corresponding authors contact: fernandesf@ist.utl.pt; lloura@ff.uc.pt 1. INTRODUCTION Förster resonance energy transfer (FRET) is a photophysical process by which an initially electronically excited fluorophore, the donor (D), transfers its excitation energy (thereby becoming quenched) to another chromophore, the acceptor (A), which electronic absorption spectrum overlaps that of the emission of D. The latter, initially in the electronic ground state, becomes excited upon transfer, and may (or not) fluoresce. FRET involves neither photon emission nor molecular contact between the two species, but is highly dependent on the distance between them. For an isolated donor-acceptor pair, the rate for the FRET interaction is proportional to the inverse sixth power of this distance [1]. The characteristic length for FRET is the Förster radius, R 0, defined as the donor/acceptor distance for which FRET within a given D/A pair is 50% efficient. In practice, the distance range for which FRET is sensitive is between 0.5 R 0 and 2 R 0. R 0 is characteristic of each D/A pair in a given environment, but usually lies in the nm range. This implies that FRET is mostly sensitive to distances in the 1-10 nm scale, which is out of the reach of conventional optical microscopy techniques, but is entirely adequate to the study of important questions in membrane biophysics, such as detection and characterization of nanodomains/rafts and lipid/protein or protein-protein interaction (Fig. 1). Different approaches can be envisaged, ranging from qualitative studies of variation of FRET steady-state efficiency, without consideration of the underlying kinetics, to analysis of time-resolved fluorescence data with appropriate formalisms, allowing the quantitative recovery of 12. canalbq_n.º 8_JULHO_2011

2 2. INTRAMOLECULAR FRET: A SPECTRO- SCOPIC RULER FRET between D/A pairs for which the D-A distance is the same, such as verified in a solution of a two-chromophore species, is often termed intramolecular FRET. A quantification of the extent of FRET is given by the FRET efficiency, E, which is calculated as E = 1 i DA (t) dt i D (t) dt 0 0 In this equation, i D (t) and i DA (t) are the D decays in absence and presence of A (respectively). The effect of FRET on the fluorescence of D is the reduction of its lifetime and quantum yield. In this simple case, the D decay law remains exponential, albeit faster than in the absence of acceptor. The relationship between the lifetime of D in absence and presence of A (τ 0 and τ, respectively) is given by E =1 τ /τ 0 = R 6 0 /(R 6 + R 6 0 ) 3. FRET IN HOMOGENEOUS AND HETEROGE- NEOUS LIPID BILAYERS 3.1. Uniform distribution of fluorophores in membranes In membranes, each D molecule is usually surrounded by a distribution of A molecules. Therefore, measurement of single D/A distances is neither meaningful nor feasible. The decay of D s emission becomes complex and dependent on the topology of the system under study, as well as the concentration of A. Analytical solutions can still be derived for uniform distribution of chromophores. For planar distributions of D and A, the decay of D in presence of A is given by [4-5]: Figure 1: Schematic representation of applications of FRET in membrane biophysics. Only one bilayer leaflet is depicted. (A): membrane heterogeneity; (B): determination of transverse location of a fluorescent residue/label; (C): protein/lipid selectivity; (D): protein oligomerization. Whereas membrane domains are currently thought to play a role in cell processes including signal transduction, endocytosis and cholesterol trafficking, protein selectivity for certain membrane lipids is an important issue regarding both protein activity and creation of local heterogeneity centered around the protein. In both cases, the acute distance/concentration dependence of FRET constitutes a unique asset, as described in the following sections. where R is the D-A separation. An expression identical to Eq. 2 can be written for the fluorescence quantum yield, or the fluorescence steady-state intensity. R 0 is calculated independently from spectroscopic data, topological information about the system 1/ 6 under study. This review describes the basic formalisms of FRET in membranes, as 0 R 0 (in nm units) = κ 2 Φ D n 4 I(λ) ε(λ) λ 4 dλ well as significant illustrative applications 1/ 6 of FRET in the important fields of membrane domain/raft characterization and function, R e In this equation, γ is the incomplete gamma R 0 (in nm units) = κ 2 Φ D n 4 I(λ) ε(λ) λ 4 dλ is the minimum D/A distance 0 protein/lipid interactions and selectivity. (exclusion distance) and n is the numerical where κ 2 is the orientation factor (see [2] for a detailed discussion), Φ D is the D quantum yield in the absence of A, n is the refractive index, λ is the wavelength (in nm units), I(λ) is the normalized D emission spectrum, and ε(λ) is the A molar absorption spectrum. In this way, from both steady-state and timeresolved data, R is easily computed. This is the basis of the use of intramolecular resonance energy transfer as a spectroscopic ruler [3]. concentration of A (molecules/unit area). Although it was originally derived for a plane of acceptors containing the donor (cis transfer), it is also valid if the D molecule is separated from the A plane by a distance R e, a situation common on membranes, as D and A are often located at different depths in the bilayer. Upon preparation of lipid vesicles, D and A molecules are frequently inserted in either of the bilayer leaflets, with equal probability. In this case, one must consider two planes of canalbq_n.º 8_JULHO_

3 it was shown that the phase diagram of a biacceptors for a given donor, one corresponding to the acceptors lying in the same bilayer leaflet as the donor, and another for those located in the opposite leaflet. The decay law in this case is obtained by simply multiplying the intrinsic donor decay by the FRET terms corresponding to each plane of acceptors. Another common occurrence in membrane systems is a complex decay of donor even in the absence of acceptors, with a sum of two or three exponentials being required for a proper description. In this case, the above equations can be still used, provided that the exponential donor intrinsic decay term is replaced by this function, and τ 0 is replaced by the intensity-average [6] decay lifetime. For steady-state applications, Eq. 4 can be integrated numerically (in a program or spreadsheet) to produce curves of FRET efficiency E (calculated using Eq. 1) as function of acceptor concentration n, with R e as a parameter. Alternatively, R e is fixed and experimental FRET decays/efficiencies are compared with theoretical expectations. Eventual failure to analyze FRET kinetics with the uniform probe distribution formalism may have relevance (e.g., addition of a new component to a given one-phase lipid bilayer system may induce compartmentalization and/or phase separation, and hence deviations to the theoretical uniform distribution expectation) [7-10] Non-uniform distribution of fluorophores contains topological information Non-uniform component distribution and phase separation are common occurrences in lipid mixtures. The molecules bearing the donor and acceptor fluorophores in a FRET experiment in such a system will naturally have non-uniform distributions, following their partition between the coexisting phase domains. Let us consider that there are Figure 2: Top: FRET efficiency E between 0.1 mol% NBD-DMPE (D) and 0.5 mol% Rh-DMPE (A) in DMPC/cholesterol at 30 ºC (which displays wide liquid ordered (lo)/liquid disordered (ld) phase separation range) large unilamellar vesicles as a function of cholesterol mole fraction (X chol ). NBD-DMPE partitions preferably to the lo phase, while Rh-DMPE prefers the ld phase. Bottom: cartoons depicting the extent of phase separation and probe distribution for different composition ranges. Adapted from [12] and [15]. only two phases or types of domains present (the most common experimental situation). If these are sufficiently large to be infinite in the FRET scale (i.e., complications resulting form FRET involving molecules in different domains, or boundary effects, are negligible; this is the case if the domains are larger than ~5-10 R 0 ), then the donor decay law is simply a linear combination of the hypothetical decay laws in each phase i DA,phase i (given by Eq. 4), weighed by the relative amount of donor in each phase A i [11,12]: i DA (t) = A 1 i DA,phase 1 (t) + A 2 i DA,phase 2 (t) Note that usually the donor lifetimes are different in the two phases, as are the acceptor surface concentrations (and possibly also the D-A exclusion distances). This introduces a large number of fitting parameters in Eq. 5. To ensure meaningful recovery of these parameters, the decay of D in presence of A is globally analyzed together with that in the absence of A, i DA (t) = A 1 exp( t /τ 1 ) + A 2 exp( t /τ 2 ) where τ 1 and τ 2 are the D lifetimes in each phase. The recovered parameters are usually τ 1, τ 2, A 2 /A 1 (from which the D partition coefficient K pd is obtained), and the A concentrations in the two phases, C 1 and C 2 (from which the A partition coefficient K pa is obtained) [11]. A particular case of this formalism is the so-called isolated donors situation, which corresponds to C 2 = 0. Being strictly valid for infinite phase separation, this model can be applied to nano-scale domain formation; in this case, the recovered A partition coefficient ( FRET K pa ) will not be equal to the true coefficient recovered from independent (fluorescence intensity, anisotropy or lifetime of A) measurements ( non- FRET K pa ), and is affected (being closer to unity than the non-fret value) by the fact that donors in one phase are sensitive to acceptors in the other. The extent of this deviation reflects the size of the nanodomains. In the very small domain size limit (< R 0 ), the FRET K pa is essentially unity. Therefore, this kind of analysis can produce insights regarding the size of the domains. This was applied to study the composition-dependent variation of domain sizes in liquid ordered/ liquid disordered phase-separated binary [12] and ternary [13,14] membrane systems (Fig. 2). In the case of infinite phase separation (which can be validated if the FRET and non-fret K pa values are indistinguishable) 14. canalbq_n.º 8_JULHO_2011

4 nary mixture could be obtained from the decay parameters [12]. An experimental method of characterization of phase separation in lipid membranes (and determination of binary and ternary phase diagrams in the infinite phase limit) based on this formalism, but relying solely on acceptor steady-state sensitized emission, was proposed by Buboltz [15], who termed it Steady- -State Probe-Partitioning FRET or SP-FRET. This method was used to characterize the phase behavior of different saturated phosphatidylcholine/unsaturated phosphatidylcholine/cholesterol systems [16,17] Numerical and simplified analytical treatments of FRET in nonhomogeneous systems No exact solution of the FRET rate or efficiency has been derived for the case of incomplete phase separation, with nm-sized domains of a given type dispersed in the continuous phase. This stems from the evident symmetry loss introduced by the presence of the domains. One way to tackle this complexity is to calculate the D decay using numerical simulation. Basically, the process starts by building a topology of the lipid matrix (i.e. define size of the simulated system, domain shape, average size and size distribution, and then place the domains on the matrix, ensuring that the overall fraction of each phase is as intended) and placing D and A molecules taking into account their domain preference. A given D is then selected and its interaction with all the acceptors (or those within a cutoff of several R0 lengths) is computed. This step is then repeated for all the donors in order to obtain an ensemble average value for all the system. One obtains the average D decay, and, by using Eq. 1, the FRET efficiency value. This kind of numerical simulations was already described by Wolber and Hudson [5] for uniform distribution in a planar geometry to test their analytical theory. The first numerical solution of FRET for non-uniform membrane probe distribution was given by Snyder and Freire [18]. In this work, heterogeneity of probe distribution was introduced by incorporating a heuristic potential function in the random placement of the probes. Therefore, no domains are actually simulated, and whereas the authors equations are suited to the analysis of probe aggregation in a single phase system, they are not useful regarding phase separation. Simulations in which probe distribution heterogeneity is introduced by building a biphasic system and taking into consideration probe partition have been presented by authors to provide tests for their analytical formalisms [12, 19-21]. Another simulation approach consists in recreating the excitation and de-excitation processes of each D or A molecule by performing stochastic simulations. These calculations take into account the probabilities of donor excitation, donor decay by non- FRET processes, FRET to a given acceptor and acceptor de-excitation. The FRET efficiency is simply calculated by the ratio between the total number of transfers and the total number of D excitations. This type of simulation has been applied to the case of FRET in a planar geometry with disk-like domains [22]. All previous works are characterized by prior fixing of the underlying lipid matrix, including domain size and shape, and the simulations concern exclusively the calculation of FRET rates and probabilities. A different approach was recently undertaken by Frazier et al. [23], who combined statistical mechanical lattice Monte-Carlo simulations (to describe the lipid matrix and to generate chromophore positions therein) with a simplified step-function FRET distance dependence (FRET was considered to occur if and only if the D-A distance in a given pair were less than R0) to analyze experimental data of FRET in a ternary, raft-model mixture. This work demonstrates that FRET and computational techniques can be combined to create a powerful combination, suited to the study of lipid phase separation. Globally, numerical simulations have the advantage of allowing the calculation of FRET in systems for which, due to their complexity, an exact solution is precluded. However, thus far they do not provide a way to analyze experimental data directly, as many degrees of freedom are expected in an actual experiment. For each simulation, values for R0, donor lifetime, D/A exclusion distance (all being possibly different in each coexisting phase), domain shape and size, and D and A partition coefficients need to be fixed. This multiplicity of variables excludes the possibility of fitting with simple empirical functions that could describe, e.g., the variation of FRET efficiency as a function of concentration of A in a general manner, which would be convenient for most researchers. The study of a particular system requires individual setup and simulation procedures. An alternative to numerical simulations of FRET in nanoheterogeneous bilayers is the use of simplified analytical treatments, which, unlike the former, could potentially be suited to direct analysis of experimental data enabling recovery of the parameters of interest. However, this kind of formalisms has been characterized by either severe simplifying approximations [24-26] or relying to some extent to numerical results [20], precluding their widespread use Application of FRET to protein lipid selectivity studies A central aspect in lipid-protein interaction is protein preference for selected lipid species or classes, which may act as the driving force for enrichment of these components in the bilayer region surrounding the protein, at the expense of other lipids. This provides a mechanism for creation of lipid distribution heterogeneity, potentially to several lipid shells around the protein. Some superficial membrane proteins demonstrate specific binding to some lipid classes, a phenomenon that can control protein canalbq_n.º 8_JULHO_

5 membrane, an exclusion distance (R e ) berecruitment to the membrane and activate signalling cascades [27]. Additionally, transmembrane proteins display differential interactions with lipids of different acylchain lengths due to packing constraints in the lipid/protein hydrophobic interface, that have a significant effect on the activity of several proteins. Membrane proteins have also been shown to present binding sites for lipids in hydrophobic pockets away from the protein/lipid interface and binding of specific lipids to such sites is essential for activity in several cases [28]. The study of protein-lipid selectivity has been generally addressed using electronspin-resonance (ESR) [29], while fluorescence collisional quenching methods [30,31] can also provide a similar structural type of information. These techniques are able to probe the lipid environment in direct contact with the protein, but are insensitive to the presence of lipids displaced from the protein/lipid interface. On the other hand, FRET is sensitive to distances up to 100 Å, and when used as a tool to the study of protein-lipid interactions is able to access information unavailable to other techniques. Due to the high sensitivity of FRET to changes in distances around R0, it is possible through the use of chemical labeling or intrinsic fluorophores, to directly measure the binding of lipids to proteins, or protein mediated enrichment of particular lipids in the membrane. The multitude of lipid fluorescent analogues commercially available as well as of fluorescent probes for protein labeling allows for a significant flexibility in experimental design. Several fluorescently-tagged lipids have been shown to mimic properties of their natural analogues and were extremely helpful in elucidating several problems in lipid traffi- Figure 3: Molecular model for the FRET analysis according to the model of Fernandes et al. (2004) ((A) side view; (B) top view). Protein-lipid organization presents a hexagonal geometry. Donor fluorophore from the mutant protein is located in the center of the bilayer, whereas the acceptors are distributed in the bilayer surface. Two different environments are available for the labeled lipids (acceptors), the annular shell surrounding the protein and the bulk lipid. Energy transfer to acceptors in direct contact with the protein has a rate coefficient dependent on the distance between donor and annular acceptor (Eq. 8). Energy transfer toward acceptors in the bulk lipid is given by Eq. 4. Adapted from [46]. cking and sorting [32]. Also, aromatic amino acid residues can be used as donors in FRET experiments avoiding in this way the need for chemical labeling of the protein. FRET has been applied to protein-lipid selectivity problems both in a qualitative and a quantitative manner. The simplest approach relies on just comparing FRET efficiencies of the protein to different fluorescent lipid analogues (or vice-versa), presenting the same chromophores and at the same concentration [33]. Additionally, selectivity for unlabeled lipids can be studied through competition experiments by measuring the effect of adding the lipids of interest on the FRET efficiencies between the protein and a membrane probe (or vice-versa). The membrane probe can be a fluorescent analogue of a lipid species or a lipophilic organic dye. Lipids with greater affinity for the protein will decrease FRET efficiency to a larger extent as the membrane probe is excluded from the vicinity of the protein at the expense of the high affinity lipid [34-36]. For quantification of protein-lipid selectivity, one faces the problem of accounting for non-interacting lipids. When studying interactions with very high affinity, it is possible to avoid the contribution of non-interacting lipids to FRET by limiting the concentration of donor/acceptor lipids in the medium. In this way, while the probabilities of noninteracting lipids to be close enough to the protein can be low enough to be neglected, it is still possible to measure FRET from interacting species as a result of the high affinity between the species. Assuming as an approximation that there is 100% FRET efficiency between the protein-bound lipid pair (i.e. complete quenching of the donor after acceptor binding), it is possible to recover a dissociation constant for the interaction. This approximation will overestimate binding and retrieved dissociation constants should be considered upper limits for its real value [37-38]. However, the application of this strategy is somewhat limited as typical protein-lipid association constants rarely diverge by more than one order of magnitude [28,39]. Typical R0 values are often comparable to protein dimensions (20-60 Å) and a significant area around the protein FRET species (either donor or acceptor) is unavailable for lipids. As a result, FRET efficiencies are smaller than what would be expected from donors and acceptors with negligible sizes. This effect is more significant for larger proteins and low R0 values. In several protein-lipid selectivity studies it is assumed that due to large protein dimensions, FRET efficiencies to or from unbound lipid species can be neglected and only bound lipid species contribute to FRET [40-42]. More detailed FRET models include geometrical parameters in the analysis of FRET efficiencies between proteins and lipids [41,43-45]. These models include a transverse distance (l) between the planes of donor and acceptor chromophores in the 16. canalbq_n.º 8_JULHO_2011

6 membrane, it can be assumed that half of the acceptors (in the same monolayer as the donor) are associated with a higher FRET rate constant, and the other half (in the opposing monolayer) is associated with a lower FRET rate constant. The FRET rate constant is always given by: k T = 1 R 0 τ D d 6 Figure 4: Model of Lactose permease/phospholipid arrangement in the membrane: sagittal view showing the lipid annular shell. The tryptophan acting as a FRET donor is located in the membrane surface while FRET acceptor pyrene from pyrene-labeled lipids positions itself in the center of the bilayer. Distance between donor and all acceptors is approximately the same, and a single FRET rate constant is considered for energy transfer to annular acceptors. From geometrical considerations and molecular areas from the phospholipids, the number of phospholipid molecules in the annular layer surrounding the protein of radius 3.0 nm was estimated to be 23 per bilayer leaflet or 46 in total [45]. tween donors and acceptors (Fig. 3), and an interaction parameter K S, which represents the apparent dissociation constant of the fluorescent lipid analogue for the lipid belt region (that is, the ratio of the dissociation constant of labeled over that of the unlabeled lipid). K S >1 implies preferential location of labeled lipid in the lipid belt region, whereas K S <1 denotes labeled lipid exclusion from this region. This method works best if good estimates are known for d and R e, and the sole optimizing parameter is K S. Additionally, the contribution of unbound acceptors can be included using eq. 4 [44,45]. In this way, and for the case of a transmembrane protein donor under FRET to a lipid acceptor, the donor decay is described by: i DA (t) =i D (t)ρ annular ρ random (t) Where ρ annular and ρ random are the FRET contributions arising from energy transfer to bound or annular labeled lipids and to randomly distributed labeled lipids outside the annular shell, respectively. The ρ random contribution is calculated from eq. 4. The simplest geometrical donor/acceptor arrangements occurs for proteins modeled as cylinders with a donor in the center of the cylinder and of the transmembrane segment of the protein, for which all acceptors in the annular region are at the same distance d (Fig. 3). In this case, FRET to each of these acceptors is associated with the same rate constant. The acceptors can be located in the center of the membrane [45] or in the surface of the membrane [44]. In case the donor is at the surface and the acceptors locate in the center of the bilayer as is common for pyrene labeled lipids, it is also reasonable to consider that all acceptors are associated with the same rate constant [45]. If both donors and acceptors are positioned in the interfacial area of the The ρ annular contribution is then calculated from: N N ρ annular = e nk T t μ n 1 μ n n= 0 ( ) N n where N is the number of phospholipid molecules in the first layer surrounding the protein, and µ is the probability of each of these phospholipids to be labeled. The probability of each of the annular sites to be occupied by an acceptor depends on the acceptor molar fraction and on K S. The value of N for a single monomeric transmembrane protein is 12 (Fig. 3), and for larger proteins can be calculated from geometrical considerations taking into account the molecular areas of lipids and protein (Fig. 4). One alternative to this model is the assumption of acceptor concentration (labeled lipid) around the donor (protein) as step-functions of the donor-acceptor distance. Three regions can be considered: (i) an exclusion region closest to the donor (R < R 1 ), reflecting the radius of the protein; (ii) the annular region (R 1 < R < R 2 ), for which there is an increased probability of finding acceptors, characterized by a parameter B; and (iii) a region for which the acceptor concentration is equal to the overall value (R > R 2 ). The analytical law for donor decay canalbq_n.º 8_JULHO_

7 chemically distinct from the donor chromophore (hetero-fret), though some experiments use FRET among like chromophores (homo-fret). FRET efficiency: The fraction of donor excitation events that leads to FRET. It can be calculated both from the extent of quenching of donor emission (most used) or from the sensitized acceptor emission (in case that the acceptor is fluorescent). Förster radius: Also known as R 0, it represents the donor-acceptor distance for which the FRET efficiency (for an isolated donor-acceptor pair) is 50%. It has a characteristic value for a donor/acceptor pair in a given environment and it can be conveniently calculated from spectroscopic data. For most donor-acceptor pairs, it lies in the 1-6 nm range. Intramolecular FRET: FRET involving donor and acceptor chromophores bound to the same molecule (for example, FRET between two fluorescent labels covalently bound to a protein molecule). The measurement of its efficiency allows determination of the donor-acceptor distance, in case that the Förster radius is known (which often is the case, as it is determined independently). By contrast, in case that donor and acceptor are not bound to the same molecule (as often is the case in membranes), FRET is termed as intermolecular. Steady-State Probe-Partitioning FRET: A method developed by Jeffrey Buboltz [15], which allows determination of donor and acceptor partition coefficients in biphasic membranes from steady-state fluorescence data (acceptor sensitized emission intensity). Although it assumes the infinite phase separation hypothesis (domains >> R 0 ) and that the phase compositions are known, the author showed that the latter hypothesis can be alleviated, as phase boundaries can be identified from maxima of the sensitized emission gradient s absolute value. ACKNOWLEDGEMENTS The authors acknowledge funding by FEDER, through the COMPETE program, and by FCT, project references FCOMP FEDER (FCT PTDC/ Donor: The chromophore from which electronic excitation energy is transferred in FRET. QUI-QUI/098198/2008), PTDC/QUI-BIQ/099947/2008 and PTDC/QUI-BIQ/112067/2009. F.F. acknowledges a Acceptor: The chromophore to which electronic excitation energy is transferred in FRET. In most uses, it is research grant (SFRH/BPD/64320/2009) from Fundacão para a Ciência e Tecnologia (FCT). in the presence off the acceptor has been numerically integrated for different exclusion distances, donor-acceptor plane spacing, acceptor concentrations, and B [47]. In this study, empirical five parameter functions were retrieved for the determination of FRET efficiency in different conditions, and the model was successfully applied to a protein-lipid interaction problem. 4. CONCLUSIONS In this review, applications of FRET in membrane biophysics, comprising studies of lateral heterogeneity (membrane domains) and protein/lipid selectivity (preference of a specific lipid for the protein vicinity) are described. The complexity of FRET in membranes was addressed, and it was shown that detailed topological information can be obtained from this methodology, once adequate modeling is taken into account. Examples of relevant works in this area were critically reviewed. On the whole, the power of FRET as a tool in membrane biophysics is emphasized. GLOSSARY Förster resonance energy transfer (FRET): Radiationless process by which an electronically excited chromophore (donor) returns to the ground state, as in turn another chromophore (the acceptor) becomes itself resonantly excited. 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