Properties of mixed DOTAP-DPPC bilayer membranes as reported by differential scanning calorimetry and dynamic light scattering measurements.

We have investigated the effect of a cationic lipid [DOTAP] on both the thermotropic phase behavior and the structural organization of aqueous dispersions of dipalmitoyl-phosphatidylcholine [DPPC] by means of high-sensitivity differential scanning calorimetry and dynamic light scattering measurements. We find that the incorporation of increasing quantities of DOTAP progressively reduces the temperature and the enthalpy of the gel-to-liquid crystalline transition. We are further showing that, in mixed DOTAP-DPPC systems, the reduction of the phase transition temperature is accompanied by a reduction of the average size of the structures present in the aqueous mixtures, whatever the DOTAP concentration is. These results, which extend a previous investigation by Campbell et al. (Campbell, R. B.; Balasubramanian, S. V.; Straubinger, R. M.; Biochim. Biosphys. Acta 2001, 27, 1512.) limited to a DOTAP concentration below 20 mol %, confirm that the insertion of cationic head groups in zwitterionic phosphatidylcholine bilayers facilitates the formation of stable, relatively small, unilamellar vesicles. This self-assembling restructuring from an aqueous multilamellar structure toward a liposomal phase is favored by decreasing the phospholipid phase transition temperature and by increasing the temperature of the system. This reduction of the average size and the appearance of a stable liposomal phase is also promoted by a heating and cooling thermal treatment.

Met-myoglobin association in dilute solution during pressure-induced denaturation: an analysis at pH 4.5 by high-pressure small-angle X-ray scattering.

In this paper, we report on the original global fit procedure of synchrotron small-angle X-ray scattering (SAXS) data applied to a model protein, met-myoglobin, in dilute solution during temperature- and pressure-induced denaturation processes at pH 4.5. Starting from the thermodynamic description of the protein unfolding pathway developed by Hawley (Hawley, S. A. Biochemistry 1971, 10, 2436), we have developed a new method for analyzing the set of SAXS curves using a global fitting procedure, which allows us to derive the form factor of all the met-myoglobin species present in the solution, their aggregation state, and the set of thermodynamic parameters, with their p and T dependence. This method also overcomes a reasonably poor quality of the experimental data, and it is found to be very powerful in analyzing SAXS data. SAXS experiments were performed at four different temperatures from hydrostatic pressures up to about 2000 bar. As a result, the presence of an intermediate, partially unfolded, dimeric state of met-myoglobin that forms during denaturation has been evidenced. The obtained parameters were then used to derive the met-myoglobin p, T phase diagram that fully agrees with the corresponding phase diagram obtained by spectroscopic measurements.

Liposome-induced DNA compaction and reentrant condensation investigated by dielectric relaxation spectroscopy and dynamic light scattering techniques.

Interaction of DNA with oppositely charged objects, such as multivalent ions, cationic surfactants, cationic liposomes, basic proteins, and alcohols, up to nano- or mesoscopic particles, gives rise to a very interesting and fascinating phenomenology, where the shape, size, and stability of the resulting aggregates depend on a delicate balance between different driving forces, mainly of electrostatic origin. We have studied the cationic liposome-DNA complexes during the whole complexation process, below, close to, and above the isoelectric condition, where the number of cationic lipids equals the number of phosphate groups on the DNA chain. We took advantage of the combined use of dynamic light scattering, laser Doppler electrophoretic mobility, and radio-wave dielectric relaxation measurements in order to characterize both the structural parameters (hydrodynamic radius) and the electrical parameters (charge and counterion concentration) of the resulting structures. These structures are fundamentally of two types, clusters of liposomes stuck together by DNA chains (cluster phase in low-density colloidal suspension) and coexisting DNA coils and DNA globules, according to the procedure through which interactions occur (liposomes in excess DNA solution or DNA in excess liposome suspension).

Calorimetric and dynamic light-scattering investigation of cationic surfactant--DNA complexes.

By means of combined calorimetric and dynamic light-scattering measurements, we have investigated the conformational behavior of DNA chains after thermal melting in the presence of a cationic surfactant at different concentrations, up to a surfactant-to-phosphate group molar ratio close to unity. Both the specific heat capacity, C(ex)(p) and the hydrodynamic radius R of the DNA chains provide support for the existence of two structural arrangements with different thermal stabilities, coexisting in the bulk solution. Although a component remains an elongated unfolded DNA chain originated in the thermal denaturation, the second component, consisting of DNA-surfactant complexes, assumes a compact structure with an average size of about 80 nm, whose thermal denaturation occurs at temperatures higher than 100 degrees C.

Temperature-dependent dynamics of water confined in nafion membranes.

We performed a neutron scattering study to investigate the dynamical behavior of water absorbed in Nafion at low hydration level as a function of temperature in the range 200-300 K. To single out the spectral contribution of the confined water, the measurements were done on samples hydrated with both H(2)O and D(2)O. Due to the strong incoherent scattering cross section of hydrogen atoms with respect to deuterium, in the difference spectra, the contribution from the Nafion membrane is subtracted out and the signal originates essentially from protons in the liquid phase. The main quantities we extracted as a function of the momentum transfer are the elastic incoherent structure factor (EISF) and the line width of the quasielastic component. Their trend suggests that the motion of hydrogen atoms can be schematized as a random jumping inside a confining region, which can be related to the boundaries of the space where water molecules move in the cluster they form around the sulfonic acid site. Through the calculated EISF, we obtained information on the size of such a region, which increases up to 260 K and then attains a constant value. Above this temperature, the number of water protons that are dynamically activated in the accessible time window increases with a faster rate. The jump diffusion dynamics is characterized by a typical jumping time which is stable at 5.3 ps up to approximately 260 K and then gradually decreases. The ensemble of the findings indicates that, within the limits of the energy resolution of the present experiment, water absorbed in the Nafion membrane undergoes a dynamical transition at around 260 K. We discuss the possible relationship of this dynamical onset with the behavior of the electrical conductivity of the membrane as a function of the temperature.

The role of water coordination in binary mixtures. A study of two model amphiphilic molecules in aqueous solutions by molecular dynamics and NMR.

Two binary aqueous mixtures which contain the small amphiphilic molecules TMAO (trimethylamine-N-oxide) and TBA (tert-butyl alcohol) have been investigated by molecular dynamics simulations and NMR chemical shift and self-diffusion measurements. TMAO is an osmolyte, while TBA is a monohydrate alcohol. Both possess bulky hydrophobic groups and polar heads, namely, NO in TMAO and OH in TBA. The hydrophilic/hydrophobic content of these isosteric molecules strongly modulates the structure and dynamics of the hydration shell, which is thought to be responsible for the effects observed on proteins and phospholipids. Simulation results, especially on hydrogen-bond networking, spatial correlations, and self-diffusivity, are consistent with NMR data and agree well with previous numerical studies on similar solutions. The methods employed allow the elucidation of the microscopic features of the solutions. For TBA solutions, the hydration shell is found to have a low density and a large spatial spread, and thus, above the molar fraction of 0.03, reduction of hydrophobic hydration drives self-aggregation of the solute. This effect does not take place in TMAO solutions, where the hydration shell is more compact and stable, maintaining its structure over a wider range of solute concentrations.

Dielectric behavior of DNA in water-organic co-solvent mixtures.

The radiowave dielectric dispersions of DNA in different water-organic co-solvent mixtures have been measured in the frequency range from 100 kHz to 100 MHz, where the polarization mechanism is generally attributed to the confinement of counterions within some specific lengths, either along tangential or perpendicular to the polyion chain. The dielectric dispersions have been analyzed on the basis of two partially different dielectric models, a continuum counterion fluctuation model proposed by Mandel and a discrete charged site model, proposed by Minakata. The influence of the quality of the solvent on the dielectric parameters has been investigated in water-methanol and water-glycerol mixtures at different composition, by varying the permittivity (m) and the viscosity eta of the solvent phase. The analysis of the dielectric spectra in solvents where electrostatic and hydrodynamic interactions vary with the solvent composition suggests that both the two models are able, in principle, to account for the observed high-frequency dielectric behavior. However, while some certain assumptions are necessary about the polyion structure within the Mandel model, no structural prerequisite is needed within the Minakata model, where the polarization mechanism invoked considers a radial counterion exchange with the outer medium, which is largely independent of the local polyion conformation.

DNA condensation induced by cationic surfactant: a viscosimetry and dynamic light scattering study.

The compaction of DNA induced by two simple amphiphiles, cetyltrimethylammonium bromide [CTAB] and dodecyldimethylamine oxide [DDAO], has been investigated by means of combined viscosity and dynamic light scattering measurements, to demonstrate the formation of soluble DNA/surfactant complexes, undergoing a coil-globule transition, upon the increase of the amphiphile concentration. In both of the two systems investigated, the complexation process reaches a maximum for a value of the surfactant to DNA phosphate groups molar ratio of about X = 1. Below this critical concentration, the coil and the globule state coexist in the solution, as clearly shown by the bimodal size distribution obtained from the light scattering intensity correlation functions. Some suggestions are given to support a molecular mechanism responsible for the complex formation, both in the case of a cationic surfactant (CTAB) and of a pH-dependent neutral or cationic amphiphile (DDAO), where the hydrophobic interactions play an important role.

Intrinsic structural differences between "tight couples" and Kaltschmidt-Wittmann ribosomes evidenced by dielectric spectroscopy and scanning microcalorimetry.

Measurements of dielectric spectroscopy (DS) and microcalorimetry (differential scanning calorimetry (DSC)) of Escherichia coli 70S, 50S and 30S were performed on particles prepared according either to the "classical" twice NH(4)Cl-washed ribosomes, also known as loose couples (LC), or to the "tight couples" preparative protocol (TC). Results show that 70S particles prepared according to the two different protocols exhibit different structural properties. Two subsequent relaxation processes occur in both samples as measured by DS. However, in LC ribosomes the first one is shifted towards a lower frequency with a higher dielectric increment. This is suggestive of a more extensive exposure of RNA to the solvent and of an overall more relaxed structure. The smaller LC subunit exhibits only one relaxation while the TC 30S shows two dielectric dispersions as well as 70S. No substantial differences were evidenced in either 50S species. Two typical melting peaks were observed by DSC both in LC and TC 70S as well as in 50S. Thermograms obtained from the TC 30S show a single well structured peak while LC particles produce a large unstructured curve. On the basis of these results we conclude that TC 70S particles are more compact than LC ribosomes and that in the former ones the rRNA is less exposed to the solvent phase. Furthermore 30S particles obtained from TC show a more stable structure with respect to LC 30S. We conclude that the 30S subunit gives a major contribution to the compact character of the whole TC 70S. These differences might be related to the intrinsic and well documented functional difference between the two ribosome species.

A spectrophotometric study of the binding of Cu(II) ions to ATP.

The interaction of copper(II) with adenosine triphosphate (ATP) has been studied as a function of pH in the range pH 3-12. Our approach is the study of the effect of binding both on the ATP ultraviolet absorption spectrum and on the optical d-d transition of copper ions. The results show that Cu(II)-ATP complexes exist in a variety of forms in equilibrium, the percentage of each species varying according to the state of ionization of the intrinsic adenine, phosphate and ribose groups. These results also show a close correlation between the rate of dephosphorylation of ATP in the presence of Cu(II) ions and Cu(II) bonding to the adenine of ATP, thus supporting the hypothesis that the metal-ion/nucleic-base interactions are crucial for the observation of a metal-ion promoted dephosphorylation of ATP (D.H. Buisson and H. Sigel, Biochim. Biophys. Acta 343 (1974) 45).

Effect of ethanol on the thermal stability of tRNA molecules.

The thermal denaturation of E. coli unfractionated tRNA in ethanol/water mixtures has been studied as a function of alcohol concentration in the water-rich region (mole fraction of co-solvent chi 2 less than 0.2). The results show that with increasing alcohol concentration the melting temperature of tRNA first reaches a minimum at an intermediate composition chi *2 approximately equal to 0.055 and then increases with increasing chi 2. The value of chi *2 is close to that at which structural changes in the mixture occur as inferred from compressibility and optical absorption measurements. The present experimental data support the assumptions that the dominant mechanism by which ethanol affects the thermal stability of tRNA molecules is through its effect on the structure of water.

Electron paramagnetic resonance and optical spectroscopic study of the Cu2+-tRNA system.

The interaction of copper ions with tRNA has been studied by optical and EPR spectroscopies. The interaction results in two different paramagnetic complexes characterized by a tetragonal symmetry of the ligand electric field sensed by the ions. The complete set of the spin Hamiltonian parameters has been extracted by computer simulation with the Monte Carlo method. Hypotheses concerning the putative ligands are put forward.

Structural stability of ribosomes subjected to RNase treatment evidenced by dielectric spectroscopy and differential scanning microcalorimetry.

Previous studies from our laboratory demonstrated the existence of at least two levels of structural complexity in E. coli 70S ribosomes. Ribosomal RNA seems to be principally involved in the overall stability of these structures. In this paper we present an investigation of ribosomes subjected to treatment with RNase. The study is based on both differential scanning microcalorimetry and dielectric spectroscopy. In the thermograms obtained on treated ribosomes only the low temperature peak of the two typical denaturation events observed in native ribosomes, is promptly eliminated by the enzyme treatment. Dielectric spectroscopy measurements carried out on the same samples indicate an alteration of the dielectric behavior previously shown to consist of two subsequent relaxation processes. In fact, only the low frequency relaxation is affected by the treatment. The second one, observed at higher frequency, remains unaltered. The same effect on the dielectric parameters is observed if the ribosome particles are heated and then cooled prior to measurement. These results are consistent with the idea that two different structures are present within the ribosome. One is very stable and withstands both temperature and RNase treatment while the second is promptly abolished by both treatments. Data presented here strongly suggest that the RNA domains exposed to the solvent play a fundamental role in the stability of the 3-D structure of the ribosome particle.

Differential stability of E. coli ribosomal particles and free RNA towards thermal degradation studied by microcalorimetry.

We investigated the thermal degradation of E. coli ribosomes by differential scanning microcalorimetry. The 70S particles show two distinctive and irreversible peaks upon thermal degradation. Free rRNA in solution produces, on the contrary, an unstructured denaturation profile. The thermal analysis of 50S particles shows a profile substantially identical to that observed in 70S, while 30S particles produce an unstructured denaturation pattern. Therefore the thermal behavior of the 70S particle is essentially attributable to the denaturation of the 50S subunit. Our data validate previous observations that the 50S has a more rigid structure as compared to 30S, which behaves as a 'floppy' particle. In addition our data suggest that protein/RNA interactions play a significant role to stabilize three-dimensional structures of the ribosome.

Instability of three-dimensional structures in ribosomal cores evidenced by microcalorimetric studies.

In this paper we show a microcalorimetric investigation carried out on the so-called cores, i.e. ribosomes deprived of select proteins by LiCl treatment. Thermal degradation of native ribosomes gives rise to two thermal transitions occurring at different temperatures. In the cores the high temperature peak persists even after treatment at very high ion strength (2 M LiCl). This strongly suggests the existence of a very stable structure that was previously observed also in particles treated with agents that hydrolyze the RNA moiety. The low temperature peak gradually but dramatically decreases even though it never disappears completely. This indicates that the treatment to obtain ribosomal cores does not cause complete unfolding of the particle but only the destabilization of a structural three-dimensional domain present in native ribosomes. These data are discussed in the light of previous results obtained by dielectric spectroscopy and microcalorimetric studies on ribosomal particles.

Lysozyme hydration in concentrated aqueous solutions. Effect of an equilibrium cluster phase.

Water close to proteins plays a key role in determining their structural and functional properties. Despite being a subject of considerable interest, the characterization of hydration water, as far as its total amount is concerned, is still controversial and its influence on protein structure and folding is not yet fully understood. In this work, we have investigated the dielectric properties of lysozyme aqueous solutions over the frequency range where the orientational polarization relaxation of the aqueous phase occurs (from 500 MHz to 50 GHz). Measurements extend over a wide concentration range, up to 300 mg/mL, corresponding to a volume fraction of the order of 0.4. The analysis of the dielectric spectra, based on the decrease of the dielectric increment of the γ-dispersion as a function of protein concentration, allows us to estimate the total amount of hydration water (both bound water and loosely bound water) present in the system investigated. We observe a decrease of the hydration number as a function of the protein concentration. This behavior is well accounted for by considering the formation of small equilibrium clusters with aggregation number of some units, as recently reported by Stradner et al. (1) on the basis of small-angle X-ray and neutron scattering measurements.

Heat-denatured lysozyme aggregation and gelation as revealed by combined dielectric relaxation spectroscopy and light scattering measurements.

The dielectric behavior of native and heat-denatured lysozyme in ethanol-water solutions was examined in the frequency range from 1 MHz to 2 GHz, using frequency-domain dielectric relaxation spectroscopy. Because of the conformational changes on unfolding, dielectric methods provide information on the denaturation process of the protein and, at protein concentration high enough, on the subsequent aggregation and gelation. Moreover, the time evolution of the protein aggregation and gelation was monitored measuring, by means of dynamic light scattering methods, the diffusion coefficient of micro-sized polystyrene particles, deliberately added to the protein solution, which act as a probe of the viscosity of the microenvironment close to the particle surface. All together, our measurements indicate that heat-induced denaturation favors, at high concentrations, a protein aggregation process which evolves up to the full gelation of the system. These findings have a direct support from IR measurements of the absorbance of the amide I band that, because of the unfolding, indicate that proteins entangle each other, producing a network structure which evolves, in long time limit, in the gel.

Dielectric relaxation spectroscopy of lysozyme aqueous solutions: analysis of the δ-dispersion and the contribution of the hydration water.

The dielectric properties of lysozyme aqueous solutions have been investigated over a wide frequency range, from 1 MHz to 50 GHz, where different polarization mechanisms, at a molecular level, manifest. The dielectric relaxation spectra show a multimodal structure, reflecting the complexity of the protein-water interactions, made even more intricate with the increase of the protein concentration. The deconvolution of the spectra into their different components is not unambiguous and is generally a delicate process which requires caution. We have analyzed the whole relaxation region, on the basis of the sum of simple Debye-type relaxation functions, considering three main contributions. Particular attention has been payed to the δ-dispersion, intermediate between the ß-dispersion (rotational dynamics of the protein) and the γ-dispersion (orientational polarization of the water molecules). This intermediate contribution to the dielectric spectrum is attributed to the orientational polarization of water molecules in the immediate vicinity of the protein surface (hydration water). Our measurements clearly demonstrate that, at least at high protein concentrations, the δ-dispersion has a bimodal structure associated with two kinds of hydration water, i.e., tightly bound and loosely bound hydration water. In the concentration range investigated, the existence of a three-modal δ-dispersion, as recently suggested, is not supported, on the basis of statistical tests, by the analysis of the dielectric relaxations we have performed and a bimodal dispersion is accurate enough to describe the experimental data. The amount of the hydration water has been evaluated both from the dielectric parameters associated with the δ-dispersion and from the decrement of the loss peak of the γ-dispersion. The relative weight of tightly bound and loosely bound hydration water is briefly discussed.