Spontaneous vesicles, disks, threadlike and spherical micelles found in the solubilization of DMPC liposomes by the detergent DTAC.

The spontaneous colloidal nanostructures formed in water by the zwitterionic phospholipid DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) with the cationic detergent DTAC (n-dodecyltrimethylammonium chloride) were investigated at a fixed DMPC concentration and variable detergent:lipid total molar ratios (D:L). Apparent (neutral-sphere-equivalent) hydrodynamic diameters (Φ(e)) of liposomes and micelles were obtained by dynamic light scattering (DLS). Fluorescence lifetime imaging microscopy (FLIM), using chlorophyll-a as a probe, showed the morphology of giant vesicles and threadlike micelles. Micro-differential scanning calorimetry (micro-DSC) detected the presence of bilayers, in vesicles and discoidal micelles (disks). Pure DMPC liposomes are multilamellar and polydisperse (Φ(e)≈100-10,000 nm). As D:L increased, smaller vesicles were found, due to the bigger spontaneous curvature of the bilayer: at D:L=1, ULVs (unilamellar vesicles; Φ(e)≈100 nm) appeared and, at D:L=2-10, ULVs coexisted with disks (Φ(e)≈30 nm). Bilayers totally disappeared at D:L≥15, giving rise to spheroidal (Φ(e)≈2-16 nm) and threadlike (Φ(e)≈100-10,000 nm) micelles. A quasi-equilibrium structural diagram for the DMPC-DTAC-water system shows equivalent diameters of the scattering nanoparticles as a function of D:L. The results obtained herein for the system DMPC-DTAC show the role of electrostatic interactions in the formation of the mixed structures.

Time evolution of the thermotropic behavior of spontaneous liposomes and disks of the DMPC-DTAC aqueous system.

In this work, solubilization of the phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) by the cationic detergent n-dodecyltrimethylammonium chloride (DTAC) was studied in aqueous solution, at a fixed DMPC concentration and variable detergent:lipid (D:L) molar ratios. The colloidal nanostructures present in different stages of the solubilization process were characterized using micro-differential scanning calorimetry (DSC) and dynamic light scattering (DLS) techniques. For total (analytical) D:L molar ratios below approximately 1, DTAC monomers incorporate into the DMPC liposome bilayers, forming smaller and more fluid vesicles than pure DMPC liposomes. At D:L approximately 1-2, vesicles begin to rupture, coexisting with intact vesicles and bilayer fragments. At D:L approximately 2-12.5, discoidal and spherical micelles are formed and coexist with vesicles; a slow structural rearrangement of the system, monitored in successive DSC heating/cooling cycles, was observed, and is reported for the first time. Finally, for D:L above approximately 15-20, the bilayers are completely dissolved, and the main aggregates in solution become spherical micelles, which slowly evolve to cylindrical (threadlike) micelles. Based on the dependence of the temperature and enthalpy of transitions on the total D:L molar ratio, at constant DMPC concentration, a schematic model, showing the different colloidal nanostructures present in the solubilization process, is proposed.

Unfolding kinetics of beta-lactoglobulin induced by surfactant and denaturant: a stopped-flow/fluorescence study.

The beta-->alpha transition of beta-lactoglobulin, a globular protein abundant in the milk of several mammals, is investigated in this work. This transition, induced by the cationic surfactant dodecyltrimethylammonium chloride (DTAC), is accompanied by partial unfolding of the protein. In this work, unfolding of bovine beta-lactoglobulin in DTAC is compared with its unfolding induced by the chemical denaturant guanidine hydrochloride (GnHCl). The final protein states attained in the two media have quite different secondary structure: in DTAC the alpha-helical content increases, leading to the so-called alpha-state; in GnHCl the amount of ordered secondary-structure decreases, resulting in a random coil-rich final state (denatured, or D, state). To obtain information on both mechanistic routes, in DTAC and GnHCl, and to characterize intermediates, the kinetics of unfolding were investigated in the two media. Equilibrium and kinetic data show the partial accumulation of an on-pathway intermediate in each unfolding route: in DTAC, an intermediate (I(1)) with mostly native secondary structure but loose tertiary structure appears between the native (beta) and alpha-states; in GnHCl, another intermediate (I(2)) appears between states beta and D. Kinetic rate constants follow a linear Chevron-plot representation in GnHCl, but show a more complex mechanism in DTAC, which acts like a stronger binding species.

Conformational transitions in beta-lactoglobulin induced by cationic amphiphiles: equilibrium studies.

The conformational transition from the native state in water ("beta-state") to a state containing a considerable amount of alpha-helices ("alpha-state") was studied for the protein beta-lactoglobulin (BLG), from bovine milk, in several colloidal solutions containing mixed micelles or spontaneous vesicles. These aggregates were formed in the bicationic system containing the surfactant dodecyltrimethylammonium chloride (DTAC) and the lipid didodecyldimethylammonium bromide (DDAB). The beta-->alpha transition in BLG, investigated by far-ultraviolet circular dichroism spectroscopy, is induced to the same protein alpha-state by pure and mixed DDAB/DTAC micelles or vesicles. This implies a similar interaction mechanism of BLG with DDAB or DTAC, once the colloidal aggregates are formed. In premicelle DTAC solutions, the fraction of alpha-helix is lower and increases with the DTAC concentration. DDAB and DTAC also promote conformational changes in the protein tertiary structure that expose the tryptophans to a less constrained environment. These unfolding transitions were investigated by near-ultraviolet circular dichroism and steady-state fluorescence spectroscopies. In equilibrium conditions, it was found that higher DTAC (and, probably, DDAB) concentrations are needed to induce the beta-->alpha transition than to unfold the protein. beta-Lactoglobulin may therefore be considered as a model for protein-surfactant and protein-lipid interactions.