Abstract: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… Show more
“…However, this kind of formalisms has been characterized by either severe simplifying approximations (Gutierrez-Merino, 1981; Brown et al, 2007a,b) or relying to some extent to numerical results (Towles et al, 2007; see Loura et al, 2010a for a detailed discussion), precluding their widespread use.…”
Section: Numerical and Simplified Analytical Treatments Of Fret In Nomentioning
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.
“…In this case FRET (or quenching) is measured from a peptide-attached fluorophore to a labeled lipid . Preferential interaction with that particular lipid will be demonstrated by a quenching efficiency higher than that expected for a random distribution of acceptors in the plane of the membrane.…”
Section: Lateral Mobility and Domain Formationmentioning
Since their initial discovery, 30 years ago, antimicrobial peptides (AMPs) have been intensely investigated as a possible solution to the increasing problem of drug-resistant bacteria. The interaction of antimicrobial peptides with the cellular membrane of bacteria is the key step of their mechanism of action. Fluorescence spectroscopy can provide several structural details on peptide-membrane systems, such as partition free energy, aggregation state, peptide position and orientation in the bilayer, and the effects of the peptides on the membrane order. However, these "low-resolution" structural data are hardly sufficient to define the structural requirements for the pore formation process. Molecular dynamics simulations, on the other hand, provide atomic-level information on the structure and dynamics of the peptide-membrane system, but they need to be validated experimentally. In this review we summarize the information that can be obtained by both approaches, highlighting their versatility and complementarity, suggesting that their synergistic application could lead to a new level of insight into the mechanism of membrane destabilization by AMPs.
“…Variations in the separation and spatial arrangement of chromophores lead to alterations in FRET, that can be monitored under steady-state and/or time-resolved conditions. On a very basic level, FRET may be employed as a phenomenological indicator of molecular proximity, and a large body of work has been reported describing such qualitative applications . However, to take full advantage of the technique's potential, models that describe the dependence of FRET observables on the structural properties of the system under study are required.…”
Section: The Biological Importance Of Electrostatic Lipid-protein Intmentioning
Electrostatics govern the association of a large number of proteins with cellular membranes. In some cases, these proteins present specialized lipid-binding modules or membrane targeting domains while in other cases association is achieved through nonspecific interaction of unstructured clusters of basic residues with negatively charged lipids. Given its spatial resolution in the nanometer range, Förster resonance energy transfer (FRET) is a powerful tool to give insight into protein-lipid interactions and provide molecular level information which is difficult to retrieve with other spectroscopic techniques. In this review we present and discuss the basic formalisms of both hetero- and homo-FRET pertinent to the most commonly encountered problems in lipid-protein interaction studies and highlight some examples of implementations of different FRET methodologies to characterize lipid/protein systems in which electrostatic interactions play a crucial role. This article is part of a Special Issue entitled: Lipid-protein interactions.
“…www.intechopen.com R 0 is an essential parameter for the quantitative description of FRET kinetics in membrane systems (Loura et al, 2010b(Loura et al, , 2010c as well as in the classic use of FRET as a spectroscopic ruler (Stryer, 1978). In the preceding equation, whereas Q D and J may be obtained from straightforward calculations from spectral data, there is no experimental technique suited to a definite measurement of 2 (though it was shown by Dale and co-workers (1979) that intervals containing its average value < 2 > can be inferred from adequate fluorescence anisotropy measurents).…”
“…The results obtained with the non‐amyloidogenic proteins were intriguing, and prompted us to study the structure of these lipid‐protein supramolecular complexes, which are typically formed when using both a low ionic strength buffer and a high protein/lipid ratio in the experiments. Our team has developed state‐of‐the‐art methods of analysis for time‐resolved Förster resonance energy transfer (FRET) measurements, which have been applied to diverse problems in membrane biophysics such as lateral membrane domains, lipid/protein selectivity and peptide‐induced morphological alterations (reviewed in Loura et al. 2010a,b; Loura and Prieto 2009, respectively).…”
J. Neurochem. (2011) 116, 696–701.
Acidic lipids are known to both catalyze amyloid fiber formation by amyloidogenic peptides/proteins and induce formation of ‘amyloid‐like’ fibrils by non‐amyloidogenic proteins. In this work, we describe the application of state‐of‐the‐art time‐resolved Förster resonance energy transfer methodologies to the characterization of the supramolecular structure of the aggregates formed by both a cationic peptide (hexalysyltryptophan) and a basic non‐amyloidogenic protein (lysozyme) upon their interaction with negatively‐charged fluid membranes (mixtures of zwitterionic phosphatidylcholine and anionic phosphatidylserine). It was concluded that both the peptide and protein induce the formation of multistacked lipid bilayers. Furthermore, upon using conditions that are described in the literature to cause the formation of amyloid‐like fibers, lysozyme was found to induce the formation of a ‘pinched lamellar’ structure, with reduced interbilayer distance in the regions where there is bound protein, and increased interbilayer distance (stabilized by hydration repulsion) outside these areas. No significant lateral domains (lipid demixing) were induced in the membrane by either the cationic peptide or lysozyme.
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