Biomedical subjects

Oscar Domènech

Publications and source records attributed to Oscar Domènech.

8 recordsLinked to original sources

PubMed2006

Supported planar bilayers from hexagonal phases.

In this work the presence of inverted hexagonal phases H(II) of 1-palmitoy-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and cardiolipin (CL) (0.8:0.2, mol/mol) in the presence of Ca(2+) were observed via (31)P-NMR spectroscopy. When suspensions of the same composition were extended onto mica, H(II) phases transformed into structures which features are those of supported planar bilayers (SPBs). When characterized by atomic force microscopy (AFM), the SPBs revealed the existence of two laterally segregated domains (the interdomain height being approximately 1 nm). Cytochrome c (cyt c), which binds preferentially to acidic phospholipids like CL, was used to demonstrate the nature of the domains. We used 1-anilinonaphtalen-8-sulfonate (ANS) to demonstrate that in the presence of cyt c, the fluorescence of ANS decreased significantly in lamellar phases. Conversely, the ANS binding to H(II) phases was negligible. When cyt c was injected into AFM fluid imaging cells, where SPBs of POPE:CL had previously formed poorly defined structures, protein aggregates ( approximately 100 nm diameter) were ostensibly observed only on the upper domains, which suggests not only that they are mainly formed by CL, but also provides evidence of bilayer formation from H(II) phases. Furthermore, a model for the nanostructure of the SPBs is herein proposed.

Aluminum Silicates↗

PubMed2006

Thermodynamic and structural study of the main phospholipid components comprising the mitochondrial inner membrane.

Cardiolipin (CL) is a phospholipid found in the energy-transducing membranes of bacteria and mitochondria and it is thought to be involved in relevant biological processes as apoptosis. In this work, the mixing properties of CL and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocoline (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) at the air-water interface, have been examined using the thermodynamic framework analysis of compression isotherms. Accordingly, the values of the Gibbs energy of mixing, the more stable monolayers assayed were: POPC:CL (0.6:0.4, mol:mol) and POPE:CL (0.8:0.2, mol:mol). The results reflect that attractive forces are the greatest contributors to the total interaction in these compositions. Supported planar bilayers (SPBs) with such compositions were examined using atomic force microscopy (AFM) at different temperatures. With the POPC:CL mixture, rounded and featureless SPBs were obtained at 4 degrees C and 24 degrees C. In contrast, the extension of the POPE:CL mixture revealed the existence of different lipid domains at 24 degrees C and 37 degrees C. Three lipid domains coexisted which can be distinguished by measuring the step height difference between the uncovered mica and the bilayer. While the low and intermediate domains were temperature dependent, the high domain was composition dependent. When cytochrome c (cyt c) was injected into the fluid cell, the protein showed a preferential adsorption onto the high domain of the POPC:CL. These results suggest that the high domain is mainly formed by CL.

Cardiolipins↗

PubMed2005

Preliminary atomic force microscopy study of two-dimensional crystals of lactose permease from Escherichia coli.

Lactose permease (LacY) of Escherichia coli is not only a paradigm for secondary transporters but also for difficulties in two-dimensional (2D) crystallization. In this work we present the progresses achieved in the observation of 2D crystals of wild-type LacY by atomic force microscopy (AFM). Crystals were obtained following reconstitution of LacY in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes. Proteolipid sheets (PLSs) 6.4 nm in height were obtained after spreading the samples onto mica. Observations were carried out in liquid medium and in contact mode (CM-AFM). When the crystalline surfaces of the PLSs were imaged regular packing arrangements were observed. The back-Fourier transformation revealed the existence of various orientations mostly consistent with crystals possessing p2 symmetry and unit-cell dimensions: a=13.15 nm, b=16.74 nm, gamma=116 degrees. The characteristics, size, and shape of the repetitive motif could be compatible with dimers of this protein. These preliminary results are compared and discussed with previously reported 2D crystals observed by electron microscopy.

Crystallization↗

PubMed2005

Effects of lactose permease on the phospholipid environment in which it is reconstituted: a fluorescence and atomic force microscopy study.

The membrane transport protein lactose permease (LacY), a member of the major facilitator superfamily containing 12 membrane-spanning segments connected by hydrophilic loops, was reconstituted in liposomes whose composition was 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol in a 3:1 molar ratio. The structural order of the lipid membranes, in the presence and absence of LacY, was assessed using steady-state fluorescence anisotropy. The features of the anisotropy curves obtained with 1,6-phenyl-1,3,5-hexatriene and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate suggest a surface effect of LacY on the membranes. Atomic force microscopy imaging of supported planar bilayers (SPBs) deposited onto mica was used to examine the effect of LacY on the nanostructure of the phospholipid matrix. Two separated domains were observed in SPBs formed from pure phospholipid mixture. Protein assemblies segregated from the rest of the matrix were observed after the extension of proteoliposomes. The effect of the protein on the electrostatic surface potential of the bilayer was also examined using a fluorescent pH indicator, 4-heptadecyl-7-hydroxycoumarin. Changes in surface potential were enhanced in the presence of the substrate (i.e., lactose). Taken together the results indicate that LacY is segregated into the phospholipid matrix and has moderate effects on the acyl chain order of the bilayers. The changes in surface electrical properties of the bilayers suggest a role for the phospholipid headgroups in proton transfer to the amino acids involved in substrate translocation.

Amino Acid Sequence↗

PubMed2005

Surface thermodynamics study of monolayers formed with heteroacid phospholipids of biological interest.

The interaction of 1-palmitoy-2-oleoyl-sn-glycero-3-phosphocoline (POPC) and 1-palmitoy-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), two of the major components in biological membranes, were investigated using the monolayer technique at the air-water interface. The pressure-area isotherms indicate that both phospholipids are miscible through all range of compositions. POPE-POPC form stable mixtures, with a minimum for the Gibbs energy of mixing at X(POPC) = 0.4. A virial equation of state was fitted to the experimental values. Positive values found for the second virial coefficient indicate repulsion between POPC and POPE. The interaction parameter was evaluated which indicated that a corresponding decrease in the repulsion occurs when POPC molar fraction is low. This effect suggests the existence of hydrogen bonds between POPE and the water beneath the interface.

Air↗

PubMed2005

Atomic force microscopy study of Escherichia coli lactose permease proteolipid sheets.

Proteolipid sheets (PLSs) obtained using the vesicle fusion technique on a convenient surface are the base to obtain transmembrane protein biosensors. In this preliminary work, we have screened several physicochemical conditions to optimize the visualization of proteolipid sheets formed between different phospholipid matrices and the membrane protein lactose permease (LacP) by atomic force microscopy (AFM). When LacP was reconstituted in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes, the proteolipid sheets were densely packed with an upper layer that protruded from a background layer. Several lipid protein molar ratios (LPR) were screened. High resolution analysis of the upper layer revealed a quasi-crystalline arrangement formed by small entities that could be attributed to the protein. The approach described here may be suitable for the rational design of biosensors based in other transmembrane proteins.

Adsorption↗

PubMed2005

Surface thermodynamic properties of monolayers versus reconstitution of a membrane protein in solid-supported bilayers.

Atomic force microscopy (AFM) was used to study the influence of a membrane protein, lactose permease of Escherichia coli (LacY), on the surface spreading behavior and the features of self-assembled phospholipids bilayers on mica. The miscibility of phospholipids used, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), was investigated by surface pressure area isotherm measurements at the air-water interface. A composition with an equimolar proportion of POPC and DMPC was used to form the liposomes. Surface layers formed with DMPC:POPC (0.5:0.5, mol/mol) or LacY reconstituted in proteoliposomes with the same phospholipid composition were imaged by using AFM. When lactose permease was reconstituted in DMPC:POPC (0.5:0.5, mol/mol), self-assembled structures that remained firmly adsorbed onto the mica surface were observed. These sheets had an irregular shape and their upper layer was more corrugated than that obtained for the phospholipid matrix.

Adsorption↗

PubMed2004

Preliminary studies of the 2D crystallization of Omp1 of Serratia marcescens: observation by atomic force microscopy in native membranes environment and reconstituted in proteolipid sheets.

In this work the porin Omp1 of Serratia marcescens was expressed in a porin deficient mutant (Escherichia coli UH302) and its functionality studied following the accumulation of ciprofloxacin in bacteria. The protein was extracted, purified and reconstituted in proteoliposomes of different composition (lipopolysaccharide (LPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)). Maximum extraction of the detergent was achieved applying different steps of dialysis and centrifugation. Proteolipid sheets with different composition were spread onto mica and observed by atomic force microscopy. Two-dimensional crystal of Omp1 was not observed in any case due to low resolution achieved. Judging from the images features POPC is the most suitable phospholipid to enhance 2D lattice formation for Omp1.

Anti-Infective Agents↗