Pressure jump relaxation investigations of lipid membranes using FTIR spectroscopy

The relaxation kinetics of aqueous lipid dispersions after a pressure jump (p-jump) were investigated using time-resolved FTIR spectroscopy. The methylene stretching vibrational band and the carbonyl band were analyzed to detect changes in conformational order of the hydrocarbon chains and to follow the degree of hydration of the head group, respectively. The kinetics of the transition was found to consist of multiple processes with relaxation constants from seconds down to milliseconds. Faster processes are also present, but could not be resolved by our instrument. This is the first investigation showing directly the time resolved change in chain order in lipid bilayers induced by a p-jump. The results obtained with this IR detection method support previous results that the change in chain order after a perturbation is a multi-step process with the initial molecular events occurring with time constants shorter than milliseconds.     

Relaxation dynamics of the gel to liquid-crystalline transition of phosphatidylcholine bilayers. Effects of chainlength and vesicle size.

The relaxation kinetics of the gel to liquid-crystalline transition of five phosphatidylcholine (DC14PC to DC18PC) bilayer dispersions have been investigated using volume perturbation calorimetry, a steady-state technique which subjects a sample to sinusoidal changes in volume. Temperature and pressure responses to the volume perturbation are measured to monitor the relaxation to a new equilibrium position. The amplitude demodulation and phase shift of these observables are analyzed with respect to the perturbation frequency to yield relaxation times and amplitudes. In the limit of low perturbation frequency, the temperature and pressure responses are proportional to the equilibrium excess heat capacity and bulk modulus, respectively. At all temperatures, the thermal response data are consistent with a single primary relaxation process of the lipid. The less accurate bulk modulus data exhibit two relaxation times, but it is not clear whether they reflect lipid processes or are characteristic of the instrument. The observed thermal relaxation behavior of all multilamellar vesicles are quantitatively similar. The relaxation times vary from approximately 50 ms to 4 s, with a pronounced maximum at a temperature just greater than Tm, the temperature of the excess heat capacity maximum. Large unilamellar vesicles also exhibit a single relaxation process, but without a pronounced maximum in the relaxation time. Their relaxation time is approximately 80 ms over most of the transition range.     

The protein glass transition as measured by dielectric spectroscopy and differential scanning calorimetry

The glass transition and its related dynamics of myoglobin in water and in a water–glycerol mixture have been investigated by dielectric spectroscopy and differential scanning calorimetry (DSC). For all samples, the DSC measurements display a glass transition that extends over a large temperature range. Both the temperature of the transition and its broadness decrease rapidly with increasing amount of solvent in the system. The dielectric measurements show several dynamical processes, due to both protein and solvent relaxations, and in the case of pure water as solvent the main protein process (which most likely is due to conformational changes of the protein structure) exhibits a dynamic glass transition (i.e. reaches a relaxation time of 100 s) at about the same temperature as the calorimetric glass transition temperature T_g is found. This glass transition is most likely caused by the dynamic crossover and the associated vanishing of the α-relaxation of the main water relaxation, although it does not contribute to the calorimetric T_g. This is in contrast to myoglobin in water–glycerol, where the main solvent relaxation makes the strongest contribution to the calorimetric glass transition. For all samples it is clear that several proteins processes are involved in the calorimetric glass transition and the broadness of the transition depends on how much these different relaxations are separated in time.     

Gel-liquid crystalline transition of some multilamellar lipid bilayers follows classical kinetics with a fractional dimensionality of approximately two.

The relaxation kinetics of the gel-liquid crystalline transition of phosphatidylcholine (DC14PC, DC16PC, and DC18PC) multilamellar vesicles have been examined using volume-perturbation calorimetry. The time-dependent temperature and pressure changes associated with a periodic volume perturbation are monitored in real time. Data collected in the time domain are transformed to the frequency domain using Fourier series representations of the perturbation and response functions. Because a very small perturbation is imposed during the experiment, linear response theory is suitable for analysis of the relaxation process. The Laplace transform of the classical Kolmogorov-Avrami relation of transition kinetics is used to describe the dynamic response in the frequency domain. For DC14PC and DC16PC, the relaxation process is better fit with an effective dimensionality of n = 2 rather than n = 1. For DC18PC, we estimate that an effective dimensionality of approximately 1.5 will best fit the data. These results indicate that the gel-liquid crystalline transition of these lipid bilayers follows the classical Kolmogorov-Avrami kinetic model with an effective dimensionality greater than 1 and the assumption of simple exponential decay (n = 1) commonly used in data analysis may not always be valid for lipid transitions. Insofar as the dimensionality of the relaxation reflects the geometry of fluctuating lipid clusters, this parameter may be useful in connecting experimental thermodynamic and kinetic results with those obtained from Monte Carlo simulations.     

Critical temperatures and a critical chain length in saturated diacylphosphatidylcholines: calorimetric, ultrasonic and Monte Carlo simulation study of chain-melting/ordering in aqueous lipid dispersions

Chain-ordering/melting transition in a series of saturated diacylphosphatidylcholines (PCs) in aqueous dispersions have been studied experimentally (calorimetric and ultrasonic techniques) and theoretically (an Ising-like lattice model). The shape of the calorimetric curves was compared with the theoretical data and interpreted in terms of the lateral interactions and critical temperatures determined for each lipid studied. A critical chain length has been found (between 16 and 17 C-atoms per chain) which subdivides PCs into two classes with different phase behavior. In shorter lipids, the transition takes place above their critical temperatures meaning that this is an intrinsically continuous transition. In longer lipids, the transition occurs below the critical temperatures of the lipids, meaning that the transition is intrinsically discontinuous (first-order). This conclusion was supported independently by the ultrasonic relaxation sensitive to density fluctuations. Interestingly, it is this length that is the most abundant among the saturated chains in biological membranes.     

Application of pressure perturbation calorimetry to lipid bilayers

Pressure perturbation calorimetry (PPC) is a new method that measures the heat consumed or released by a sample after a sudden pressure jump. The heat change can be used to derive the thermal volume expansion coefficient, alpha(V), as a function of temperature and, in the case of phase transitions, the volume change, DeltaV, occurring at the phase transition. Here we present the first report on the application of PPC to determine these quantities for lipid bilayers. We measure the volume changes of the pretransition and main transition of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and the thermal expansivity of the fluid phase of DMPC and of two unsaturated lipids, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphocholine. The high sensitivity of PPC instrumentation gives accurate data for alpha(V) and DeltaV even upon the application of relatively low pressures of approximately 5 bar.     

Lipid phase separations induced by the association of cholera toxin to phospholipid membranes containing ganglioside GM1

The interactions of cholera toxin and their isolated binding and active subunits with phospholipid bilayers containing the toxin receptor ganglioside GM1 have been studied by using high-sensitivity differential scanning calorimetry and steady-state and time-resolved fluorescence and phosphorescence spectroscopy. The results of this investigation indicate that cholera toxin associates with phospholipid bilayers containing ganglioside GM1, independent of the physical state of the membrane. In the absence of Ca2+, calorimetric scans of intact cholera toxin bound to dipalmitoylphosphatidylcholine (DPPC) large unilamellar vesicles containing ganglioside GM1 result in a broadening of the lipid phase transition peak and a slight decrease (less than 5%) in the transition enthalpy. In the presence of Ca2+ concentrations sufficient to cause ganglioside phase separation, the association of the intact toxin to the membrane results in a significant decrease of enthalpy change for the lipid transition, indicating that under these conditions the toxin molecule perturbs the hydrophobic core of the bilayer. Calorimetric scans using isolated binding subunits lacking the hydrophobic toxic subunit did not exhibit a decrease in the phospholipid transition enthalpy even in the presence of Ca2+, indicating that the binding subunits per se do not perturb the hydrophobic core of the bilayer. On the other hand, the hydrophobic A1 subunit by itself was able to reduce the phospholipid transition enthalpy when reconstituted into DPPC vesicles. These calorimetric observations were confirmed by fluorescence experiments using pyrene phospholipids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Time-resolved charge movements in the sarcoplasmatic reticulum Ca-ATPase

The time-resolved kinetics of the Ca(2+)-translocating partial reaction of the sarcoplasmatic reticulum Ca-ATPase was investigated by ATP-concentration jump experiments. ATP was released by an ultraviolet light flash from its inactive precursor and charge movements in the membrane domain of the ion pumps were detected by the fluorescent styryl dye 2BITC. Two oppositely directed cation movements were found, which were assigned to Ca(2+) release and H(+) binding. The faster process with a typical time constant of 30 ms reports the rate-limiting process before Ca(2+) release, probably the conformation transition E(1) --> E(2). The following, slow uptake of positive charge had a pH-dependent time constant, which was 1 s at low pH and approximately 3 s at pH > 8. This process is assigned to an electrically silent conformational relaxation of the state P-E(2) preceding H(+) binding. This interpretation is in agreement with the observation that the fast process was independent of the substrate concentrations (i.e., when [Ca(2+)] > 200 nM, and [ATP] > 20 micro M). The slow process was independent of the Ca(2+) concentration. The activation energy of the resolved processes was between 80 kJ/mol and 90 kJ/mol, which is comparable to the activation energy of the enzymatic activity (92 kJ/mol) and these high values point to conformational changes underlying rate-limiting steps of the pump cycle.     

Synchrotron X-ray and neutron small-angle scattering of lyotropic lipid mesophases, model biomembranes and proteins in solution at high pressure

In this review we discuss the use of X-ray and neutron diffraction methods for investigating the temperature- and pressure-dependent structure and phase behaviour of lipid and model biomembrane systems. Hydrostatic pressure has been used as a physical parameter for studying the stability and energetics of lipid mesophases, but also because high pressure is an important feature of certain natural membrane environments and because the high pressure phase behaviour of biomolecules is of importance for several biotechnological processes. Using the pressure jump relaxation technique in combination with time-resolved synchrotron X-ray diffraction, the kinetics of different lipid phase transformations was investigated. The techniques can also be applied to the study of other soft matter and biomolecular phase transformations, such as surfactant phase transitions and protein un/refolding reactions. Several examples are given. In particular, we present data on the pressure-induced unfolding and refolding of small proteins, such as Snase. The data are compared with the corresponding results obtained using other trigger mechanisms and are discussed in the light of recent theoretical approaches.     

Broadband dielectric spectroscopy and calorimetric investigations of d-lyxose

Using broadband dielectric spectroscopy, we have studied different types of relaxation processes, namely, primary (α), secondary (β), and another sub-T_g process called γ-process, in the supercooled state of d-lyxose, over a wide frequency (10^-2–) and temperature range (120–340 K). In addition, the same sample was analyzed by differential scanning calorimeter. The temperature dependence of the relaxation times as well as the dielectric strength of different processes has been critically examined. It has been observed that the slower secondary relaxation (designated as β-) process shifts to lower frequencies with increasing applied pressure, but not the faster one. This pressure dependence indicates that the observed slower secondary relaxation (β-) is Johari–Goldstein relaxation process and faster one (γ-process) is probably the rotation of hydroxymethyl (–CH₂OH) side group attached to the sugar ring, that is, of intramolecular origin.     

Kinetic investigations on the phase transition of phospholipid bilayers

Pressure-jump experiments were performed on vesicles and liposomes of dimyristoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine following the time course of solution turbidity. For both lipids two relaxation effects were evaluated the time constants of which exhibit clear maxima at the midpoint of the phase transition. The time constants lie for vesicles in the 100 μs and 1 ms ranges and for liposomes in the 1 ms and 10 ms ranges. The processes are slightly faster for dimyristoyl phosphatidylcholine than for dipalmitoyl phosphatidylcholine. All relaxation times are concentration-independent. The time constant and amplitude behaviours indicate that all processes are cooperative in agreement with previous interpretations. It is demonstrated that cooperative units can be evaluated from the relaxation amplitudes. These are of the same order of magnitude as those obtained from static experiments. On the grounds of the present kinetic investigation we can state that the application of the linear Ising model to two-dimensional processes as attempted for the static lipid phase transition is inadequate.     

NMR and optical activity observations on the helix-coil transition in poly(gamma-benzyl L-glutamate) solutions

The helix–coil transition of poly(γ-benzyl L -glutamate) was studied by comparing proton magnetic relaxation behavior with optical activity studies. The transition temperature as determined by magnetic relaxation was lower than that obtained by optical activity. The concentration dependence of the transition was also studied. The relationship of these experiments with previous NMR studies and also with calorimetric investigation of the transition is developed.     

Interaction between perdeuterated dimyristoylphosphatidylcholine and low molecular weight pulmonary surfactant protein SP-C

A low molecular weight hydrophobic protein was isolated from porcine lung lavage fluid using silicic acid and Sephadex LH-20 chromatography. The protein migrated with an apparent molecular weight of 5000-6000 on SDS-PAGE under reducing and nonreducing conditions. Gels run under reducing conditions also showed a minor band migrating with a molecular weight of 12,000. Amino acid compositional analysis and sequencing data suggest that this protein preparation contains intact surfactant protein SP-C and about 30% of truncated SP-C (N-terminal leucine absent). The surfactant protein was combined with perdeuterated dimyristoylphosphatidylcholine (DMPC-d54) in multilamellar vesicles. The protein enhanced the rate of adsorption of the lipid at air-water interfaces. The ability of the protein to alter normal lipid organization was examined by using high-sensitivity differential scanning calorimetry (DSC) and 2H nuclear magnetic resonance spectroscopy (2H NMR). The calorimetric measurements indicated that the protein caused a decrease in the temperature maximum (Tm) and a broadening of the phase transition. At a protein concentration of 8% (w/w), the enthalpy change of transition was reduced to 4.4 kcal/mol compared to 6.3 kcal/mol determined for the pure lipid. NMR spectral moment studies indicated that protein had no effect on lipid chain order in the liquid-crystal phase but reduced orientational order in the gel phase. Two-phase coexistence in the presence of protein was observed over a small temperature range below the pure lipid transition temperature. Spin-lattice relaxation times (T1) were not substantially affected by the protein. Transverse relaxation time (T2e) studies suggest that the protein influences slow lipid motions.     

DMPG gel-fluid thermal transition monitored by a phospholipid spin labeled at the acyl chain end

Low ionic strength aqueous dispersion of dimyristoyl phosphatidylglycerol (DMPG) presents a rather peculiar gel-fluid thermal transition behavior. The lipid main phase transition occurs over a large temperature interval (ca. 17 degrees C), along which several calorimetric peaks are observed. Using lipids spin labeled at the acyl chain end, a two-peak electron spin resonance (ESR) spectrum is observed along that temperature transition region (named intermediate phase), at three different microwave frequencies: L-, X- and Q-bands. The intermediate phase ESR spectra are analyzed, and shown to be most likely due to spin labels probing two distinct types of lipid organization in the DMPG bilayer. Based on the ESR spectra parameters, a model for the DMPG intermediate phase is proposed, where rather fluid and hydrated domains, possibly high curvature regions, coexist with patches that are more rigid and hydrophobic.     

Effects of the anesthetic dibucaine on the kinetics of the gel-liquid crystalline transition of dipalmitoylphosphatidylcholine multilamellar vesicles.

The effects of the anesthetic dibucaine on the relaxation kinetics of the gel-liquid crystalline transition of dipalmitoylphosphatidylcholine (DC16PC) multilamellar vesicles have been investigated using volume-perturbation calorimetry. The temperature and pressure responses to a periodic volume perturbation were measured in real time. Data collected in the time domain were subsequently converted into and analyzed in the frequency domain using Fourier series representations of the perturbation and response functions. The Laplace transform of the classical Kolmogorov-Avrami kinetic relation was employed to describe the relaxation dynamics in the frequency domain. The relaxation time of anesthetic-lipid mixtures, as a function of the fractional degree of melting, appears to be qualitatively similar to that of pure lipid systems, with a pronounced maximum, tau max, observed at a temperature corresponding to greater than 75% melting. The tau max decreases by a factor of approximately 2 as the nominal anesthetic/lipid mole ratio increases from 0 to 0.013 and exhibits no further change as the nominal anesthetic/lipid mole ratio is increased. However, the fractional dimensionality of the relaxation process decreases monotonically from slightly less than two to approximately one as the anesthetic/lipid mole ratio increases from 0 to 0.027. At higher ratios, the dimensionality appears to be less than one. These results are interpreted in terms of the classical kinetic theory and related to those obtained from Monte Carlo simulations. Specifically, low concentrations of dibucaine appear to reduce the average cluster size and cause the fluctuating lipid clusters to become more ramified. At the highest concentration of dibucaine, where n < 1, the system must be kinetically heterogeneous.     

The effect of pressure on the lipid microviscosity and phase transition of lung surfactant

The effect of pressure on the lipid dynamics of the rat lung surfactant was studied in liposomes made of the natural lung surfactant of the rat and of model phospholipid mixture. The determined parameter was the lipid microviscosity, monitored by the fluorescence polarization of the probe 1,6-diphenyl-1,3,5-hexatriene. Osmotic pressure of up to 47 atm, as well as hydrostatic pressure of up to 1.4 kbar, were applied at a constant temperature. The effect of pressure was monitored by the change in the lipid microviscosity of the system. The maximal change achieved with osmotic pressure at a constant temperature was only 30%. This suggests that the conversion of melted lipid to its solid phase above the lipid critical temperature requires several hundred atmospheres. Similarly, measurements of lipid microviscosity under increased hydrostatic pressure revealed transitions which occurred at above 400 atm. Since such pressures are far beyond the physiological scale, it excludes the possibility that pressure alone can be responsible for a full phase transition of the lung surfactant during respiration. Upon decompression, microviscosity of the examined lipid system was found to return to its original values, confirming the reversibility of the process.     

Electrostatic interactions at charged lipid membranes Kinetics of the electrostatically triggered phase transition

Charged lipid membranes of dimyristoylmethylphosphatidic acid were mixed rapidly in a stopped-flow cell with protons or Ca²⁺ to compensate the charges and thereby trigger the ordered-fluid phase transition. The kinetics of the transition was studied by following the time development of the fluorescence anisotropy of diphenylhexatriene. A relaxation process was observed with a characteristic time in the range 10–200 ms. By comparison with existing theories of non-equilibrium relaxation it was concluded that the relaxation process is governed by a nucleation step.     

Interaction of fluorinated ether anesthetics with artificial membranes

Fluorine-19 nuclear magnetic resonance spectroscopy is applied to the study of the environment of dipalmitoyl phosphatidylcholine-bound fluorinated ether anesthetics (enflurane, fluoroxene and methoxyflurane) both below and above the lipid gel to liquid crystal phase transition temperature. Line widths and spin-lattice relaxation time (T₁) measurements are consistent with substantial immobilization of the lipid-bound anesthetic molecules. Heating anesthetic/lipid mixtures above the lipid transition temperature leads to narrowing of the lipid-bound anesthetic fluorine resonances accompanied by little or no change in anesthetic fluorine-19 chemical shifts, suggesting that although the mobility of the bound anesthetic increases at the higher temperature, the nature of the anesthetic-lipid interaction changes little as a result of this phase change. Differential scanning calorimetric studies of the effects of these anesthetics on the phase transition behavior of the phospholipid indicate that the regions of the bilayer in which volatile anesthetics partition at lower concentrations are different from the regions in which they partition at higher concentrations.     

Electron diffraction study of hydrated phospholipid single bilayers. Effects of temperature,hydration and surface pressure of the “precursor” monolayer

The molecular packing and phase transition of hydrated dipalmitoylphosphatidylcholine single bilayers are studied by electron diffraction, using an electron microscope equipped with a hydration stage. The phase transition and area per molecule are measured as functions of temperature, hydration and the surface pressure of the monolayer from which the bilayer is formed. The transition temperature of a bilayer agrees with calorimetric measurements on bulk lipid/water mixtures. The molecular packing of a bilayer corresponds to that of the precursor monolayer at a surface pressure of 47 dyne/cm.     

Ultrafast excited-state dynamics of phenyleneethynylene oligomers in solution.

The excited-state dynamics of oligomeric phenyleneethynylenes (OPEs) of various length and substitution has been investigated by femtosecond time-resolved spectroscopy. The fluorescence lifetime of the OPEs decreases with the number of phenyleneethynylene units up to about 9. This effect is due to an increase of the oscillator strength for the S(1)-S(0) transition. Dynamic features occurring within a few tens of picoseconds and ascribed to structural relaxation directly after population of the S(1) state can be observed in non-viscous solvents. The effect of torsional disorder on the fluorescence intensity is shown to depend strongly on the nature of the substituent on the phenyl groups. All these effects are qualitatively discussed with a simple exciton model.