Stopped-flow study of anesthetic effect on water-transport kinetics through phospholipid membranes. Interfacial versus lipid core ligands

We have compared ligand effects between polar and apolar anesthetic molecules upon water transport across phospholipid membranes by kinetic analysis of the osmotic swelling rate, using a stopped-flow technique. Chloroform and 1-hexanol were used as interfacial ligands, and carbon tetrachloride and n-hexane were used as their counterparts, representing lipid core action. Because anesthetics transform the solid-gel membrane into a liquid-crystalline state, and because phospholipid membranes display an anomaly in permeability at the phase transition, dimyristoylphosphatidylcholine vesicles were studied at temperatures above the main phase transition to avoid this anomaly. All these molecules increased the osmotic swelling rate. However, a significant difference was observed in the activation energy, delta Ep, between polar and apolar molecules; delta Ep was almost unaltered by the addition of polar molecules (chloroform and 1-hexanol), whereas it was decreased by apolar molecules (carbon tetrachloride and n-hexane). The obtained results were analyzed in terms of the dissolution-diffusion mechanism for water permeation across the lipid membrane. It is suggested that polar molecules affect water permeability by altering the partition of water between the membrane interior and water phase, and apolar molecules affect it by altering both the partition and the diffusion of water within the membrane interior.     

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.     

The influence of cholesterol on the main phase transition of unilamellar dipalmytoylphosphatidylcholine vesicles. A differential scanning calorimetry and iodine laser T-jump study.

The influence of cholesterol (CHOL) on the main phase transition in single shell dipalmytoylphosphatidylcholine (DPPC) vesicles was investigated in equilibrium and kinetic experiments. CHOL increases the optical density and causes a slight hysteresis in turbidity transition curves. Static fluorescence anisotropy measurements showed interesting differences for three probes sensing different parts in the hydrophobic region of the phospholipid bilayer. Differential scanning calorimetry (DSC) peaks can be separated into a narrow and a broad component. The narrow component, which decreases linearly with increasing CHOL content and disappears at 20 mol %, is attributed to the transition of free phospholipid, while the broad component, being associated with the transition of CHOL-lipid units, increases monotoniously from 0 to 20%. Kinetic experiments were performed on our iodine-laser T-jump arrangement with turbidity detection. Three cooperative relaxation signals in the microsecond and millisecond time range were detected for pure DPPC vesicles as well as vesicles containing 7.5 and 16.5 mol % CHOL. All three relaxation processes were changed by CHOL: the superposition of the three relaxation amplitudes can be separated into a narrow and a broad component, as in DSC experiments. A speculative model is presented which assumes an inhomogeneous CHOL distribution fluctuating on a millisecond time scale in the temperature region of the main phase transition.     

Interaction of tryptophan with lecithin liposomes: NMR and turbidity studies

A study on the interactions between tryptophan (Trp) and dipalmitoylphosphatidyl choline (DPPC) liposomes conducted with the NMR technique and taking turbidity measurements is reported. Trp is shown to be incorporated into the bilayer only when interaction occurs above gel-liquid transition. Disappearance of turbidity changes at the phase transition temperatures are shown to occur with Trp incorporation. 1H and 13C NMR relaxation times T1 of DPPC are seen to be reduced. Acyl chain signal intensity is shown to decrease and the corresponding line-width to increase as a function of Trp concentration. DPPC 31P [1H] Nuclear Overhauser Effect (NOE) is depressed by the presence of Trp above gel-liquid transition temperature whereas NOE remains high below phase transition temperature when Trp is present in the bilayer. Effects are shown to be the same in both H2O and in 2H2O. A membrane modification that may account for the previously observed inhibition of polysaccharide induced cell aggregation is hypothesized.     

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.     

Effect of short-chain primary alcohols on fluidity and activity of sarcoplasmic reticulum membranes

Intramolecular excimer formation with the fluorescent probe 1,3-di(1-pyrenyl)propane, differential scanning calorimetry, and X-ray diffraction were used to assess the effect of ethanol, 1-butanol, and 1-hexanol on the bilayer organization in model membranes, sarcoplasmic reticulum (SR) lipids and native SR membranes. These alcohols have fluidizing effects on membranes and lower the main transition temperature of dimyristoylphosphatidylcholine (DMPC), but only 1-hexanol alters the cooperativity of the phase transition and significantly increases the thickness of DMPC bilayers. The interaction of the three alcohols with the SR Ca2+ pump was also investigated. Hydrolysis of ATP and coupled Ca2+ uptake are differently sensitive to the three alcohols. Whereas ethanol and 1-butanol inhibited the Ca2+ uptake, 1-hexanol stimulated it. Nevertheless, the energetic efficiency of the pump (Ca2+/ATP) is not significantly affected by ethanol or 1-hexanol, but uncoupling was observed with 1-butanol at high concentrations. The different effects of alcohols on the activity of SR membranes rule out an unitary mechanism of action on the basis of fluidity changes induced in the lipid bilayer. Depending on the chain length, the alcohols interact with the SR membranes in different domains, perturbing differently the Ca2+-pump activity.     

Phase transition kinetics of phosphatidic acid bilayers A stopped-flow study of the electrostatically induced transition

The kinetics of the electrostatically induced phase transition of dimyristoyl phosphatidic acid bilayers was followed using the stopped-flow technique. The phase transition was triggered by a fast change in the pH or the magnesium ion concentration and followed by recording the time dependence of the absorbance. When the phase transition was induced by a pH jump the time course of the absorbance could be described by two exponentials, their time constants displaying the for cooperative processes characteristic maximum at the transition midpoint. The time constants are in the 10 and 100 ms range for the H⁺ triggered transition from the fluid to the ordered state. A third slower process shows no appreciable temperature dependence and is probably caused by vesicle aggregation. For the OH^--induced transition fron the ordered to the fluid state the time constants are in the 100 and 1000 ms range. The fluid-ordered transition could also be triggered by addition of magnesium ions. Of the several observed processes only the fastest in the 10–100 ms time range could definitely be assigned to the fluid-ordered transition while the others are due to aggregation phenomena. The experimental data were compared with results obtained from pressure jump experiments and could be interpreted on the basis of theories for non-equilibrium relaxation.     

Molecular motions and phase transitions. NMR relaxation times studies of several lecithins.

The spin-lattice relaxation time, T1, and the dipolar energy relaxation time, TD, were measured as a function of temperature. The materials studied were samples of anhydrous L-dipalmitoyl lecithin, DL-dipalmitoyl lecithin, L-dimyristoyl lecithin, DL-dimyristoyl lecithin and their monohydrates, and of anhydrous egg yolk lecithin. It is shown that TD is a much more sensitive parameter than T1 for the determination of the Chapman phase transition. Comparison between T1 and TD provides information about new types of slow molecular motions below and above the phase transition temperature. It is suggested that the relaxation mechanisms for T1 and TD in the gel phase are governed by segmental motion in the phospholipid molecule. A new metastable phase was detected in dimyristoyl lecithin monohydrates. This phase could only be detected from the dipolar energy relaxation times.     

Equilibrium and kinetics of the allosteric transition of GroEL studied by solution X-ray scattering and fluorescence spectroscopy

We have studied the ATP-induced allosteric structural transition of GroEL using small angle X-ray scattering and fluorescence spectroscopy in combination with a stopped-flow technique. With X-ray scattering one can clearly distinguish the three allosteric states of GroEL, and the kinetics of the transition of GroEL induced by 85 microM ATP have been observed directly by stopped-flow X-ray scattering for the first time. The rate constant has been found to be 3-5s(-1) at 5 degrees C, indicating that this process corresponds to the second phase of the ATP-induced kinetics of tryptophan-inserted GroEL measured by stopped-flow fluorescence. Based on the ATP concentration dependence of the fluorescence kinetics, we conclude that the first phase represents bimolecular non-cooperative binding of ATP to GroEL with a bimolecular rate constant of 5.8 x 10(5)M(-1)s(-1) at 25 degrees C. Considering the electrostatic repulsion between negatively charged GroEL (-18 of the net charge per monomer at pH 7.5) and ATP, the rate constant is consistent with a diffusion-controlled bimolecular process. The ATP-induced fluorescence kinetics (the first and second phases) at various ATP concentrations (< 400 microM) occur before ATP hydrolysis by GroEL takes place and are well explained by a kinetic allosteric model, which is a combination of the conventional transition state theory and the Monod-Wyman-Changeux model, and we have successfully evaluated the equilibrium and kinetic parameters of the allosteric transition, including the binding constant of ATP in the transition state of GroEL.     

A kinetic folding intermediate probed by native state hydrogen exchange

Stopped-flow fluorescence studies on the N-terminal domain of rat CD2 (CD2.d1) have demonstrated that folding from the fully denatured state (U) proceeds via the transient accumulation of an apparent intermediate (I) in a so-called burst phase that precedes the rate-limiting transition leading to the native state (N). A previous pH-dependent equilibrium hydrogen exchange (HX) study identified a subset of amides in CD2.d1 which, under EX2 conditions, exchange from N with free energies greater than or equal to the free energy difference between the N and I states calculated from the stopped-flow data. Under EX1 conditions the rates of HX for these amides tend towards an asymptote that matches the global unfolding rate calculated from the stopped-flow data, suggesting that exchange for these amides requires traversing the N-to-I transition state barrier. Exchange for these amides presumably occurs from exchange-competent forms comprising the kinetic burst phase therefore. To explore this idea further, native state HX (NHX) data have been collected for CD2.d1 under EX2 conditions using denaturant concentrations which span either side of the denaturant concentration where, according to the stopped-flow data, the apparent U and I states are iso-energetic. The data fit to a two-component, sub-global (sg)/global (g) NHX mechanism, yielding Delta G and m value parameters (where the m value is a measure of hydrocarbon solvation). Regression analysis demonstrates that the (m(sg), Delta G(sg)) and (m(g), Delta G(g)) values calculated for this subset of amides correspond with those describing the kinetic burst phase transition. This result confirms the ability of the NHX technique to explore the structural and energetic properties of kinetic folding intermediates.     

X-ray analysis of the kinetics of Escherichia coli lipid and membrane structural transitions

Synchrotron radiation was used to follow the time course of the transitions, induced by temperature jump, in Escherichia coli membranes and their lipid extracts isolated from a fatty acid auxotroph grown with different fatty acids. We measured the relaxation times associated with the phase transitions as well as with the conformational transition of the hydrocarbon chains and observed different behavior as a function of chemical composition. Relaxation times of about 1-2 s were found at a hexagonal to lamellar phase transition and within a lamellar phase whose parameters display important variations with temperature when the conformational transition takes place. On the other hand, no delay was observed for a phase transition where large lipid or water diffusion was not needed. We have shown that phase transitions and conformational transitions are, to a large extent, uncoupled and that the relaxation times corresponding to the latter transition could be related to the size of the ordered domains. In all cases, the order to disorder conformational transition is more rapid than the disorder to order transition. Finally, the relaxation times of the disorder to order transition observed with the membranes and with their lipid extracts were found to be strongly correlated, indicating that the proteins do not play a role in this transition.     

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.     

Substrate entropy in enzyme enantioselectivity: an experimental and molecular modeling study of a lipase

The temperature dependence of the enantioselectivity of Candida antarctica lipase B for 3-hexanol, 2-butanol, 3-methyl-2-butanol, 3,3-dimethyl-2-butanol, and 1-bromo-2-butanol revealed that the differential activation entropy, deltaR-SdeltaS, was as significant as the differential activation enthalpy, DeltaR-SdeltaH, to the enantiomeric ratio, E. 1-Bromo-2-butanol, with isosteric substituents, displayed the largest deltaR-SdeltaS. 3-Hexanol displayed, contrary to other sec-alcohols, a positive deltaR-SdeltaS. In other words, for 3-hexanol the preferred R-enantiomer is not only favored by enthalpy but also by entropy. Molecular dynamics (MD) simulations and systematic search calculations of the substrate accessible volume within the active site revealed that the (R)-3-hexanol transition state (TS) accessed a larger volume within the active site than the (S)-3-hexanol TS. This correlates well with the higher TS entropy of (R)-3-hexanol. In addition, this enantiomer did also yield a higher number of allowed conformations, N, from the systematic search routines, than did the S-enantiomer. The substrate accessible volume was greater for the enantiomer preferred by entropy also for 2-butanol. For 3,3-dimethyl-2-butanol, however, neither MD-simulations nor systematic search calculations yielded substrate accessible volumes that correlate to TS entropy. Ambiguous results were achieved for 3-methyl-2-butanol.     

The influence of Ca2+ on the turbidity of DPPC-DMPA vesicles within the temperature range of the phase transition

The influence of the addition of Ca2+ on the phase behaviour of vesicles, composed of dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidic acid (DMPA) in a ratio of 4 to 1, has been investigated by means of turbidity measurements. As expected one single phase transition for the mixed phospholipids was observed in the absence of Ca2+. Passing through the temperature range of this transition after the addition of Ca2+, conditions appeared to favor fusion of the vesicles. A possible reason for this is that during the transition Ca2+ may permeate through the vesicle membranes and gain access to the inside DMPA binding sites. Therefore it is not unambiguously possible to determine phase transition temperatures from the turbidity changes that occur under these conditions. However, when within the temperature range of the phase transition of the mixed phospholipids the influence of Ca2+ addition to the vesicles was recorded isothermally, at each temperature separately, the final plot of turbidity versus temperature turned out to be far less confused by fusion events and adopted the form of two separate phase transitions. The temperatures at which these two transitions occur closely resemble the phase transition temperatures that may be observed in the absence of Ca2+ for DMPA and DPPC alone, 39 degrees C and 43 degrees C respectively. The results of this study suggest that when Ca2+ has only access to the outside of the vesicle membranes it may segregate the neutral and the acidic phospholipids into separate domains, both domains adopting their proper phase condition at the actual temperature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phase transition of dimyristoylphosphatidylglycerol bilayers induced by electric field pulses

The phase transition of dimyristoylphosphatidylglycerol (DMPG) bilayers has been studied by measurements of light scattering under high electric field pulses. Midpoints of phase transitions have been identified by a clear discontinuity of field induced relaxation amplitudes. We show that the phase transition of DMPG suspensions in monovalent salt is virtually independent of the electric field strength up to approx. 35 kV/cm. A shift of the lipid phase by electric field pulses has been observed, however, for DMPG suspensions in the presence of Ca2+ ions. DMPG suspensions exhibit a jump of the phase transition temperature from 17 degrees C at Ca/DMPG molar ratios r less than 1/7 to 32 degrees C at r greater than 1/7. Field pulses of 60 to 100 microseconds applied to DMPG suspensions with Ca2+ at r greater than 1/7 induce discontinuities of relaxation amplitudes in the temperature range 15 to 22 degrees C in addition to the 'standard' one at 32 degrees C, when the electric field strength is above 15 kV/cm. These results indicate that electric field pulses induce a transition from the phase formed at 'high' Ca(2+)- to the one formed at 'low' Ca(2+)-ion concentrations. Our results are consistent with a dissociation field effect on Ca(2+)-lipid complexes which drives the phase transition.     

Kinetics of the co-operative reaction of Helix pomatia hemocyanin with oxygen. Oxygen binding at low and intermediate oxygen saturations

The kinetics of oxygen binding of Helix pomatia α-hemocyanin has been studied at low and intermediate levels of ligand saturation, under conditions in which oxygen binding is highly co-operative. Temperature-jump relaxation spectra are heterogeneous and can be resolved into a slow and a fast phase. The latter is related to a bimolecular reaction, i.e. the binding of oxygen. At very low degrees of fractional saturation (<0.15) the reactant concentration-dependence of the faster relaxation rate allows the combination and dissociation rate constants of the low affinity or T-state to be estimated as 1.3 × 10⁶m^?1 s^?1 and 300 s^?1, respectively. A possible interpretation of the slow component in the relaxation spectrum is discussed.In stopped-flow experiments, after mixing deoxyhemocyanin with oxygen-containing buffer, most of the binding process to the T-state is lost in the dead time. The observed initial rates of oxygen binding are between 15 and 120 s^?1. depending on the oxygen concentration, and may reflect the rate of the allosteric change from a low to a high affinity state (T→R transition), which is slower than oxygen binding.Similarities and differences in the overall kinetic properties of small and giant respiratory proteins, i.e. hemoglobin and hemocyanin, are discussed.     

Effect of Confinement on Melting in Slit-Shaped Pores: Experimental and Simulation Study of Aniline in Activated Carbon Fibers

Abstract

We report both experimental and molecular simulation studies of the melting behavior of aniline confined within an activated carbon fiber having slit-shaped pores. Dielectric relaxation spectroscopy is used to determine the transition temperatures and also the dielectric relaxation times over the temperature range 240 to 340 K. For the confined system two transitions were observed, one at 298 K and a second transition at 324 K. The measured relaxation times indicate that the low temperature phase (below 298 K) is a crystalline or partially crystalline solid phase, while that above 324 K is a liquid-like phase; for the intermediate phase, in the range 298–324 K, the relaxation times are of the order 10^?5s, which is typical of a hexatic phase. The melting temperature of the confined system is well above that of bulk aniline, which is 267 K. The simulations are carried out using the Grand Canonical Monte Carlo method together with Landau free energy calculations, and phase transitions are located as state points where the grand free energies of two confined phases are equal. The nature of these phases is determined by analysis of in-plane pair positional and orientational correlation functions. The simulations also show two transitions. The first is a transition from a two-dimensional hexagonal crystal phase to a hexatic phase at 296 K; the second transition is from the hexatic to a liquid-like phase at 336 K. Confinement within the slit-shaped pores appears to stabilize the hexatic phase, which is the stable phase over a wider temperature range than for quasi-two-dimensional thin films.     

Sequence-dependent mechanics of single DNA molecules

Atomic force microscope-based single-molecule force spectroscopy was employed to measure sequence-dependent mechanical properties of DNA by stretching individual DNA double strands attached between a gold surface and an AFM tip. We discovered that in lambda-phage DNA the previously reported B-S transition, where 'S' represents an overstretched conformation, at 65 pN is followed by a nonequilibrium melting transition at 150 pN. During this transition the DNA is split into single strands that fully recombine upon relaxation. The sequence dependence was investigated in comparative studies with poly(dG-dC) and poly(dA-dT) DNA. Both the B-S and the melting transition occur at significantly lower forces in poly(dA-dT) compared to poly(dG-dC). We made use of the melting transition to prepare single poly(dG-dC) and poly(dA-dT) DNA strands that upon relaxation reannealed into hairpins as a result of their self-complementary sequence. The unzipping of these hairpins directly revealed the base pair-unbinding forces for G-C to be 20 +/- 3 pN and for A-T to be 9 +/- 3 pN.     

Ultrafast events in the folding of ferrocytochrome c

Laser flash photolysis and stopped-flow methods have been used to study the dynamic events in the micro- to millisecond time bin in the refolding of horse ferrocytochrome c in the full range of guanidine hydrochloride concentration at pH 12.8 (+/-0.1), 22 degrees C. Under the absolute refolding condition, the earliest relaxation time of the unfolded protein chain is less than 1 micros. The chain then undergoes diffusive dynamics-mediated contraction and expansion, in which intrapolypeptide ligands make transient contacts with the heme iron, giving rise to two distinct kinetic phases of approximately 0.4 and approximately 3 micros. Under moderate to absolute refolding conditions, the rates of these processes show little dependence on the denaturant concentration, indicating the absence of structural element in the incipient or the relaxed state. Chain expansion and contraction events continue until the polypeptide finds a stable and supportive transition state. The crossing of this transition barrier, which rate-limits the folding of alkaline ferrocytochrome c, is characterized by a stopped-flow measured time constant of approximately 3 ms in aqueous solvent. Observed kinetics thus implicate no submillisecond folding structure. The folding kinetics is effectively two state in which the unfolded polypeptide first relaxes to an unstructured chain and then crosses over a late rate-limiting barrier to achieve the native conformation. The experimentally observed rates as a function of guanidine hydrochloride concentration have been simulated by numerically calculated microscopic rates of a simple kinetic model that captures the essential features of folding.     

A stopped-flow kinetic study of the interaction of potential-sensitive oxonol dyes with lipid vesicles

The interaction of the dyes oxonol V and oxonol VI with unilamellar dioleoylphosphatidylcholine vesicles was investigated using a fluorescence stopped-flow technique. On mixing with the vesicles, both dyes exhibit an increase in their fluorescence, which occurs in two phases. According to the dependence of the reciprocal relaxation time on vesicle concentration, the rapid phase appears to be due to a second-order binding of the dye to the lipid membrane, which is very close to being diffusion-controlled. The slow phase is almost independent of vesicle concentration, and it is suggested that this may be due to a change in dye conformation or position within the membrane, possibly diffusion across the membrane to the internal monolayer. The response times of the dyes to a rapid jump in the membrane potential has also been investigated. Oxonol VI was found to respond to the potential change in less than 1 s, whereas oxonol required several minutes. This has been attributed to lower mobility of oxonol V within the lipid membrane.