Biomedical subjects

Manuel Prieto

Publications and source records attributed to Manuel Prieto.

25 records · Page 2Linked to original sources

PubMed2003

Dependence of M13 major coat protein oligomerization and lateral segregation on bilayer composition.

M13 major coat protein was derivatized with BODIPY (n-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-yl)methyl iodoacetamide), and its aggregation was studied in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and DOPC/1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DOPG) or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)/DOPG (model systems of membranes with hydrophobic thickness matching that of the protein) using photophysical methodologies (time-resolved and steady-state self-quenching, absorption, and emission spectra). It was concluded that the protein is essentially monomeric, even in the absence of anionic phospholipids. The protein was also incorporated in pure bilayers of lipids with a strong mismatch with the protein transmembrane length, 1,2-dierucoyl-sn-glycero-3-phosphocholine (DEuPC, longer lipid) and 1,2-dimyristoleoyl-sn-glycero-3-phosphocholine (DMoPC, shorter lipid), and in lipidic mixtures containing DOPC and one of these lipids. The protein was aggregated in the pure vesicles of mismatching lipid but remained essentially monomeric in the mixtures as detected from BODIPY fluorescence emission self-quenching. From fluorescence resonance energy transfer (FRET) measurements (donor-n-(iodoacetyl)aminoethyl-1-sulfonaphthylamine (IAEDANS)-labeled protein; acceptor-BODIPY labeled protein), it was concluded that in the DEuPC/DOPC and DMoPC/DOPC lipid mixtures, domains enriched in the protein and the matching lipid (DOPC) are formed.

Boron Compounds↗

PubMed2003

Characterization of DNA/lipid complexes by fluorescence resonance energy transfer.

Fluorescence resonance energy transfer (FRET) is a potential method for the characterization of DNA-cationic lipid complexes (lipoplexes). In this work, we used FRET models assuming a multilamellar lipoplex arrangement. The application of these models allows the determination of the distance between the fluorescent intercalator on the DNA and a membrane dye on the lipid, and/or the evaluation of encapsulation efficiencies of this liposomal vehicle. The experiments were carried out in 1,2-dioleoyl-3-trimethylammonium-propane/pUC19 complexes with different charge ratios. We used 2-(3-(diphenylhexatrienyl)propanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (DPH-PC) and 2-(4,4-difluoro-5-octyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (BODIPY-PC) as membrane dyes, and ethidium bromide (EtBr) and BOBO-1 as DNA intercalators. In cationic complexes (charge ratios (+/-) >or= 2), we verified that BOBO-1 remains bound to DNA, and FRET occurs to the membrane dye. This was also confirmed by anisotropy and lifetime measurements. In complexes with all DNA bound to the lipid (charge ratio (+/-) = 4), we determined 27 A as the distance between the donor and acceptor planes (half the repeat distance for a multilamellar arrangement). In complexes with DNA unbound to the lipids (charge ratio (+/-) = 0.5 and 2), we calculated the encapsulation efficiencies. The presented FRET methodology is, to our knowledge, the first procedure allowing quantification of lipid-DNA contact.

Binding Sites↗

PubMed2003

Interaction of peptides with binary phospholipid membranes: application of fluorescence methodologies.

The application of fluorescence methodologies to obtain information about the extent, dynamics and topology of peptide interaction with binary phospholipid (mainly zwitterionic/anionic) mixtures is reviewed. First, general approaches based on peptide (tryptophan residues) fluorescence properties that give information about its partition, location and dynamics will be presented. Then, methodologies based on membrane probes fluorescence that report the influence of peptide binding and/or incorporation on the lateral organization (phase separation) of membrane phospholipids will be described. Specific examples taken from the literature that illustrate both situations are presented as well as formalisms for data analysis. It is shown that steady-state and time-resolved fluorescence data (particularly important in the case of fluorescence resonance energy transfer studies) give complementary information, allowing a molecular picture of peptide interaction with biphasic systems to be drawn.

Amino Acid Sequence↗

PubMed2002

Nonequilibrium phenomena in the phase separation of a two-component lipid bilayer.

Lipid bilayers composed of two phospholipids with significant acyl-chain mismatch behave as nonideal mixtures. Although many of these systems are well characterized from the equilibrium point of view, studies concerning their nonequilibrium dynamics are still rare. The kinetics of lipid demixing (phase separation) was studied in model membranes (large unilamellar vesicles of 1:1 dilauroylphosphatidylcholine (C(12) acyl chain) and distearoylphosphatidylcholine (C(18) acyl chain)). For this purpose, photophysical techniques (fluorescence intensity, anisotropy, and fluorescence resonance energy transfer) were applied using suitable probes (gel phase probe trans-parinaric acid and fluid phase probe N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-dilauroylphosphatidylethanolamine). The nonequilibrium situation was induced by a sudden thermal quench from a one-fluid phase equilibrium situation (higher temperature) to the gel/fluid coexistence range (lower temperature). We verified that the attainment of equilibrium is a very slow process (occurs in a time scale of hours), leading to large domains at infinite time. The nonequilibrium structure stabilization is due essentially to temporarily rigidified C(12) chains in the interface between gel/fluid domains, which decrease the interfacial tension by acting as surfactants. The relaxation process becomes faster with the increase of the temperature drop. In addition, heterogeneity is already present in the supposed homogeneous fluid mixture at the higher temperature.

Anisotropy↗

PubMedDate not supplied

Ceramide-platform formation and -induced biophysical changes in a fluid phospholipid membrane.

To understand the formation and properties of ceramide-enriched domains in cell membranes, the biophysical properties of the binary system palmitoyloleoylphosphatidylcholine (POPC)/palmitoylceramide were thoroughly characterized. Diverse fluorescent probes and parameters were necessary to unravel the complexity of this apparently simple system. For the first time, a complete phase diagram is reported, characterizing the lamellar phases of these mixtures, and providing a quantitative framework integrating biophysical and biological studies. The diagram suggests that in resting cells no ceramide domains exist, but upon apoptotic stimuli, platforms may form. Moreover, our data show that 2 mol% of Cer strongly affects the POPC fluid matrix, suggesting that a small increase in Cer levels can significantly affect cell membrane properties. In this work, we also show that Cer domains, formed in conditions similar to physiological, are extremely ordered and rigid. The domains composition is estimated from the phase diagram and their large size was concluded from fluorescence resonance energy transfer. Dynamic light scattering and electron microscopy were used to characterize the system morphology, which is highly dependent on ceramide content and includes vesiculation and tubular structure formation.

Anisotropy↗

PubMedDate not supplied

Structure and dynamics of the gammaM4 transmembrane domain of the acetylcholine receptor in lipid bilayers: insights into receptor assembly and function.

A 28-mer peptide (gammaM4) corresponding to the fourth transmembrane segment of the nicotinic acetylcholine receptor (AChR) gamma-subunit, with a single tryptophan residue (Trp6), was reconstituted into lipid bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), loaded with either high or low amounts of cholesterol, i.e., in the conjugated liquid-ordered and liquid-disordered phases, respectively, at room temperature. By making use of the Trp intrinsic fluorescence, both steady-state and time-resolved fluorescence techniques were employed, namely, red-edge excitation shift effect, decay-associated spectra (DAS), and time-resolved anisotropy. The results obtained here, together with previous studies on the same reconstituted peptide, indicate that: (i) Trp6 is strongly anchored in the bilayer with a defined transverse location; (ii) the modifications in the measured DAS are related to the complex result of a self-quenching process on the decay parameters; (iii) the wobbling movement of the indole moiety of Trp6 is fast but severely restricted in amplitude; and, (iv) in the liquid-ordered phase, the bilayer properties and the tilt angle of the peptide enhance peptide-peptide interactions, with the formation of peptide rich patches and possibly some anti-parallel helix-helix aggregates, showing different dynamics from that of the peptide in the liquid-disordered phase where the peptide is randomly distributed.

Animals↗

PubMedDate not supplied

Photophysical behavior of a dimeric cyanine dye (BOBO-1) within cationic liposomes.

This study is aimed at establishing optimal conditions for the use of 2,2'-[1,3-propanediylbis[(dimethyliminio)-3,1-propanediyl-1(4H)-pyridinyl-4-ylidenemethy-lidyne]]bis[3-methyl]-tetraiodide (BOBO-1) as a fluorescent probe in the characterization of lipid/DNA complexes (lipoplexes). The fluorescence spectra, anisotropy, fluorescence lifetimes and fluorescence quantum yields of this dimeric cyanine dye in plasmid DNA (2694 base pairs) with and without cationic liposomes (1,2-dioleoyl-3-trimethylammonium-propane [DOTAP]), are reported. The photophysical behavior of the dye in the absence of lipid was studied for several dye/DNA ratios using both supercoiled and relaxed plasmid. At dye/DNA ratios (d/b) below 0.01 the fluorescence intensity increases linearly, whereas lifetime and anisotropy values of the dye are constant (tau approximately 2.5 ns and = 0.20). By agarose gel electrophoresis it was verified that up to d/b = 0.01 DNA conformation is not considerably modified, whereas for d/b = 0.05-0.06 a single heavy band appears on the gel. For these and higher dye/DNA ratios the fluorescence intensity, anisotropy and average lifetime values decrease with an increase in BOBO-1 concentration. When cationic liposomes are added to the BOBO-1/DNA complex, an additional effect is noticed: The difference in the environment probed by BOBO-1 bound to DNA leads to a decrease in quantum yield and average lifetime values, and a redshift is apparent in the emission spectrum. For fluorescence measurements including energy transfer (FRET), a d/b ratio of 0.01 seems to be adequate because no considerable change on DNA conformation is detected, a considerable fluorescent signal is still measured after lipoplex formation, and energy migration is not efficient.

Benzothiazoles↗