31P NMR and X-ray diffraction study of the effect of photopolymerization on lipid polymorphism.

It was recently shown that oligolamellar vesicles of 3:1 mixtures of dioleoylphosphatidylethanolamine (DOPE) and the photopolymerizable lipid 1,2-bis[10-(2',4'-hexadienoyloxy)decanoyl]-sn-glycero-3-phosphocho line (SorbPC) are destabilized by polymerization of the SorbPC [Lamparski, H., Liman, U., Frankel, D.A., Barry, J.A., Ramaswami, V., Brown, M.F., & O'Brien, D.F. (1992) Biochemistry 31, 685-694]. The current work describes the polymorphic phase behavior of these mixtures in extended bilayers, as studied by 31P NMR spectroscopy and X-ray diffraction. In the NMR experiments, samples with varying degrees of polymerization were slowly raised in temperature, with spectra acquired every 2.5-10 degrees C. In the unpolymerized mixiture, and in those photopolymerized samples where the monomeric SorbPC was decreased by 33% and 51%, an isotropic signal grew progressively until no signal from the lamellar liquid-crystalline (L alpha) phase remained. In the highly polymerized sample with a 90% loss of monomeric SorbPC, less than 20% of the lipids underwent this transition. In none of the samples was an inverted hexagonal phase (HII) observed, under conditions of slow heating to almost 100 degrees C. The X-ray diffraction studies indicated that samples which exhibit the isotropic NMR signal corresponded to a structure exhibiting no well-defined crystalline order, which upon thermal cycling became an inverted cubic phase belonging to either the Pn3m or Pn3 space groups. The temperature of the transition to the cubic precursor decreased as the extent of polymerization increased, demonstrating that photopolymerization of these lipid bilayers can significantly alter the composition and thermotropic phase behavior of the mixture.

Kinetics of the lamellar-inverse hexagonal phase transition determined by time-resolved X-ray diffraction.

The kinetics of the lamellar (L alpha)-inverse hexagonal (HII) phase transition in diacylphosphatidylethanolamine (PE)--water systems were probed with time-resolved X-ray diffraction. Transition kinetics in the fast time regime (approximately 100 ms) were studied by initiating large temperature jumps (up to 30 degrees C) with a 50-ms electrical current pulse passed through a lipid-salt water dispersion, resulting in ohmic heating of the sample. Diffraction with a time resolution to 10 ms was acquired at the National Synchrotron Light Source. The time constant for the phase transition for 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) was on the order of 100 ms for the largest temperature jumps recorded. Faster transition behavior was found for a 1,2-dielaidoyl-sn-glycero-3-PE mixture. The HII lattice parameters for both systems were seen to swell from an initial value commensurate with the lamellar lattice to the final equilibrium value. The rate of swelling was seen to be independent of the magnitude of the temperature jump. For small temperature jumps (less than 10 degrees C), the phase transition kinetics slow dramatically, and transition studies can readily be performed on a conventional rotating anode X-ray source. At 4 degrees C, a DOPE sample was observed to slowly convert to the hexagonal phase over the course of a week, with the decay in the lamellar intensity fitting a power law behavior over four decades of time. This power law behavior is shown to have interesting consequences to the determination of the phase transition temperature of lipid-water dispersions by conventional methods such as calorimetry.

Nonbilayer phases of membrane lipids.

Numerous liquid crystalline biomembrane lipids are known to exhibit non-lamellar phases characterized by curvature of their component lipid monolayers. An understanding of the phase stability of these systems begins with analysis of the energy of bending the monolayers, the interactions which lead to the bending energy, and the geometrical constraints which lead to competing energy terms which arise when the monolayers are bent and packed onto lattices with different structures. Diffraction and other techniques suitable for probing lipid phase structure are described. A phenomenological model is reviewed which successfully explains many of the qualitative features of lipid mesomorphic phase behavior. A key result of this model is that lipid bilayer compositions which are close to the non-lamellar phase boundaries of their phase diagrams are characterized by a frustrated elastic stress which may modulate the activity of imbedded membrane proteins and which may provide a rationale for the prevalence of non-lamellar-tending lipid species in biomembrane bilayers. Areas in need of future research are discussed.

Is the mechanism of general anesthesia related to lipid membrane spontaneous curvature?

Lipid bilayers in biomembranes may exist in a state of elastic curvature stress which may couple to the conformation of integral membrane proteins in a logical, and energetically significant, fashion. Many biomembranes contain sufficiently large fractions of nonlamellar-prone lipids to have monolayers under substantial curvature stress. Although very few experiments have been performed that can be used to correlate protein activity with curvature stress, the literature does contain a small number of studies that indicate that some protein function is nonspecifically modulated by the amounts of nonlamellar-prone lipid in the imbedding bilayers. The spontaneous curvature, is altered by the presence of anesthetics in physiologically relevant concentrations. This leads us to suggest that anesthetic action may be coupled to protein function via alteration of the tensions leading to the spontaneous curvature of biomembrane layers. The spontaneous curvature is also sufficiently sensitive to pressure that a mechanism for the pressure reversal of anesthesia follows if the effects of pressure are to counter changes in membrane lateral tension induced by anesthetics. It is emphasized that many more experimental data must be acquired to determine whether the ideas presented in this paper have validity. In particular, there is a need for data on the effects of different anesthetics and pressure on the spontaneous curvature and, more generally, lipid monolayer lateral tensions. Most importantly, experiments must be performed to investigate whether protein function correlates with these quantities.

Unusual lipid structures selectively reduce the toxicity of amphotericin B.

Ribbon-like structures result when amphotericin B interacts with lipid in an aqueous environment. At high ratios of amphotericin to lipid these structures, which are lipid-stabilized amphotericin aggregates, become prevalent resulting in a dramatic attenuation of amphotericin-mediated mammalian cell, but not fungal cell, toxicity. Studies utilizing freeze-etch electron microscopy, differential scanning calorimetry, 31P NMR, x-ray diffraction, and optical spectroscopy revealed that this toxicity attenuation is related to the macromolecular structure of the complexes in a definable fashion. It is likely that amphotericin in this specific form will have a much improved therapeutic utility.

Observation of inverted cubic phase in hydrated dioleoylphosphatidylethanolamine membranes.

We report the observation of an inverted cubic phase in aqueous dispersions of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) by small-angle X-ray diffraction. DOPE is a paradigm in the study of nonlamellar phases in biological systems: it exhibits a well-known phase transition from the lamellar (L alpha) to the inverted hexagonal phase (HII) as the temperature is raised. The transition is observed to occur rapidly when a DOPE dispersion is heated from 2 degrees C, where the L alpha phase is stable, to 15 degrees C, where the HII phase is stable. We report on the induction of a crystallographically well-defined cubic lattice that is slowly formed when the lipid dispersion is rapidly cycled between -5 and 15 degrees C hundreds of times. Once formed, the cubic lattice is stable at 4 degrees C for several weeks and exhibits the same remarkable metastability that characterizes other cubic phases in lipid-water systems. X-ray diffraction indicates that the cubic lattice is most consistent with either the Pn3m or Pn3 space group. Tests of lipid purity after induction of the cubic indicate the lipid is at least 98% pure. The cubic lattice can be destroyed and the system reset by cycling the specimen several times between -30 and 2 degrees C. The kinetics of the formation of the cubic are dependent on the thermal history of the sample, overall water concentration, and the extreme temperatures of the cycle.(ABSTRACT TRUNCATED AT 250 WORDS)

Recombination of carbon monoxide to ferrous horseradish peroxidase types A and C.

The recombination of carbon monoxide to isoenzymes A2 and C of horseradish peroxidase (HRP) was studied as a function of temperature (2 to 320 K) and pH (5 to 8.3) with flash photolysis and infrared difference absorption. At low temperatures three geminate recombination processes are observed. One of these internal processes, denoted by I*, is exponential in time with a rate coefficient that deviates strongly from an Arrhenius behavior below 100 K, implying phonon-assisted tunneling. The two other processes, denoted by I, are non-exponential in time and related to different carbonyl isomers, as shown by the infrared difference spectra. The existence of three internal processes indicates that HRP differs considerably from myoglobin where only one internal process, I, is seen. Moreover, the internal processes in HRP are faster than process I in myoglobin. At 300 K, only one recombination process from the solvent is observed and it is very slow (lambda s approximately 1 s-1 at 1 atm CO (1 atm = 101,325 Pa)), much slower than the corresponding association process in myoglobin. Since process I is fast, but binding from the solvent is slow, the barrier at the heme cannot be responsible for the small association rate. The infrared absorption difference spectra of the amide I/II bands indicate that photolysis and recombination trigger a two-step structural change. The slow recombination rate at 300 K can thus be explained by the large Gibbs energy of the conformational transition that is necessary to let CO move into the heme pocket. The partition coefficient for the CO in the heme pocket and the solvent is extremely small, while bond formation with the heme iron occurs in less than 100 nanoseconds.

Ligand binding to heme proteins: relevance of low-temperature data.

Binding of carbon monoxide to the beta chain of adult human hemoglobin has been studied by flash photolysis over the time range from about 100 ps to seconds and the temperature range from 40 to 300 K. Below about 180 K, binding occurs directly from the pocket (process I) and is nonexponential in time. Above about 180 K, some carbon monoxide molecules escape from the pocket into the protein matrix. Above about 240 K, escape into the solvent becomes measurable. Process I can be observed up to 300 K. The low-temperature data extrapolate smoothly to 300 K, proving that the results obtained below 180 K provide functionally relevant information. The experiments show again that the binding process even at physiological temperatures is regulated by the final binding step at the heme iron and that measurements at high temperatures are not sufficient to fully understand the association process.

Protein states and proteinquakes.

After photodissociation of carbon monoxide bound to myoglobin, the protein relaxes to the deoxy equilibrium structure in a quake-like motion. Investigation of the proteinquake and of related intramolecular equilibrium motions shows that states and motions have a hierarchical glass-like structure.

Dynamics of dioxygen and carbon monoxide binding to soybean leghemoglobin.

The association of dioxygen and carbon monoxide to soybean leghemoglobin (Lb) has been studied by laser flash photolysis at temperatures from 10 to 320 K and times from 50 ns to 100 s. Infrared spectra of the bound and the photodissociated state were investigated between 10 and 20 K. The general features of the binding process in leghemoglobin are similar to the ones found in myoglobin. Below about 200 K, the photodissociated ligands stay in the heme pocket and rebinding is not exponential in time, implying a distributed enthalpy barrier between pocket and heme. At around 300 K, ligands migrate from the solvent through the protein to the heme pocket, and a steady state is set up between the ligands in the solvent and in the heme pocket. The association rate, lambda on, is mainly controlled by the final binding step at the heme, the bond formation with the heme iron. Differences between Lb and other heme proteins show up in the details of the various steps. The faster association rate in Lb compared to sperm whale myoglobin (Mb) is due to a faster bond formation. The migration from the solvent to the heme pocket is much faster in Lb than in Mb. The low-temperature binding (B----A) and the infrared spectra of CO in the bound state A and the photodissociated state B are essentially solvent-independent in Mb, but depend strongly on solvent in Lb. These features can be correlated with the x-ray structure.

Comparison of the magnetic properties of deoxy- and photodissociated myoglobin.

The magnetic susceptibility of photodissociated carbon monoxy myoglobin has been measured over the temperature range from 1.7 to 25 K at 10 and 50 kG with a superconducting susceptometer. The spin and the crystal field parameters of the iron ion were extracted by a spin Hamiltonian approach. Under equivalent conditions the magnetic susceptibility of deoxy myoglobin was measured. In both experiments the CO-bound protein was used as a diamagnetic reference. Above about 5 K the metastable photolysed state and the equilibrium deoxy form of myoglobin are magnetically indistinguishable and can be fitted with S = 2 and g = 2. The transition from spin 0 to spin 2 and the conformational changes known to accompany the electronic change thus also occur after photolysis at low temperature. At temperatures below 5 K, differences become apparent, indicating a somewhat smaller zero-field splitting in the photoproduct as compared to the ligand-free state at equilibrium. In qualitative agreement with observations made by other techniques, the data imply that even at 1.7 K substantial structural relaxation occurs in the heme region of myoglobin after photodissociation. The results are important for the interpretation of the ligand binding kinetics after flash photolysis at low temperature and contribute to the understanding of the relationship between electronic structure and function in heme proteins.

Control and pH dependence of ligand binding to heme proteins.

The recombination after flash photolysis of dioxygen and carbon monoxide with sperm whale myoglobin (Mb), and separated beta chains of human hemoglobin (beta A) and hemoglobin Zürich (beta ZH), has been studied as a function of pH and temperature from 300 to 60 K. At physiological temperatures, a preequilibrium is established between the ligand molecules in the solvent and in the heme pocket. The ligand in the pocket binds to the heme iron by overcoming a barrier at the heme. The association rate is controlled by this final binding step. The association rate of CO to Mb and beta A is modulated by a single titratable group with a pK at 300 K of 5.7. The binding of CO to beta ZH, in which the distal histidine is replaced by arginine, does not depend on pH. Oxygen recombination is independent of pH in all three proteins. Comparison of the binding of CO at 300 K and at low temperatures shows that pH does not affect the preequilibrium but changes the barrier height at the heme. The pH dependence and the difference between O2 and CO binding can be explained by a charge-dipole interaction between the distal histidine and CO.

Infrared spectroscopy of photodissociated carboxymyoglobin at low temperatures.

We have studied the infrared spectra of the bound and photodissociated states of Mb-12CO and Mb-13CO from 5.2 to 300 K. The absorbance peaks seen between 1800 and 2200 cm-1 correspond to CO stretching vibrations. In the bound state of Mb-12CO, the known lines A0 at 1969, A1 at 1945, and A2 at 1927 cm-1, have center frequencies, widths, and absorbances that are independent of temperature between 5.2 and 160 K. Above 160 K, A2 gradually shifts to 1933 cm-1. The low-temperature photodissociated state (Mb) shows three lines (B0, B1, B2) at 2144, 2131, and 2119 cm-1 for 12CO. The absorbances of the three lines depend on temperature. B0 is tentatively assigned to free CO in the heme pocket and B1 and B2, to CO weakly bound to the heme or heme pocket wall. The data are consistent with a model in which photodissociation of MbCO leads to B1 and B2. B2 decays thermally to B1 above 13 K; rebinding to A occurs from B1. The barriers between B2 and B1 and between B1 and A are described by activation enthalpy spectra. Heme and the central metal atom in state Mb have near-infrared, EPR, and Mössbauer spectra that differ slightly from those of deoxyMb. The observation of essentially free CO in state B implies that the difference between Mb and deoxyMb is not due to an interaction of the flashed-off ligand with the protein but is caused by an incomplete relaxation of the protein structure at low temperatures.