Phase transition kinetics of lipid bilayer membranes studied by time-resolved pressure perturbation calorimetry

The relaxation kinetics of aqueous lipid dispersions after a pressure jump (p-jump) was investigated using time-resolved pressure perturbation calorimetry (PPC). Analysis of the calorimetric response curves by deconvolution with the instrumental response function gives information about slow processes connected with the lipid phase transition. The lipid transition from the gel to the liquid-crystalline state was found to be a multi-step process with relaxation constants in the seconds range resolvable by time-resolved PPC and faster processes with relaxation times shorter than ca. 5 s that could not be resolved by the instrument. The faster processes comprise ca. 50% of the total heat uptake at the transition midpoint. This is the first calorimetric measurement showing the multi-step nature of the transition. The results are in good agreement with data obtained with other detection methods and with molecular modelling experiments describing the transition as a multi-step process with nucleation and growth steps.     

A kinetic study of the subunit dissociation and reassembly of rabbit muscle phosphofructokinase.

The kinetics of dissociation and reassembly of rabbit skeletal muscle phosphofructokinase has been studied using fluorescence, stopped-flow fluorescence and enzyme activity measurements. The dissociation of the fully active tetramer in 0.8 M guanidine hydrochloride (0.1 M potassium phosphate, pH 8.0) occurs in three kinetic phases as measured by changes in the protein fluorescence emission intensity: dissociation of tetramer to dimer with a relaxation time of a few milliseconds; dissociation of dimer to monomer with a relaxation time of a few seconds; and a conformational change of the monomer with a relaxation time of a few minutes. All three phases exhibit first-order kinetics; ATP (0.05 mM) retards the second step but does not influence the rate of the other two processes. The rate of the second process increases with decreasing temperature; this may be due to the involvement of hydrophobic interactions in the stabilization of the dimeric enzyme. A further unfolding of the monomer polypeptide chain occurs at higher guanidine concentrations, and the relaxation time associated with this process was found to be 83 ms in 2.5 M guanidine, 0.1 M potassium phosphate (pH 8.0) at 23 degrees C. The phosphofructokinase monomers were reassembled from 0.8 M guanidine chloride by 1:10 dilution of the guanidine hydrochloride concentration and yielded a protein with 70-94% of the original activity, depending on the protein concentration. The reactivation process follows second-order kinetics; ATP (5 mM) increases the rate of reactivation without altering the reaction order, while fructose 6-phosphate does not influence the rate of reaction. The rate-determining step is probably the association of monomers to form the dimer.     

The kinetics of binding of o-methyl red to the outer surface of unilamellar spherical phospholipid vesicles

The kinetics of adsorption of the proton carrier o-methyl red to the surface of unilamellar spherical phospholipid vesicles have been investigated by means of the temperature-jump relaxation technique with absorbance detection. Single-exponential relaxation curves were observed with time constants in the range 30-130 microseconds. o-Methyl red binds in both its anionic form A- and protonated form AH. Adsorption-desorption of the two species is coupled by two fast protolytic reactions, occurring in the aqueous bulk phase and in the surface region of the membrane. The rate constants for adsorption and desorption of the two species were obtained from the dependences of the relaxation time on lipid concentration at different pH values. The analysis yielded apparent adsorption rate constants of kasAH = 9.8 X 10(6) M-1 s-1 and kasA = 1.3 X 10(6) M-1 s-1 (expressed in terms of monomeric lipid), and kasAH = 1.2 X 10(11) M-1 s-1 and kasA = 1.6 X 10(10) M-1 s-1 (expressed in terms of vesicle concentration). From the order of these rate constants it is concluded that adsorption of both species is actually diffusion-controlled. The peculiar pH dependence of the relaxation time is a consequence of the protolytic reaction in the surface region of the membrane. Its implication for the kinetics of adsorption-desorption processes are discussed.     

Kinetics of the subtransition of asymmetrically substituted phosphatidylcholines

The thermodynamics and kinetics of the subtransition L epsilon----P beta of sonicated unilamellar vesicles of 1-myristoyl-2-stearoylphosphatidylcholine (1M-2S-PC) and of 1-stearoyl-2-myristoylphosphatidylcholine (1S-2M-PC) were studied by equilibrium cooling curves and temperature-jump relaxation spectrometry with an anthracenophane cryptand as a mobile fluorescent probe. The unilamellar vesicles exhibit the midpoint temperature TsII of the subtransition about 10 degrees C below the respective main transition. The kinetics of the subtransition in the time range between 10(-4) and 10(3) s is characterized by a cooperative relaxation process in the range of milliseconds and a further noncooperative process in the range of seconds. The slow process is assigned to the rearrangement of lattice defects. The fast process is evaluated in terms of a cyclic reaction scheme that consists of two pathways for the biomolecular association of probe and vesicle coupled with the conformational change of the lipid matrix during the subtransition. The analysis reveals that the fast process comprises the nucleation and growth of cluster. The cooperative lattice transformation of the subtransition follows a first-order rate law. The rate constants at TsII are 70 s-1 for 1S-2M-PC and 170 s-1 for 1M-2S-PC. Since the plots of the relaxation time vs. the degree of transition are in accordance with the predictions of the linear Ising model, it is concluded that clusters are propagated anisotropically in a linear fashion; e.g., fluidlike P beta conformations grow along the ripple.     

Multiple geminate ligand recombinations in human hemoglobin

The geminate ligand recombination reactions of photolyzed carbonmonoxyhemoglobin were studied in a nanosecond double-excitation-pulse time-resolved absorption experiment. The second laser pulse, delayed by intervals as long as 400 ns after the first, provided a measure of the geminate kinetics by rephotolyzing ligands that have recombined during the delay time. The peak-to-trough magnitude of the Soret band photolysis difference spectrum measured as a function of the delay between excitation pulses showed that the room temperature kinetics of geminate recombination in adult human hemoglobin are best described by two exponential processes, with lifetimes of 36 and 162 ns. The relative amounts of bimolecular recombination to T- and R-state hemoglobins and the temperature dependence of the submicrosecond kinetics between 283 and 323 K are also consistent with biexponential kinetics for geminate recombination. These results are discussed in terms of two models: geminate recombination kinetics modulated by concurrent protein relaxation and heterogeneous kinetics arising from alpha and beta chain differences.     

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.     

Voltage-induced thickness changes of lipid bilayer membranes and the effect of an electrin field on gramicidin A channel formation.

The thickness changes of black lipid membranes of different composition after a voltage jump were investigated. In a second series of electrical relaxation experiments the kinetics of channel formation by gramicidin A were measured. The time course of the membrane current was compared with the time course of the thickness change of the membranes. We found that the time course of the current as a consequence of channel formation by gramicidin A did not correlate with the thickness change of the lipid membranes. A possible direct influence of the electric field is discussed.     

Determination of oxygen emission and uptake in leaves by pulsed, time resolved photoacoustics

Pulsed, time resolved photoacoustics has sufficient sensitivity to determine oxygen emission and uptake by single turnover flashes to leaves. The advantage over previous methodologies is that when combined with single turnover flashes the kinetics of the thermal and the gas signals can be resolved to 0.1 millisecond and separated. The S-state oscillations of oxygen formation are readily observed. The gas signal from common spongy leaves such as spinach (Spinacia sp.), Japanese andromeda (Pieris japonica), mock orange (Philadelphus coronarius) and viburnum (Viburnum tomentosum), after correction for instrumental rise time, show a lag of only 1 millisecond and a rise time of 5 milliseconds in the formation of oxygen. Thus a recent proposal that the formation of oxygen requires over 100 milliseconds cannot be true for choroplasts in vivo. The rapid emission is correlated with structure of the leaf. At low light flash energies a rapid gas uptake is observed. The uptake has slightly slower kinetics than oxygen evolution, and its magnitude increases with damage to the leaf. The pulse methodology shows that the uptake begins with the very first flash after dark adaption, and allows the detection of a positive signal (oxygen) on the third flash. These observations, the long wavelength of excitation (695 nanometers) and the magnitude of the signal support the contention that the gas uptake is oxygen reduction by electrons from photosystem I. These results show that important physiological aspects of a leaf can be studied by pulsed, time resolved photoacoustics.     

Temperature-jump experiments on thin lipid membranes in the presence of valinomycin

Summary Temperature jump relaxation experiments on planar lipid membranes in the presence of valinomycin were performed using the absorption of a strong light flash as an energy source for the generation of the T-jump. The relaxation of the current carried by valinomycin/Rb⁺ complexes was measured. The results were interpreted on the basis of a transport model which was also analyzed by voltage jump relaxation experiments. The study shows that the application of the T-jump technique provides valuable information about transport kinetics as well as the dynamics of the membrane structure. At the given experimental conditions the relaxation of the current is believed to reflect a temperature-dependent transition of the membrane to a new conformational state of lower order. The relaxation could be resolved with the present technique only at low temperatures and for membranes of high microviscosity.     

Kinetics and stoichiometry of light-induced proton release and uptake from purple membrane fragments, Halobacterium halobium cell envelopes, and phospholipid vesicles containing oriented purple membrane

We have used flash spectroscopy and pH indicator dyes to measure the kinetics and stoichiometry of light-induced proton release and uptake by purple membrane in aqueous suspension, in cell envelope vesicles and in lipid vesicles. The preferential orientation of bacteriorhodopsin in opposite directions in the envelope and lipid vesicles allows us to show that uptake of protons occurs on the cytoplasmic side of the purple membrane and release on the exterior side.

In suspensions of isolated purple membrane, approximately one proton per cycling bacteriorhodopsin molecule appears transiently in the aqueous phase with a half-rise time of 0.8 ms and a half-decay time of 5.4 ms at 21 °C.

In cell envelope preparations which consist of vesicles with a preferential orientation of purple membrane, as in whole cells, and which pump protons out, the acidification of the medium has a half-rise time of less than 1.0 ms, which partially relaxes in approx. 10 ms and fully relaxes after many seconds.

Phospholipid vesicles, which contain bacteriorhodopsin preferentially oriented in the opposite direction and pump protons in, show an alkalinization of the medium with a time constant of approximately 10 ms, preceded by a much smaller and faster acidification. The alkalinization relaxes over many seconds.

The initial fast acidification in the lipid vesicles and the fast relaxation in the envelope vesicles are accounted for by the misoriented fractions of bacteriorhodopsin. The time constants of the main effects, acidification in the envelopes and alkalinization in the lipid vesicles correlate with the time constants for the release and uptake of protons in the isolated purple membrane, and therefore show that these must occur on the outer and inner surface respectively. The slow relaxation processes in the time range of several seconds must be attributed to the passive back diffusion of protons through the vesicle membrane.     

The use of pressure-jump relaxation kinetics to study protein folding landscapes

Pressure-jump induced relaxation kinetics can be used to study both protein unfolding and refolding. These processes can be initiated by upward and downward pressure-jumps of amplitudes of a few 10 to 100 MPa, with a dead-time on the order of milliseconds. In many cases, the relaxation times can be easily determined when the pressure cell is connected to a spectroscopic detection device, such as a spectrofluorimeter. Adiabatic heating or cooling can be limited by small pressure-jump amplitudes and a special design of the sample cell. Here, we discuss the application of this method to four proteins: 33-kDa and 23-kDa proteins from photo-system II, a variant of the green fluorescent protein, and a fluorescent variant of ribonuclease A. The thermodynamically predicted equivalency of upward and downward pressure-jump induced protein relaxation kinetics for typical two-state folders was observed for the 33-kDa protein, only. In contrast, the three other proteins showed significantly different kinetics for pressure-jumps in opposite directions. These results cannot be explained by sequential reaction schemes. Instead, they are in line with a more complex free energy landscape involving multiple pathways.     

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.     

Valinomycin-mediated ion transport through neutral lipid membranes: Influence of hydrocarbon chain length and temperature

Summary Stationary electrical conductance experiments together with nonstationary relaxation experiments allow a quantitative determination of rate constants describing carrier-mediated ion transport. Valinomycin-induced ion transport across neutral lipid membranes was studied. The dependence of the transport parameters on the chain length of the lipid molecules, on the kind of alkali ion, and on the temperature was determined. The relaxation time the current following a voltage jump shows a marked increase with decreasing temperature or with increasing chain length of the lipid molecules. This variation of is interpreted on the basis of a varying membrane fluidity. It is shown that under favorable circumstances the equilibrium constant of complex formation in the aqueous phase may be obtained from membrane experiments. Furthermore, the kinetics of exchange of valinomycin between membrane and water was studied. We found a marked influence of the totus surrounding the black film on the kinetics as well as on the total amount of valinomycin molecules in the membrane. The problem of location of the free carrier molecules inside the membrane is discussed.     

Spin equilibrium relaxation kinetics of cytochrome P450 LM2

The relaxation kinetics of the temperature-dependent spin equilibrium of cytochrome P450 LM2 has been resolved by means of a laser temperature-jump device. The Nd laser system in the Q-switched mode delivers 3 J/25 ns pulses which were Raman shifted to 1.89 μm by use of a high pressure H₂-gas cell. The LM2 equilibrium relaxation proceeds in the ns-range, the rate constants of the on- and off-reactions amount to about 10⁶ s^?1 in correspondence with respective processes of other hemoproteins and protein-free model compounds. Benzphetamine increases the on-rate constant (low-spin → high-spin) about 5 fold, the off-process is nearly unchanged.     

Ligand-Induced Protein Responses and Mechanical Signal Propagation Described by Linear Response Theories

In this study, a general linear response theory (LRT) is formulated to describe time-dependent and -independent protein conformational changes upon CO binding with myoglobin. Using the theory, we are able to monitor protein relaxation in two stages. The slower relaxation is found to occur from 4.4 to 81.2 picoseconds and the time constants characterized for a couple of aromatic residues agree with those observed by UV Resonance Raman (UVRR) spectrometry and time resolved x-ray crystallography. The faster “early responses”, triggered as early as 400 femtoseconds, can be best described by the theory when impulse forces are used. The newly formulated theory describes the mechanical propagation following ligand-binding as a function of time, space and types of the perturbation forces. The “disseminators”, defined as the residues that propagate signals throughout the molecule the fastest among all the residues in protein when perturbed, are found evolutionarily conserved and the mutations of which have been shown to largely change the CO rebinding kinetics in myoglobin.     

Model calculations on the kinetics of oligonucleotide double helix coil transitions. Evidence for a fast chain sliding reaction

Relaxation data obtained previously for the double helix coil transition of oligoriboadenylates and oligoribouridylates are compared to the results of numerical calculations according to various models. In these models the helix coil transition is described by individual rate constants for the first steps of helix formation, whereas the rate constants of the following steps of helix chain growth are assumed to be uniform. The existence of various helix intermediates containing the same number of base pairs is accounted for by statistical factors. First a quasistationary treatment of a zipper model is used for an analysis of the influence of various model parameters. Then relaxation spectra are calculated including helix coil intermediates explicitly without any assumption of quasistationarity. The relaxation spectrum calculated for any chain length N comprises N—1 fast processes with time constants in the range of 0.1 to 0.5 μs and one slow process with a time constant τ depending upon the nucleotide concentration (τ is usually in the ms time range). The fast processes are associated mainly with the unzippering at helix ends and are usually characterized by relatively small amplitudes, whereas the slow process represents the overall helix coil transition usually characterized by a very large amplitude.Consideration of staggered helix series (where the different helix scries are coupled to each other by the single stranded state) leads to a spectrum of slow relaxation processes with one separate relaxation process for each helix series. It is shown that this “non-sliding” staggering zipper model is not consistent with the experimental results. The measured relaxation curves can be represented by single exponentials for nucleotide chain lengths 8 to 11 (within experimental accuracy). This is also true for conditions where several, clearly separated time constants should be expected according to the theoretical model. The experimental data suggest the existence of a direct coupling between different series of staggered helices by a chain sliding mechanism with a time constant < 1ms. Chain sliding may be explained by diffusion of helix defects along the double helix such as diffusion of small loops. A simple model calculation for the diffusion of a bulge loop assuming quasistationarity suggests a sliding time constant around 100 μs for a helix comprising 10 base pairs.Finally some thermodynamic and kinetic parameters are evaluated according to the “sliding” staggering zipper model: The negative activation enthalpy observed for helix recombination can he described using a series of nucleation parameters indicating reduced stability constants for the first three base pairs. Nucleation may usually be achieved with the formation of the third or fourth base pair depending upon the magnitude of the chain growth parameter. The rate constant of helix chain growth is around 10⁶ s^?1 at 0.05 M [Na⁺] and increases to about 4 × 10⁶ s^?1 at 0.17 M [Na⁺].     

The effect of water on the rate of conformational change in protein allostery

The influence of solvation on the rate of quaternary structural change is investigated in human hemoglobin, an allosteric protein in which reduced water activity destabilizes the R state relative to T. Nanosecond absorption spectroscopy of the heme Soret band was used to monitor protein relaxation after photodissociation of aqueous HbCO complex under osmotic stress induced by the nonbinding cosolute poly(ethylene glycol) (PEG). Photolysis data were analyzed globally for six exponential time constants and amplitudes as a function of osmotic stress and viscosity. Increases in time constants associated with geminate rebinding, tertiary relaxation, and quaternary relaxation were observed in the presence of PEG, along with a decrease in the fraction of hemes rebinding CO with the slow rate constant characteristic of the T state. An analysis of these results along with those obtained by others for small cosolutes showed that both osmotic stress and solvent viscosity are important determinants of the microscopic R --> T rate constant. The size and direction of the osmotic stress effect suggests that at least nine additional water molecules are required to solvate the allosteric transition state relative to the R-state hydration, implying that the transition state has a greater solvent-exposed area than either end state.     

Pressure-jump-induced kinetics reveals a hydration dependent folding/unfolding mechanism of ribonuclease A

Pressure-jump (p-jump)-induced relaxation kinetics was used to explore the energy landscape of protein folding/unfolding of Y115W, a fluorescent variant of ribonuclease A. Pressure-jumps of 40 MPa amplitude (5 ms dead-time) were conducted both to higher (unfolding) and to lower (folding) pressure, in the range from 100 to 500 MPa, between 30 and 50 degrees C. Significant deviations from the expected symmetrical protein relaxation kinetics were observed. Whereas downward p-jumps resulted always in single exponential kinetics, the kinetics induced by upward p-jumps were biphasic in the low pressure range and monophasic at higher pressures. The relative amplitude of the slow phase decreased as a function of both pressure and temperature. At 50 degrees C, only the fast phase remained. These results can be interpreted within the framework of a two-dimensional energy surface containing a pressure- and temperature-dependent barrier between two unfolded states differing in the isomeric state of the Asn-113-Pro-114 bond. Analysis of the activation volume of the fast kinetic phase revealed a temperature-dependent shift of the unfolding transition state to a larger volume. The observed compensation of this effect by glycerol offers an explanation for its protein stabilizing effect.     

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.     

The activation energy for insertion of transmembrane alpha-helices is dependent on membrane composition

The physical mechanisms that govern the folding and assembly of integral membrane proteins are poorly understood. It appears that certain properties of the lipid bilayer affect membrane protein folding in vitro, either by modulating helix insertion or packing. In order to begin to understand the origin of this effect, we investigate the effect of lipid forces on the insertion of a transmembrane alpha-helix using a water-soluble, alanine-based peptide, KKAAAIAAAAAIAAWAAIAAAKKKK-amide. This peptide binds to preformed 1,2-dioleoyl-l-alpha-phosphatidylcholine (DOPC) vesicles at neutral pH, but spontaneous transmembrane helix insertion directly from the aqueous phase only occurs at high pH when the Lys residues are de-protonated. These results suggest that the translocation of charge is a major determinant of the activation energy for insertion. Time-resolved measurements of the insertion process at high pH indicate biphasic kinetics with time constants of ca 30 and 430 seconds. The slower phase seems to correlate with formation of a predominantly transmembrane alpha-helical conformation, as determined from the transfer of the tryptophan residue to the hydrocarbon region of the membrane. Temperature-dependent measurements showed that insertion can proceed only above a certain threshold temperature and that the Arrhenius activation energy is of the order of 90 kJ mol(-1). The kinetics, threshold temperature and the activation energy change with the mole fraction of 1,2-dioleoyl-l-alpha-phosphatidylethanolamine (DOPE) introduced into the DOPC membrane. The activation energy increases with increasing DOPE content, which could reflect the fact that this lipid drives the bilayer towards a non-bilayer transition and increases the lateral pressure in the lipid chain region. This suggests that folding events involving the insertion of helical segments across the bilayer can be controlled by lipid forces.