Manuel Prieto - Academia.edu (original) (raw)

Papers by Manuel Prieto

Journal of the Chemical Society, Faraday Transactions 2: Molecular and Chemical Physics, 1987

Journal of Biological Chemistry, 2000

The topography of nicotinic acetylcholine receptor (AChR) membrane-embedded domains and the relat... more The topography of nicotinic acetylcholine receptor (AChR) membrane-embedded domains and the relative affinity of lipids for these protein regions were studied using fluorescence methods. Intact Torpedo californica AChR protein and transmembrane peptides were derivatized with N-(1-pyrenyl)maleimide (PM), purified, and reconstituted into asolectin liposomes. Fluorescence mapped to proteolytic fragments consistent with PM labeling of cysteine residues in ␣M1, ␣M4, ␥M1, and ␥M4. The topography of the pyrene-labeled Cys residues with respect to the membrane and the apparent affinity for representative lipids were determined by differential fluorescence quenching with spin-labeled derivatives of fatty acids, phosphatidylcholine, and the steroids cholestane and androstane. Different spin label lipid analogs exhibit different selectivity for the whole AChR protein and its transmembrane domains. In all cases labeled residues were found to lie in a shallow position. For M4 segments, this is compatible with a linear ␣-helical structure, but not so for M1, for which "classical" models locate Cys residues at the center of the hydrophobic stretch. The transmembrane topography of M1 can be rationalized on the basis of the presence of a substantial amount of non-helical structure, and/or of kinks attributable to the occurrence of the evolutionarily conserved proline residues. The latter is a striking feature of M1 in the AChR and all members of the rapid ligand-gated ion channel superfamily.

Methods in Membrane Lipids

Cholesterol is a major component of mammalian cell membranes. It has remarkable effects on the pr... more Cholesterol is a major component of mammalian cell membranes. It has remarkable effects on the properties of phospholipids bilayers, and is implicated in the lipid raft model. Depending on the membrane composition, cholesterol-containing bilayers can exist either as single phase or as mixture of coexisting phases. These are organized in domains of variant size determined by composition, but can be smaller than the optical microscopy resolution limit. This chapter describes a methodology based on fluorescence resonance energy transfer, which is sensitive to phase separation and can provide estimates of domain size in these usually hard to characterize systems.

Photochemistry and Photobiology, 1988

ABSTRACT

Chemistry and Physics of Lipids, 2008

Biophysical Journal, 1995

The interaction between Nystatin and small unilamellar vesicles of 1,2-dipalmitoyl-sn-glycero-3-p... more The interaction between Nystatin and small unilamellar vesicles of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, both in gel (T = 210C) and in liquid-crystalline (T = 450C) phases, was studied by steady-state and time-resolved fluorescence measurements by taking advantage of the intrinsic tetraene fluorophore present in this antibiotic. It was shown that Nystatin aggregates in aqueous solution with a critical concentration of 3 puM. The enhancement in the fluorescence intensity of the antibiotic was applied to study the membrane binding of Nystatin, and it was shown that the antibiotic had an almost fivefold higher partition coefficient for the vesicles in a gel (P = (1.4 ± 0.1) x 103) than in a liquid-crystalline phase (P = (2.9 ± 0.1) x 102). Moreover, a time-resolved fluorescence study was used to examine Nystatin aggregation in the membrane. The emission decay kinetics of Nystatin was described by three and two exponentials in the lipid membrane at 210C and 450C, respectively. Nystatin mean fluorescence lifetime is concentration-dependent in gel phase lipids, increasing steeply from 11 to 33 ns at an antibiotic concentration of 5-6 ,uM, but the fluorescence decay parameters of Nystatin were unvarying with the antibiotic concentration in fluid lipids. These results provide evidence for the formation of strongly fluorescent antibiotic aggregates in gel-phase membrane, an interpretation that is at variance with a previous study. However, no antibiotic self-association was detected in a liquid-crystalline lipid bilayer within the antibiotic concentration range studied (0-14 ,uM).

Biophysical journal, Apr 30, 2004

A 28-mer γM4 peptide, obtained by solid-state synthesis and corresponding to the fourth transmemb... more A 28-mer γM4 peptide, obtained by solid-state synthesis and corresponding to the fourth transmembrane segment of the nicotinic acetylcholine receptor γ-subunit, possesses a single tryptophan residue (Trp453), making it an excellent model for studying peptide-lipid interactions in membranes by fluorescence spectroscopy. The γM4 peptide was reconstituted with synthetic lipids (vesicles of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, ie, POPC) rich and poor in cholesterol and analyzed using steady-state ...

Biomolecules

Despite its low abundance, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a key modulator o... more Despite its low abundance, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a key modulator of membrane-associated signaling events in eukaryotic cells. Temporal and spatial regulation of PI(4,5)P2 concentration can achieve localized increases in the levels of this lipid, which are crucial for the activation or recruitment of peripheral proteins to the plasma membrane. The recent observation of the dramatic impact of physiological divalent cation concentrations on PI(4,5)P2 clustering, suggests that protein anchoring to the plasma membrane through PI(4,5)P2 is likely not defined solely by a simple (monomeric PI(4,5)P2)/(protein bound PI(4,5)P2) equilibrium, but instead depends on complex protein interactions with PI(4,5)P2 clusters. The insertion of PI(4,5)P2-binding proteins within these clusters can putatively modulate protein–protein interactions in the membrane, but the relevance of such effects is largely unknown. In this work, we characterized the impact of Ca2+ on the org...

International Journal of Molecular Sciences, 2021

Alkylammonium salts have been used extensively to study the structure and function of potassium c... more Alkylammonium salts have been used extensively to study the structure and function of potassium channels. Here, we use the hydrophobic tetraoctylammonium (TOA+) to shed light on the structure of the inactivated state of KcsA, a tetrameric prokaryotic potassium channel that serves as a model to its homologous eukaryotic counterparts. By the combined use of a thermal denaturation assay and the analysis of homo-Förster resonance energy transfer in a mutant channel containing a single tryptophan (W67) per subunit, we found that TOA+ binds the channel cavity with high affinity, either with the inner gate open or closed. Moreover, TOA+ bound at the cavity allosterically shifts the equilibrium of the channel’s selectivity filter conformation from conductive to an inactivated-like form. The inactivated TOA+–KcsA complex exhibits a loss in the affinity towards permeant K+ at pH 7.0, when the channel is in its closed state, but maintains the two sets of K+ binding sites and the W67–W67 inters...

International Journal of Molecular Sciences, 2021

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is an essential plasma membrane component invol... more Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is an essential plasma membrane component involved in several cellular functions, including membrane trafficking and cytoskeleton organization. This function multiplicity is partially achieved through a dynamic spatiotemporal organization of PI(4,5)P2 within the membrane. Here, we use a Förster resonance energy transfer (FRET) approach to quantitatively assess the extent of PI(4,5)P2 confinement within the plasma membrane. This methodology relies on the rigorous evaluation of the dependence of absolute FRET efficiencies between pleckstrin homology domains (PHPLCδ) fused with fluorescent proteins and their average fluorescence intensity at the membrane. PI(4,5)P2 is found to be significantly compartmentalized at the plasma membrane of HeLa cells, and these clusters are not cholesterol-dependent, suggesting that membrane rafts are not involved in the formation of these nanodomains. On the other hand, upon inhibition of actin polymeriza...

Journal of Lipid Research, 2012

Supplementary key words ceramide • sphingolipids • acyl chain • lipid domains • tubules • membran... more Supplementary key words ceramide • sphingolipids • acyl chain • lipid domains • tubules • membrane order Sphingolipids (SLs) are important components of biological membranes and are involved in a variety of biological functions. SL synthesis starts at the endoplasmic reticulum

Journal of Biological Chemistry, 2010

Ceramide is an important lipid signaling molecule that plays critical roles in regulating cell be... more Ceramide is an important lipid signaling molecule that plays critical roles in regulating cell behavior. Ceramide synthesis is surprisingly complex and is orchestrated by six mammalian ceramide synthases, each of which produces ceramides with restricted acyl chain lengths. We have generated a CerS2 null mouse and characterized the changes in the long chain base and sphingolipid composition of livers from these mice. Ceramide and downstream sphingolipids were devoid of very long (C22-C24) acyl chains, consistent with the substrate specificity of CerS2 toward acyl-CoAs. Unexpectedly, C16-ceramide levels were elevated, and as a result, total ceramide levels were unaltered; however, C16-ceramide synthesis in vitro was not increased. Levels of sphinganine were also significantly elevated, by up to 50-fold, reminiscent of the effect of the ceramide synthase inhibitor, fumonisin B1. With the exceptions of glucosylceramide synthase and neutral sphingomyelinase 2, none of the other enzymes tested in either the sphingolipid biosynthetic or degradative pathways were significantly changed. Total glycerophospholipid and cholesterol levels were unaltered, although there was a marked elevation in C18:1 and C18:2 fatty acids in phosphatidylethanolamine, concomitant with a reduction in C18:0 and C20:4 fatty acids. Finally, differences were observed in the biophysical properties of lipid extracts isolated from liver microsomes, with membranes from CerS2 null mice displaying higher membrane fluidity and showing morphological changes. Together, these results demonstrate novel modes of cross-talk and regulation between the various branches of lipid metabolic pathways upon inhibition of very long acyl chain ceramide synthesis.

Biopolymers, 1997

The structure of a new supramolecular drug carrier (named Biovectors-BV) was studied using light ... more The structure of a new supramolecular drug carrier (named Biovectors-BV) was studied using light scattering and scanning electronic microscopy techniques. This system consists of a polysaccharide core of chemically cross-linked maltodextrins to which phospholipids (and, in some cases, cholesterol) are added. Both polysaccharide cores and BV crosslinked with phosphate (negatively charged) and epichlorhydrin (no net charge) are spherical particles. The increase in the ionic strength of the medium increases the density of the charged polysaccharide cores. The lipid strongly interacts with neutral and negatively charged cores, decreasing both intra-and interparticle interactions. The results (mainly, r Å R g /R h õ 0.775 in some cases) suggest that BV are gel-like particles of variable density, referred to as microgels or soft spheres. Neutral polysaccharides have a strong tendency to self-aggregate. This selfaggregation of polysaccharide neutral cores is prevented by the addition of lipid or dimethylsulfoxide.

Biophysical Journal, 2012

Biophysical Journal, 2004

A 28-mer gM4 peptide, obtained by solid-state synthesis and corresponding to the fourth transmemb... more A 28-mer gM4 peptide, obtained by solid-state synthesis and corresponding to the fourth transmembrane segment of the nicotinic acetylcholine receptor g-subunit, possesses a single tryptophan residue (Trp 453), making it an excellent model for studying peptide-lipid interactions in membranes by fluorescence spectroscopy. The gM4 peptide was reconstituted with synthetic lipids (vesicles of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, i.e., POPC) rich and poor in cholesterol and analyzed using steady-state and time-resolved fluorescence techniques. The decrease in gM4 intrinsic fluorescence lifetime observed upon incorporation into a cholesterol-rich lo phase could be rationalized on the basis of a dynamic self-quenching owing to the formation of peptide-rich patches in the membrane. This agrees with the low Fö rster type resonance energy transfer efficiency from the Trp 453 residue to the fluorescent cholesterol analog, dehydroergosterol, in the lo phase. In the absence of cholesterol the gM4 nicotinic acetylcholine receptor peptide is randomly distributed in the POPC bilayer with its hydrophobic moiety matching the membrane thickness, whereas in the presence of cholesterol the increase in the membrane thickness and variation of the material properties favor the formation of peptide-enriched patches, i.e., interhelix interaction energy is essential for obtaining a stabilized structure. Thus, the presence of a cholesterol-rich, ordered POPC phase drives the organization of peptide-enriched patches, in which the gM4 peptide occupies ;30% of the patch area.

Biophysical Journal, 2015

Biochimica et Biophysica Acta (BBA) - Biomembranes, 2012

Characterization of phase coexistence in biologically relevant lipid mixtures is often carried ou... more Characterization of phase coexistence in biologically relevant lipid mixtures is often carried out through confocal microscopy of giant unilamellar lipid vesicles (GUVs), loaded with fluorescent membrane probes. This last analysis is generally limited to the vesicle hemisphere further away from the coverslip, in order to avoid artifacts induced by the interaction with the solid surface, and immobilization of vesicles is in many cases required in order to carry out intensity, lifetime or single-molecule based microscopy. This is generally achieved through the use of membrane tethers adhering to a coverslip surface. Here, we aimed to determine whether GUV immobilization through membrane tethers induces changes in lipid domain distribution within liposomes displaying coexistence of lipid lamellar phases. Confocal imaging and a Förster resonance energy transfer (FRET) methodology showed that biotinylated phospholipids present significantly different membrane phase partition behavior upon protein binding, depending on the presence or absence of a linker between the lipid headgroup and the biotinyl moiety. Membrane phases enriched in a membrane tether displayed in some cases a dramatically increased affinity for the immobilization surface, effectively driving sorting of lipid domains to the adherent membrane area, and in some cases complete sequestering of a lipid phase to the interaction surface was observed. On the light of these results, we conclude that tethering of lipid membranes to protein surfaces has the potential to drastically reorganize the distribution of lipid domains, and this reorganization is solely dictated by the partition properties of the protein-tether complex.

International Journal of Molecular Sciences

The allosteric coupling between activation and inactivation processes is a common feature observe... more The allosteric coupling between activation and inactivation processes is a common feature observed in K+ channels. Particularly, in the prokaryotic KcsA channel the K+ conduction process is controlled by the inner gate, which is activated by acidic pH, and by the selectivity filter (SF) or outer gate, which can adopt non-conductive or conductive states. In a previous study, a single tryptophan mutant channel (W67 KcsA) enabled us to investigate the SF dynamics using time-resolved homo-Förster Resonance Energy Transfer (homo-FRET) measurements. Here, the conformational changes of both gates were simultaneously monitored after labelling the G116C position with tetramethylrhodamine (TMR) within a W67 KcsA background. At a high degree of protein labeling, fluorescence anisotropy measurements showed that the pH-induced KcsA gating elicited a variation in the homo-FRET efficiency among the conjugated TMR dyes (TMR homo-FRET), while the conformation of the SF was simultaneously tracked (W6...

Journal of the Chemical Society, Faraday Transactions 2: Molecular and Chemical Physics, 1987

Journal of Biological Chemistry, 2000

The topography of nicotinic acetylcholine receptor (AChR) membrane-embedded domains and the relat... more The topography of nicotinic acetylcholine receptor (AChR) membrane-embedded domains and the relative affinity of lipids for these protein regions were studied using fluorescence methods. Intact Torpedo californica AChR protein and transmembrane peptides were derivatized with N-(1-pyrenyl)maleimide (PM), purified, and reconstituted into asolectin liposomes. Fluorescence mapped to proteolytic fragments consistent with PM labeling of cysteine residues in ␣M1, ␣M4, ␥M1, and ␥M4. The topography of the pyrene-labeled Cys residues with respect to the membrane and the apparent affinity for representative lipids were determined by differential fluorescence quenching with spin-labeled derivatives of fatty acids, phosphatidylcholine, and the steroids cholestane and androstane. Different spin label lipid analogs exhibit different selectivity for the whole AChR protein and its transmembrane domains. In all cases labeled residues were found to lie in a shallow position. For M4 segments, this is compatible with a linear ␣-helical structure, but not so for M1, for which "classical" models locate Cys residues at the center of the hydrophobic stretch. The transmembrane topography of M1 can be rationalized on the basis of the presence of a substantial amount of non-helical structure, and/or of kinks attributable to the occurrence of the evolutionarily conserved proline residues. The latter is a striking feature of M1 in the AChR and all members of the rapid ligand-gated ion channel superfamily.

Methods in Membrane Lipids

Cholesterol is a major component of mammalian cell membranes. It has remarkable effects on the pr... more Cholesterol is a major component of mammalian cell membranes. It has remarkable effects on the properties of phospholipids bilayers, and is implicated in the lipid raft model. Depending on the membrane composition, cholesterol-containing bilayers can exist either as single phase or as mixture of coexisting phases. These are organized in domains of variant size determined by composition, but can be smaller than the optical microscopy resolution limit. This chapter describes a methodology based on fluorescence resonance energy transfer, which is sensitive to phase separation and can provide estimates of domain size in these usually hard to characterize systems.

Photochemistry and Photobiology, 1988

ABSTRACT

Chemistry and Physics of Lipids, 2008

Biophysical Journal, 1995

The interaction between Nystatin and small unilamellar vesicles of 1,2-dipalmitoyl-sn-glycero-3-p... more The interaction between Nystatin and small unilamellar vesicles of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, both in gel (T = 210C) and in liquid-crystalline (T = 450C) phases, was studied by steady-state and time-resolved fluorescence measurements by taking advantage of the intrinsic tetraene fluorophore present in this antibiotic. It was shown that Nystatin aggregates in aqueous solution with a critical concentration of 3 puM. The enhancement in the fluorescence intensity of the antibiotic was applied to study the membrane binding of Nystatin, and it was shown that the antibiotic had an almost fivefold higher partition coefficient for the vesicles in a gel (P = (1.4 ± 0.1) x 103) than in a liquid-crystalline phase (P = (2.9 ± 0.1) x 102). Moreover, a time-resolved fluorescence study was used to examine Nystatin aggregation in the membrane. The emission decay kinetics of Nystatin was described by three and two exponentials in the lipid membrane at 210C and 450C, respectively. Nystatin mean fluorescence lifetime is concentration-dependent in gel phase lipids, increasing steeply from 11 to 33 ns at an antibiotic concentration of 5-6 ,uM, but the fluorescence decay parameters of Nystatin were unvarying with the antibiotic concentration in fluid lipids. These results provide evidence for the formation of strongly fluorescent antibiotic aggregates in gel-phase membrane, an interpretation that is at variance with a previous study. However, no antibiotic self-association was detected in a liquid-crystalline lipid bilayer within the antibiotic concentration range studied (0-14 ,uM).

Biophysical journal, Apr 30, 2004

A 28-mer γM4 peptide, obtained by solid-state synthesis and corresponding to the fourth transmemb... more A 28-mer γM4 peptide, obtained by solid-state synthesis and corresponding to the fourth transmembrane segment of the nicotinic acetylcholine receptor γ-subunit, possesses a single tryptophan residue (Trp453), making it an excellent model for studying peptide-lipid interactions in membranes by fluorescence spectroscopy. The γM4 peptide was reconstituted with synthetic lipids (vesicles of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, ie, POPC) rich and poor in cholesterol and analyzed using steady-state ...

Biomolecules

Despite its low abundance, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a key modulator o... more Despite its low abundance, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a key modulator of membrane-associated signaling events in eukaryotic cells. Temporal and spatial regulation of PI(4,5)P2 concentration can achieve localized increases in the levels of this lipid, which are crucial for the activation or recruitment of peripheral proteins to the plasma membrane. The recent observation of the dramatic impact of physiological divalent cation concentrations on PI(4,5)P2 clustering, suggests that protein anchoring to the plasma membrane through PI(4,5)P2 is likely not defined solely by a simple (monomeric PI(4,5)P2)/(protein bound PI(4,5)P2) equilibrium, but instead depends on complex protein interactions with PI(4,5)P2 clusters. The insertion of PI(4,5)P2-binding proteins within these clusters can putatively modulate protein–protein interactions in the membrane, but the relevance of such effects is largely unknown. In this work, we characterized the impact of Ca2+ on the org...

International Journal of Molecular Sciences, 2021

Alkylammonium salts have been used extensively to study the structure and function of potassium c... more Alkylammonium salts have been used extensively to study the structure and function of potassium channels. Here, we use the hydrophobic tetraoctylammonium (TOA+) to shed light on the structure of the inactivated state of KcsA, a tetrameric prokaryotic potassium channel that serves as a model to its homologous eukaryotic counterparts. By the combined use of a thermal denaturation assay and the analysis of homo-Förster resonance energy transfer in a mutant channel containing a single tryptophan (W67) per subunit, we found that TOA+ binds the channel cavity with high affinity, either with the inner gate open or closed. Moreover, TOA+ bound at the cavity allosterically shifts the equilibrium of the channel’s selectivity filter conformation from conductive to an inactivated-like form. The inactivated TOA+–KcsA complex exhibits a loss in the affinity towards permeant K+ at pH 7.0, when the channel is in its closed state, but maintains the two sets of K+ binding sites and the W67–W67 inters...

International Journal of Molecular Sciences, 2021

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is an essential plasma membrane component invol... more Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is an essential plasma membrane component involved in several cellular functions, including membrane trafficking and cytoskeleton organization. This function multiplicity is partially achieved through a dynamic spatiotemporal organization of PI(4,5)P2 within the membrane. Here, we use a Förster resonance energy transfer (FRET) approach to quantitatively assess the extent of PI(4,5)P2 confinement within the plasma membrane. This methodology relies on the rigorous evaluation of the dependence of absolute FRET efficiencies between pleckstrin homology domains (PHPLCδ) fused with fluorescent proteins and their average fluorescence intensity at the membrane. PI(4,5)P2 is found to be significantly compartmentalized at the plasma membrane of HeLa cells, and these clusters are not cholesterol-dependent, suggesting that membrane rafts are not involved in the formation of these nanodomains. On the other hand, upon inhibition of actin polymeriza...

Journal of Lipid Research, 2012

Supplementary key words ceramide • sphingolipids • acyl chain • lipid domains • tubules • membran... more Supplementary key words ceramide • sphingolipids • acyl chain • lipid domains • tubules • membrane order Sphingolipids (SLs) are important components of biological membranes and are involved in a variety of biological functions. SL synthesis starts at the endoplasmic reticulum

Journal of Biological Chemistry, 2010

Ceramide is an important lipid signaling molecule that plays critical roles in regulating cell be... more Ceramide is an important lipid signaling molecule that plays critical roles in regulating cell behavior. Ceramide synthesis is surprisingly complex and is orchestrated by six mammalian ceramide synthases, each of which produces ceramides with restricted acyl chain lengths. We have generated a CerS2 null mouse and characterized the changes in the long chain base and sphingolipid composition of livers from these mice. Ceramide and downstream sphingolipids were devoid of very long (C22-C24) acyl chains, consistent with the substrate specificity of CerS2 toward acyl-CoAs. Unexpectedly, C16-ceramide levels were elevated, and as a result, total ceramide levels were unaltered; however, C16-ceramide synthesis in vitro was not increased. Levels of sphinganine were also significantly elevated, by up to 50-fold, reminiscent of the effect of the ceramide synthase inhibitor, fumonisin B1. With the exceptions of glucosylceramide synthase and neutral sphingomyelinase 2, none of the other enzymes tested in either the sphingolipid biosynthetic or degradative pathways were significantly changed. Total glycerophospholipid and cholesterol levels were unaltered, although there was a marked elevation in C18:1 and C18:2 fatty acids in phosphatidylethanolamine, concomitant with a reduction in C18:0 and C20:4 fatty acids. Finally, differences were observed in the biophysical properties of lipid extracts isolated from liver microsomes, with membranes from CerS2 null mice displaying higher membrane fluidity and showing morphological changes. Together, these results demonstrate novel modes of cross-talk and regulation between the various branches of lipid metabolic pathways upon inhibition of very long acyl chain ceramide synthesis.

Biopolymers, 1997

The structure of a new supramolecular drug carrier (named Biovectors-BV) was studied using light ... more The structure of a new supramolecular drug carrier (named Biovectors-BV) was studied using light scattering and scanning electronic microscopy techniques. This system consists of a polysaccharide core of chemically cross-linked maltodextrins to which phospholipids (and, in some cases, cholesterol) are added. Both polysaccharide cores and BV crosslinked with phosphate (negatively charged) and epichlorhydrin (no net charge) are spherical particles. The increase in the ionic strength of the medium increases the density of the charged polysaccharide cores. The lipid strongly interacts with neutral and negatively charged cores, decreasing both intra-and interparticle interactions. The results (mainly, r Å R g /R h õ 0.775 in some cases) suggest that BV are gel-like particles of variable density, referred to as microgels or soft spheres. Neutral polysaccharides have a strong tendency to self-aggregate. This selfaggregation of polysaccharide neutral cores is prevented by the addition of lipid or dimethylsulfoxide.

Biophysical Journal, 2012

Biophysical Journal, 2004

A 28-mer gM4 peptide, obtained by solid-state synthesis and corresponding to the fourth transmemb... more A 28-mer gM4 peptide, obtained by solid-state synthesis and corresponding to the fourth transmembrane segment of the nicotinic acetylcholine receptor g-subunit, possesses a single tryptophan residue (Trp 453), making it an excellent model for studying peptide-lipid interactions in membranes by fluorescence spectroscopy. The gM4 peptide was reconstituted with synthetic lipids (vesicles of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, i.e., POPC) rich and poor in cholesterol and analyzed using steady-state and time-resolved fluorescence techniques. The decrease in gM4 intrinsic fluorescence lifetime observed upon incorporation into a cholesterol-rich lo phase could be rationalized on the basis of a dynamic self-quenching owing to the formation of peptide-rich patches in the membrane. This agrees with the low Fö rster type resonance energy transfer efficiency from the Trp 453 residue to the fluorescent cholesterol analog, dehydroergosterol, in the lo phase. In the absence of cholesterol the gM4 nicotinic acetylcholine receptor peptide is randomly distributed in the POPC bilayer with its hydrophobic moiety matching the membrane thickness, whereas in the presence of cholesterol the increase in the membrane thickness and variation of the material properties favor the formation of peptide-enriched patches, i.e., interhelix interaction energy is essential for obtaining a stabilized structure. Thus, the presence of a cholesterol-rich, ordered POPC phase drives the organization of peptide-enriched patches, in which the gM4 peptide occupies ;30% of the patch area.

Biophysical Journal, 2015

Biochimica et Biophysica Acta (BBA) - Biomembranes, 2012

Characterization of phase coexistence in biologically relevant lipid mixtures is often carried ou... more Characterization of phase coexistence in biologically relevant lipid mixtures is often carried out through confocal microscopy of giant unilamellar lipid vesicles (GUVs), loaded with fluorescent membrane probes. This last analysis is generally limited to the vesicle hemisphere further away from the coverslip, in order to avoid artifacts induced by the interaction with the solid surface, and immobilization of vesicles is in many cases required in order to carry out intensity, lifetime or single-molecule based microscopy. This is generally achieved through the use of membrane tethers adhering to a coverslip surface. Here, we aimed to determine whether GUV immobilization through membrane tethers induces changes in lipid domain distribution within liposomes displaying coexistence of lipid lamellar phases. Confocal imaging and a Förster resonance energy transfer (FRET) methodology showed that biotinylated phospholipids present significantly different membrane phase partition behavior upon protein binding, depending on the presence or absence of a linker between the lipid headgroup and the biotinyl moiety. Membrane phases enriched in a membrane tether displayed in some cases a dramatically increased affinity for the immobilization surface, effectively driving sorting of lipid domains to the adherent membrane area, and in some cases complete sequestering of a lipid phase to the interaction surface was observed. On the light of these results, we conclude that tethering of lipid membranes to protein surfaces has the potential to drastically reorganize the distribution of lipid domains, and this reorganization is solely dictated by the partition properties of the protein-tether complex.

International Journal of Molecular Sciences

The allosteric coupling between activation and inactivation processes is a common feature observe... more The allosteric coupling between activation and inactivation processes is a common feature observed in K+ channels. Particularly, in the prokaryotic KcsA channel the K+ conduction process is controlled by the inner gate, which is activated by acidic pH, and by the selectivity filter (SF) or outer gate, which can adopt non-conductive or conductive states. In a previous study, a single tryptophan mutant channel (W67 KcsA) enabled us to investigate the SF dynamics using time-resolved homo-Förster Resonance Energy Transfer (homo-FRET) measurements. Here, the conformational changes of both gates were simultaneously monitored after labelling the G116C position with tetramethylrhodamine (TMR) within a W67 KcsA background. At a high degree of protein labeling, fluorescence anisotropy measurements showed that the pH-induced KcsA gating elicited a variation in the homo-FRET efficiency among the conjugated TMR dyes (TMR homo-FRET), while the conformation of the SF was simultaneously tracked (W6...