FRET in Membrane Biophysics: An Overview

Förster resonance energy transfer (FRET), in most applications used as a “spectroscopic ruler,” allows an easy determination of the donor-acceptor intermolecular distance. However, the situation becomes complex in membranes, since around each donor there is an ensemble of acceptors at non-correlated distances. In this review, state-of-the-art methodologies for this situation are presented, usually involving time-resolved data and model fitting. This powerful approach can be used to study the occurrence of phase separation (“rafts” or other type of domains), allowing their detection as well as size evaluation. Formalisms for studying lipid–protein and protein–protein interactions according to specific topologies are also addressed. The advantages and added complexity of a specific type of FRET (energy homotransfer or energy migration) are described, as well as applications of FRET under the microscope

Quantification of protein–lipid selectivity using FRET

Membrane proteins exhibit different affinities for different lipid species, and protein–lipid selectivity regulates the membrane composition in close proximity to the protein, playing an important role in the formation of nanoscale membrane heterogeneities. The sensitivity of Förster resonance energy transfer (FRET) for distances of 10 Å up to 100 Å is particularly useful to retrieve information on the relative distribution of proteins and lipids in the range over which protein–lipid selectivity is expected to influence membrane composition. Several FRET-based methods applied to the quantification of protein–lipid selectivity are described herein, and different formalisms applied to the analysis of FRET data for particular geometries of donor–acceptor distribution are critically assessed

Influence of the sterol aliphatic side chain on membrane properties: a molecular dynamics study

Following a recent experimental investigation of the effect of the length of the alkyl side chain in a series of cholesterol analogues (Angew. Chem., Int. Ed., 2013, 52, 12848–12851), we report here an atomistic molecular dynamics characterization of the behaviour of methyl-branched side chain sterols (iso series) in POPC bilayers. The studied sterols included androstenol (i-C0-sterol) and cholesterol (i-C8-sterol), as well as four other derivatives (i-C5, i-C10, i-C12 and i-C14-sterol). For each sterol, both subtle local effects and more substantial differential alterations of membrane properties along the iso series were investigated. The location and orientation of the tetracyclic ring system is almost identical in all compounds. Among all the studied sterols, cholesterol is the sterol that presents the best matching with the hydrophobic length of POPC acyl chains, whereas longer-chained sterols interdigitate into the opposing membrane leaflet. In accordance with the experimental observations, a maximal ordering effect is observed for intermediate sterol chain length (i-C5, cholesterol, i-C10). Only for these sterols a preferential interaction with the saturated sn-1 chain of POPC (compared to the unsaturated sn-2 chain) was observed, but not for either shorter or longer-chained derivatives. This work highlights the importance of the sterol alkyl chain in the modulation of membrane properties and lateral organization in biological membranes

Analysis of the Equilibrium Distribution of Ligands in Heterogeneous Media–Approaches and Pitfalls

The equilibrium distribution of small molecules (ligands) between binding agents in heterogeneous media is an important property that determines their activity. Heterogeneous systems containing proteins and lipid membranes are particularly relevant due to their prevalence in biological systems, and their importance to ligand distribution, which, in turn, is crucial to ligand’s availability and biological activity. In this work, we review several approaches and formalisms for the analysis of the equilibrium distribution of ligands in the presence of proteins, lipid membranes, or both. Special attention is given to common pitfalls in the analysis, with the establishment of the validity limits for the distinct approaches. Due to its widespread use, special attention is given to the characterization of ligand binding through the analysis of Stern–Volmer plots of protein fluorescence quenching. Systems of increasing complexity are considered, from proteins with single to multiple binding sites, from ligands interacting with proteins only to biomembranes containing lipid bilayers and membrane proteins. A new formalism is proposed, in which ligand binding is treated as a partition process, while considering the saturation of protein binding sites. This formalism is particularly useful for the characterization of interaction with membrane proteins

Analysis of the equilibrium distribution of Ligands in Heterogeneous Media – Approaches and pitfalls

The equilibrium distribution of small molecules (ligands) between binding agents in heterogeneous media is an important property that determines their activity. Heterogeneous systems containing proteins and lipid membranes are particularly relevant due to their prevalence in biological systems, and their importance to ligand distribution, which, in turn, is crucial to ligand’s availability and biological activity. In this work, we review several approaches and formalisms for the analysis of the equilibrium distribution of ligands in the presence of proteins, lipid membranes, or both. Special attention is given to common pitfalls in the analysis, with the establishment of the validity limits for the distinct approaches. Due to its widespread use, special attention is given to the characterization of ligand binding through the analysis of Stern–Volmer plots of protein fluorescence quenching. Systems of increasing complexity are considered, from proteins with single to multiple binding sites, from ligands interacting with proteins only to biomembranes containing lipid bilayers and membrane proteins. A new formalism is proposed, in which ligand binding is treated as a partition process, while considering the saturation of protein binding sites. This formalism is particularly useful for the characterization of interaction with membrane proteins.info:eu-repo/semantics/publishedVersio

Behaviour of NBD-head group labelled phosphatidylethanolamines in POPC bilayers: a molecular dynamics study

A complete homologous series of fluorescent phosphatidylethanolamines (diCnPE), labelled at the head group with a 7-nitrobenz-2-oxa-1,3-diazo-4-yl(NBD) fluorophore and inserted in 1-palmitoyl, 2-oleoyl-snglycero- 3-phosphocholine (POPC) bilayers, was studied using atomistic molecular dynamics simulations. The longer-chained derivatives of NBD-diCnPE, with n = 14, 16, and 18, are commercially available, and widely used as fluorescent membrane probes. Properties such as location of atomic groups and acyl chain order parameters of both POPC and NBD-diCnPE, fluorophore orientation and hydrogen bonding, membrane electrostatic potential and lateral diffusion were calculated for all derivatives in the series. Most of these probes induce local disordering of POPC acyl chains, which is on the whole counterbalanced by ordering resulting from binding of sodium ions to lipid carbonyl/glycerol oxygen atoms. An exception is found for NBD-diC16PE, which displays optimal matching with POPC acyl chain length and induces a slight local ordering of phospholipid acyl chains. Compared to previously studied fatty amines, acyl chain-labelled phosphatidylcholines, and sterols bearing the same fluorescent tag, the chromophore in NBD-diCnPE locates in a similar region of the membrane (near the glycerol backbone/carbonyl region) but adopts a different orientation (with the NO2 group facing the interior of the bilayer). This modification leads to an inverted orientation of the P–N axis in the labelled lipid, which affects the interface properties, such as the membrane electrostatic potential and hydrogen bonding to lipid head group atoms. The implications of this study for the interpretation of the photophysical properties of NBD-diCnPE (complex fluorescence emission kinetics, differences with other NBD lipid probes) are discussed

Molecular Dynamics Simulation of HIV Fusion Inhibitor T-1249: Insights on Peptide-Lipid Interaction

T-1249 is a peptide that inhibits the fusion of HIV envelope with the target cell membrane. Recent results indicate that T-1249, as in the case of related inhibitor peptide T-20 (enfuvirtide), interacts with membranes, more extensively in the bilayer liquid disordered phase than in the liquid ordered state, which could be linked to its effectiveness. Extensive molecular dynamics simulations (100 ns) were carried out to investigate the interaction between T-1249 and bilayers of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and POPC/cholesterol (1 : 1). It was observed that T-1249 interacts to different extents with both membrane systems and that peptide interaction with the bilayer surface has a local effect on membrane structure. Formation of hydrogen bonding between certain peptide residues and several acceptor and donor groups in the bilayer molecules was observed. T-1249 showed higher extent of interaction with bilayers when compared to T-20. This is most notable in POPC/Chol membranes, owing to more peptide residues acting as H bond donors and acceptors between the peptide and the bilayer lipids, including H-bonds formed with cholesterol. This behavior is at variance with that of T-20, which forms no H bonds with cholesterol. This higher ability to interact with membranes is probably correlated with its higher inhibitory efficiency

Avaliação de propriedades estruturais de membranas lipídicas após substituição do colesterol por análogos fluorescentes

A espectroscopia e a microscopia de fluorescência têm sido usadas em biofísica de membranas há décadas. Como a unidade estrutural básica das membranas biológicas é a bicamada de lípidos e estes não fluorescem, o uso de sondas extrínsecas de membrana é uma necessidade. Contudo, duas questões preocupantes se levantam quanto ao uso de sondas extrínsecas de fluorescência em estudos de membranas. Em primeiro lugar, o comportamento das moléculas de sonda na bicamada (que região da bicamada elas reportam, as suas dinâmicas translacional e rotacional) é frequentemente mal conhecido. Em segundo lugar, na interpretação de resultados de experiências de fluorescência, pode ser difícil distinguir entre propriedades legítimas da membrana e efeitos de perturbação resultantes da incorporação da sonda. Para este efeito, as simulações por dinâmica molecular (MD), ao providenciarem informação detalhada à escala atómica, representam um meio valioso para caracterizar a localização e dinâmica de sondas na bicamada, assim como a magnitude de perturbação que elas induzem na estrutura lipídica [1]. Neste contexto, optimizaram-se, com recurso ao programa Firefly, as estruturas do colesterol e de dois análogos fluorescentes (desidroergoesterol e colestatrienol) ao nível de teoria DFT/R-B3LYP/6-31G(d) e submeteram-se em seguida ao servidor de topologias ATB, inscrevendo simultaneamente as cargas parciais calculadas na topologia molecular. Estas topologias foram utilizadas na construção de modelos de membranas lipídicas constituídas por POPC, colesterol e uma das sondas fluorescentes acima identificadas. Os modelos assim obtidos foram hidratados e sujeitos a simulações de MD, donde se calculou a área por lípido, a espessura e densidade da bicamada, os coeficientes de difusão lateral para as espécies presentes e os parâmetros de ordem das cadeias acilo. As simulações foram efectuadas em ensemble NPT através do pacote de software GROMACS. Análises preliminares permitiram a comparação dos comportamentos na bicamada dos esteróis fluorescentes com o do colesterol, informação vital para validar o uso dos primeiros como análogos fluorescentes do segundo. REFERÊNCIAS [1] Loura, L.M.S.; Prates Ramalho, J.P. Biophys. Rev. 1 (2009), 141

Simple Estimation of Förster Resonance Energy Transfer (FRET) Orientation Factor Distribution in Membranes

Because of its acute sensitivity to distance in the nanometer scale, Förster resonance energy transfer (FRET) has found a large variety of applications in many fields of chemistry, physics, and biology. One important issue regarding the correct usage of FRET is its dependence on the donor-acceptor relative orientation, expressed as the orientation factor κ2. Different donor/acceptor conformations can lead to κ2 values in the 0 ≤ κ2 ≤ 4 range. Because the characteristic distance for FRET, R0, is proportional to (κ2)1/6, uncertainties in the orientation factor are reflected in the quality of information that can be retrieved from a FRET experiment. In most cases, the average value of κ2 corresponding to the dynamic isotropic limit (<κ2> = 2/3) is used for computation of R0 and hence donor-acceptor distances and acceptor concentrations. However, this can lead to significant error in unfavorable cases. This issue is more critical in membrane systems, because of their intrinsically anisotropic nature and their reduced fluidity in comparison to most common solvents. Here, a simple numerical simulation method for estimation of the probability density function of κ2 for membrane-embedded donor and acceptor fluorophores in the dynamic regime is presented. In the simplest form, the proposed procedure uses as input the most probable orientations of the donor and acceptor transition dipoles, obtained by experimental (including linear dichroism) or theoretical (such as molecular dynamics simulation) techniques. Optionally, information about the widths of the donor and/or acceptor angular distributions may be incorporated. The methodology is illustrated for special limiting cases and common membrane FRET pairs