Microviscosity parameters and protein mobility in biological membranes.

A fluorescence polarization technique with 1,6-diphenyl 1,3,5-hexatriene as a probe were employed to determine the microviscosity, n, in liposomes and biological membranes of different cholesterol to phospholipid mol ratio. From the temperature profile of n the flow activation energy, deltaE, and the unit flow volume, V, were derived. The increase of cholesterol/phospholipid ratio in liposomes is followed by a marked increase in n and a decrease in both deltaE and V. Liposomes of the same phospholipid composition as human erythrocyte membranes display in the extreme cases of cholesterol/phospholipid ratios 0 and 1.4 the values of n(25 degrees C) = 1.8 and 9.1 P, and deltaE = 15.0 and 6.5 kcal/mol, respectively. For most membranes studied the fluorescence polarization characteristics and the corresponding n values are similar to those obtained with these liposomes when the cholesterol/phospholipid level of the liposomes and the membranes were the same. However, unlike in liposomes deltaE of all membranes is in the narrow range of 6.5-8.5 kcal/mol, regardless of its cholesterol/phospholipid level. It is plausible that this is a general characteristic of biological membranes which originates from the vertical movement of membrane proteins to an equilibrium position which maintains constant deltaE and V values. This type of movement should affect the interrelation between lipid fluidity and protein mobility. Lipid microviscosity and the degree of rotational mobility of concanavalin A receptor sites in cell membranes were therefore determined. The examined cells were normal and malignant fibroblasts, as an example of cells that form solid tumours in vivo, and normal and malignant lymphocytes, as an example of cells that form ascites tumours in vivo. In both cell systems, opposite correlations between the lipid fluidity and the mobility of concanavalin A receptors were observed. In the fibroblasts the malignant cells possess a lower lipid fluidity but a higher receptor mobility, whereas in the lymphocytes the malignant cells possess a higher lipid fluidity but a lower receptor mobility. Thus, in these cell systems the degree of rotational mobility of concanavalin A receptors increases upon decreasing the lipid fluidity and decreases upon increasing the fluidity of the lipid core. This dynamic feature is in line with the above proposal according to which the concanavalin A receptor sites become more exposed to the aqueous surrounding upon increasing the microviscosity of the lipid layer and vice versa.     

Metal-activated histidine carbon donor hydrogen bonds contribute to metalloprotein folding and function

Carbon donor hydrogen bonds are typically weak interactions that contribute less than 2 kcal/mol, and provide only modest stabilization in proteins. One exception is the class of hydrogen bonds donated by heterocyclic side chain carbons. Histidine is capable of particularly strong interactions through the Cε¹ and Cδ² carbons when the imidazole is protonated or bound to metal. Given the frequent occurrence of metal-bound histidines in metalloproteins, we characterized the energies of these interactions through DFT calculations on model compounds. Imidazole-water hydrogen bonding could vary from −11.0 to −17.0 kcal/mol, depending on the metal identity and oxidation state. A geometric search of metalloprotein structures in the PDB identified a number of candidate His C-H···O hydrogen bonds which may be important for folding or function. DFT calculations on model complexes of superoxide reductase show a carbon donor hydrogen bond positioning a water molecule above the active site.     

ESR studies on the membrane properties of a moderately halophilic bacterium. I. Physical properties of lipid bilayers in whole cells

The lipid membrane properties of a moderately halophilic gram-negative bacterium, Pseudomonas halosaccharolytica ATCC 29423, were studied by the use of stearate spin labels, 5NS, 12NS, and 16NS, changing the temperature of ESR measurement from 15 to 50 degrees C. The order parameter and the rotational correlation time of the spin labels incorporated into intact cell membranes of this bacterium grown at various temperatures in media containing different NaCl concentrations were calculated. The activation energy of rotational microviscosity was obtained from Andrade plots. At low growth temperature and low NaCl concentration in the medium, extractable lipids of this bacterium contained comparatively large amounts of unsaturated fatty acids, but as the growth temperature and NaCl concentration in the medium increased, the contents of saturated and cyclopropanoic fatty acids increased to more than half of the total fatty acids. 5NS gave the highest order parameters for the intact cells of this bacterium, while 12NS gave lower and 16NS gave the lowest results. The order parameters of 5NS, 12NS, and 16NS were completely separated, and all order parameters decreased gradually as the measuring temperature was increased. In contrast, the rotational correlation times of the intact cells with 12NS were as large as those with 5NS, while those with 16NS were distinctly smaller. Increasing NaCl concentrations in the growth medium caused an increase of the rotational correlation times, that is, stiffened the lipid bilayers. The Andrade plot for 16NS was approximately a straight line, whereas 5NS and 12NS gave two straight lines crossing at a temperature near the growth temperature, indicating phase transition from solid to liquid. The microviscosity activation energies were 5--10 kcal/mol in the liquid phase and 15--25 kcal/mol in the solid phase.     

On the rate of genetic adaptation under natural selection.

Unsaturated fatty acids are constituents of nearly all biological membranes. They are always present in membranes which possess transmembrane potentials. Two completely different biosynthetic routes have evolved (aerobic and anaerobic) for placing cis double bonds in the 9 position on the fatty acids of membrane lipids. Bacterial membranes contain primarily monounsaturated fatty acids, whereas eukaryote membranes contain a significant fraction of polyunsaturated fatty acids. The polyunsaturated fatty acids are concentrated in organelles, such as chloroplasts and mitochondria that are known to manipulate transmembrane potentials. I propose that the function of the unsaturated fatty acids is to facilitate the transmission of a local compaction of the membrane (in response to a transmembrane potential) laterally through the membrane. The role of the cis double bond at position 9 is twofold: first to create a kink in the chains of a large fraction of membrane fatty acids enhancing the separation of two regions in the membrane and second to enhance the rigidity of the membrane in the region between the head group and the 9 double bond. This ordered region contains those carbons proximal to the 9 carbon and which are in a regular array of trans conformations. The presence of a reasonable proportion of cis double bonds at position 9 will tend to maintain these trans conformations utilizing pi-pi (van der Waals) interactions between adjacent hydrocarbon chains at position 9. The disordered region contains the carbons distal to the 9 carbon. These have greater degrees of freedom and considerable gauche conformations. The role of the double bonds in the polyunsaturated fatty acids distal to carbon 9 is to facilitate trans bilayer pi-pi (van der Waals) interactions enhancing compaction of the bilayer during the electrostriction. I further propose that it is the function of the ionic headgroups to form an interlocking polyionic network which constitutes an elastic sheet. These ionic interactions would serve as the restoring force converting the compaction into a wave. The facilitation of the compaction of the bilayer together with the polyionic restoring force permits the membrane to transmit conformational changes from one transmembrane protein to another. Since transmembrane potentials are created and responded to by proteins each in a single location, it is thus proposed that a potential compaction wave emanates from the first protein in all directions in the plane of the membrane. The proposed wave would have both physical and electrical components. The electrohydrodynamic wave would require that the compaction oscillations be coupled to an oscillating electrical field. These proposals are applied to mitochondrial oxidative phosphorylation, and to transport across biological membranes.     

Effects of acyl chain length, unsaturation, and pH on thermal stability of model discoidal HDLs

HDLs prevent atherosclerosis by removing excess cell cholesterol. Lipid composition affects HDL functions in cholesterol removal, yet its effects on the disk stability remain unclear. We hypothesize that reduced length or increased cis-unsaturation of phosphatidylcholine acyl chains destabilize discoidal HDL and promote protein dissociation and lipoprotein fusion. To test this hypothesis, we determined thermal stability of binary complexes reconstituted from apoC-I and diacyl PCs containing 12-18 carbons with 0-2 cis-double bonds. Kinetic analysis using circular dichroism shows that, for fully saturated PCs, chain length increase by two carbons stabilizes lipoprotein by deltaDeltaG* (37 degrees C) congruent with 1.4 kcal/mol, suggesting that hydrophobic interactions dominate the disk stability; distinct effects of pH and salt indicate contribution of electrostatic interactions. Similarly, apoA-I-containing disks show increased stability with increasing chain length. Acyl chain unsaturation reduces disk stability. In summary, stability of discoidal HDL correlates directly with fatty acyl chain length and saturation: the longer and more fully saturated are the chains, the more extensive are the stabilizing lipid-protein and lipid-lipid interactions and the higher is the free energy barrier for protein dissociation and lipoprotein fusion. This sheds new light on the existing data of cholesterol efflux to discoidal HDL and suggests that moderate lipoprotein destabilization facilitates cholesterol insertion.     

Phase fluorometric studies of spectral relaxation at the lipid-water interface of phospholipid vesicles

We examined the dynamic properties of the lipid-water interface region of model membranes using the probe 2-p-toluidinylnaphthalene-6-sulfonic acid (TNS). For comparison we also examined the temperature-dependent spectral properties of TNS in the viscous solvent glycerol. Fluorescence phase shift and demodulation measurements were used to prove that the membranes relax around the excited state of TNS on the ns time scale. The rate of spectral relaxation is thought to reflect the mobility of the polar interface region of the membranes on this same time scale. The spectral relaxation times were estimated by the use of phase-sensitive detection of fluorescence. Using this method one may directly record, in an approximate fashion, the emission spectra of the relaxed and the initially excited states of TNS. The relative intensities of these phase-sensitive spectra, in combination with the measured phase and modulation values on the short and long wavelength sides of the emission, yield the spectral relaxation times. For saturated and unsaturated phosphatidylcholines, at temperatures ranging from 5 to 50°C, the relaxation times ranged from 5 to 1 ns. The activation energies for spectral relaxation were near 4 kcal/mol. Surprisingly, the relaxation times decreased smoothly with increasing temperature, and did not change abruptly at the phase transition temperatures. These results indicate that the small molecular motions of the interface region of membranes, which are responsible for spectral relaxation, are not dramatically influenced by the phase state of the acyl side chain region of the membranes.     

Energetics and cooperativity of tertiary hydrogen bonds in RNA structure.

Tertiary interactions that allow RNA to fold into intricate three-dimensional structures are being identified, but little is known about the thermodynamics of individual interactions. Here we quantify the tertiary structure contributions of individual hydrogen bonds in a "ribose zipper" motif of the recently crystallized Tetrahymena group I intron P4-P6 domain. The 2'-hydroxyls of P4-P6 nucleotides C109/A184 and A183/G110 participate in forming the "teeth" of the zipper. These four nucleotides were substituted in all combinations with their 2'-deoxy and (separately) 2'-methoxy analogues, and thermodynamic effects on the tertiary folding DeltaG degrees ' were assayed by the Mg2+ dependence of electrophoretic mobility in nondenaturing gels. The 2'-deoxy series showed a consistent trend with an average contribution to the tertiary folding DeltaG degrees' of -0.4 to -0.5 kcal/mol per hydrogen bond. Contributions were approximately additive, reflecting no cooperativity among the hydrogen bonds. Each "tooth" of the ribose zipper (comprising two hydrogen bonds) thus contributes about -1.0 kcal/mol to the tertiary folding DeltaG degrees'. Single 2'-methoxy substitutions destabilized folding by approximately 1 kcal/mol, but the trend reversed with multiple 2'-methoxy substitutions; the folding DeltaG degrees' for the quadruple 2'-methoxy derivative was approximately unchanged relative to wild-type. On the basis of these data and on temperature-gradient gel results, we conclude that entropically favorable hydrophobic interactions balance enthalpically unfavorable hydrogen bond deletions and steric clashes for multiple 2'-methoxy substitutions. Because many of the 2'-deoxy derivatives no longer have the characteristic hydrogen-bond patterns of the ribose zipper motif but simply have individual long-range ribose-base or ribose-ribose hydrogen bonds, we speculate that the energetic value of -0.4 to -0.5 kcal/mol per tertiary hydrogen bond may be more generally applicable to RNA folding.     

Subject index

Heats of fusion and heat capacities have been measured for saturated, unsaturated and hydroxy fatty acids, differing in degree of unsaturation, geometric isomerism, and position of unsaturated and hydroxy groups. Entropies of fusion are used to draw conclusions concerning molecular structure of fatty acid chains and lateral chain-chain interactions. Position of the functional group on the chain does not seem to significantly affect the entropy values for trans and cis single double bonds and single triple bonds, but differences are noted with hydroxy group position. Whereas single acid triglycerides of saturated acids have entropies which are about three times that of the corresponding acid, cis and trans single acid triglycerides do not show the same relationship with their corresponding acids. Comparing entropies of fusion for certain groups of fatty acids, only differing in carbon number, allows the estimation of chain equivalence with saturated fatty acids. Hence, for example it is shown that a 22 to 23-carbon cis mono-unsaturated fatty acid is equivalent to an 18-carbon saturated fatty acid.     

Ring-opening polymerisation of beta-butyrolactone catalysed by distannoxane complexes: study of the mechanism.

The mechanism of the ring-opening polymerisation of beta-butyrolactones was studied. The ring-opening polymerisation of BL catatlysed by distannoxane complexes is of a living nature. The polymerisation of racemic BL gave a predominantly syndiotactic P(3HB). The temperature effect on syndiospecificity was used to determine the activation energy (deltaE = Esyndiotactic - Eisotactic) for syndiotactic versus isotactic diad placement. The deltaE value was obtained as -1.49 kcal/mol. The steric control leading to the observed syndiospecificity is due predominantly to diastereomeric interactions between the Sn-coordinated P(3HB) chain end. having a specific chain end stereochemistry, and the incoming BL enantiomeric monomers. The catalytic cycle derived from the mechanism of the polymerisation was proposed.     

Increasing protein stability by altering long-range coulombic interactions.

It is difficult to increase protein stability by adding hydrogen bonds or burying nonpolar surface. The results described here show that reversing the charge on a side chain on the surface of a protein is a useful way of increasing stability. Ribonuclease T1 is an acidic protein with a pI approximately 3.5 and a net charge of approximately -6 at pH 7. The side chain of Asp49 is hyperexposed, not hydrogen bonded, and 8 A from the nearest charged group. The stability of Asp49Ala is 0.5 kcal/mol greater than wild-type at pH 7 and 0.4 kcal/mol less at pH 2.5. The stability of Asp49His is 1.1 kcal/mol greater than wild-type at pH 6, where the histidine 49 side chain (pKa = 7.2) is positively charged. Similar results were obtained with ribonuclease Sa where Asp25Lys is 0.9 kcal/mol and Glu74Lys is 1.1 kcal/mol more stable than the wild-type enzyme. These results suggest that protein stability can be increased by improving the coulombic interactions among charged groups on the protein surface. In addition, the stability of RNase T1 decreases as more hydrophobic aromatic residues are substituted for Ala49, indicating a reverse hydrophobic effect.     

In vivo mobility of fatty acid end groups of Bacillus thuringiensis plasma membrane lipids during growth and sporulation

The mobility of 13C specifically labeled branched chain end groups of iso-even fatty acids in intact, live Bacillus thuringiensis cells was studied by 13C nuclear magnetic resonance spectroscopy. This study apparently represents the first direct observation of branched chain carbon atoms in living cells. End groups were labeled using DL-[beta, delta, delta'-13C]valine as a precursor chain initiator for iso-even fatty acid synthesis after using L-[delta, delta'-14C]L-valine to determine optimal conditions for labeling of the membrane fatty acid end groups. Cell survival in the NMR was determined for various lengths of time at 28 and 39 degrees C. Subsequently, 13C-labeled vegetative cells, sporulating cells (three stages of development), and purified mature spores were analyzed by 13C NMR using corresponding unlabeled cells as controls. Spin lattice relaxation times (T1) were obtained for the enriched iso-branched region at 23.3 ppm and for the natural abundance peak for the glycerol backbone (carbons 1 and 3) of the membrane lipids at 61.7 ppm. The T1 of the glycerol carbons (0.08 s) did not change significantly with stage of development or temperature. The T1 of the iso-even enriched end group changed dramatically from vegetative cells (0.70s) to sporulating cells (0.28 s) at 28 degrees C. A decrease in the T1 was also observed at 39 degrees C from 0.91 s for vegetative cells to 0.54 s for sporulating cells. Accompanying the reduced mobility indicated by the T1 values, there was a general decline in the signal-to-noise ratios of identically acquired spectra as sporulation continued which culminated in the lack of discernible plasma membrane lipid resonances in purified mature spores. The progressive loss of signal appeared to have resulted from a continuous decline in the fraction of plasma membrane fatty acids with sufficient mobility to give signals above background.     

The role of membrane thickness in charged protein-lipid interactions

Charged amino acids are known to be important in controlling the actions of integral and peripheral membrane proteins and cell disrupting peptides. Atomistic molecular dynamics studies have shed much light on the mechanisms of membrane binding and translocation of charged protein groups, yet the impact of the full diversity of membrane physico-chemical properties and topologies has yet to be explored. Here we have performed a systematic study of an arginine (Arg) side chain analog moving across saturated phosphatidylcholine (PC) bilayers of variable hydrocarbon tail length from 10 to 18 carbons. For all bilayers we observe similar ion-induced defects, where Arg draws water molecules and lipid head groups into the bilayers to avoid large dehydration energy costs. The free energy profiles all exhibit sharp climbs with increasing penetration into the hydrocarbon core, with predictable shifts between bilayers of different thickness, leading to barrier reduction from 26 kcal/mol for 18 carbons to 6 kcal/mol for 10 carbons. For lipids of 10 and 12 carbons we observe narrow transmembrane pores and corresponding plateaus in the free energy profiles. Allowing for movements of the protein and side chain snorkeling, we argue that the energetic cost for burying Arg inside a thin bilayer will be small, consistent with recent experiments, also leading to a dramatic reduction in pK(a) shifts for Arg. We provide evidence that Arg translocation occurs via an ion-induced defect mechanism, except in thick bilayers (of at least 18 carbons) where solubility-diffusion becomes energetically favored. Our findings shed light on the mechanisms of ion movement through membranes of varying composition, with implications for a range of charged protein-lipid interactions and the actions of cell-perturbing peptides. This article is part of a Special Issue entitled: Membrane protein structure and function.     

Carbon-13 nuclear magnetic resonance study of spin labelled cholesteryl ester in model membranes

Carbon-13 NMR longitudinal relaxation times for unilamellar vesicles of egg phosphatidyl-choline (PC) in aqueous dispersion have been measured following the incorporation of spin labelled cholesteryl palmitate. The spin label induced relaxation rates. 1/T_1.5L, for fatty acyl chain carbons show that the C5 segment of the cholesteryl ester acyl chain is located near the C1 and C2 segments of the phospholipid acyl chains. A greater spin label induced enhancement of relaxation rate was observed for the inner vesicle layer than for the outer, and is attributed to a higher ester incorporation and/or tighter lipid packing in the inner layer.     

The role of membrane thickness in charged protein–lipid interactions

Charged amino acids are known to be important in controlling the actions of integral and peripheral membrane proteins and cell disrupting peptides. Atomistic molecular dynamics studies have shed much light on the mechanisms of membrane binding and translocation of charged protein groups, yet the impact of the full diversity of membrane physico-chemical properties and topologies has yet to be explored. Here we have performed a systematic study of an arginine (Arg) side chain analog moving across saturated phosphatidylcholine (PC) bilayers of variable hydrocarbon tail length from 10 to 18 carbons. For all bilayers we observe similar ion-induced defects, where Arg draws water molecules and lipid head groups into the bilayers to avoid large dehydration energy costs. The free energy profiles all exhibit sharp climbs with increasing penetration into the hydrocarbon core, with predictable shifts between bilayers of different thickness, leading to barrier reduction from 26 kcal/mol for 18 carbons to 6 kcal/mol for 10 carbons. For lipids of 10 and 12 carbons we observe narrow transmembrane pores and corresponding plateaus in the free energy profiles. Allowing for movements of the protein and side chain snorkeling, we argue that the energetic cost for burying Arg inside a thin bilayer will be small, consistent with recent experiments, also leading to a dramatic reduction in pK_a shifts for Arg. We provide evidence that Arg translocation occurs via an ion-induced defect mechanism, except in thick bilayers (of at least 18 carbons) where solubility-diffusion becomes energetically favored. Our findings shed light on the mechanisms of ion movement through membranes of varying composition, with implications for a range of charged protein–lipid interactions and the actions of cell-perturbing peptides. This article is part of a Special Issue entitled: Membrane protein structure and function.     

Effect of alkyl chain unsaturation and cholesterol intercalation on oxygen transport in membranes: a pulse ESR spin labeling study.

Transport and diffusion of molecular oxygen in phosphatidylcholine (PC)-cholesterol membranes and their molecular mechanism were investigated. A special attention was paid to the molecular interaction involving unsaturated alkyl chains and cholesterol. Oxygen transport was evaluated by monitoring the bimolecular collision rate of molecular oxygen and the lipid-type spin labels, tempocholine phosphatidic acid ester, 5-doxylstearic acid, and 16-doxylstearic acid. The collision rate was determined by measuring the spin-lattice relaxation times (T1's) in the presence and absence of molecular oxygen with long-pulse saturation-recovery ESR techniques. In the absence of cholesterol, incorporation of either a cis or trans double bond at the C9-C10 position of the alkyl chain decreases oxygen transport at all locations in the membrane. The activation energy for the translational diffusion of molecular oxygen in the absence of cholesterol is 3.7-6.5 kcal/mol, which is comparable to the activation energy theoretically estimated for kink migration or C-C bond rotation of alkyl chains [Tr?uble, H. (1971) J. Membr. Biol. 4, 193-208; Pace, R. J., & Chan, S. I. (1982) J. Chem. Phys. 76, 4241-4247]. Intercalation of cholesterol in saturated PC membranes reduces oxygen transport in the headgroup region and the hydrophobic region near the membrane surface but little affects the transport in the central part of the bilayer. In unsaturated PC membranes, intercalation of cholesterol also reduces oxygen transport in and near the headgroup regions. In contrast, it increases oxygen transport in the middle of the bilayer. On the basis of these observations, a model for the mechanism of oxygen transport in the membrane is proposed in which oxygen molecules reside in vacant pockets created by gauche-trans isomerization of alkyl chains and the structural nonconformability of neighboring lipids, unsaturated PC and cholesterol in particular, and oxygen molecules jump from one pocket to the adjacent one or move along with the movement of the pocket itself. The presence of cholesterol decreases oxygen permeability across the membrane in all membranes used in this work in spite of the increase in oxygen transport in the central part of unsaturated PC-cholesterol membranes because cholesterol decreases oxygen transport in and near the headgroup regions, where the major barriers for oxygen permeability are located. Oxygen gradients across the membranes of the cells and the mitochondria are evaluated. Arguments are advanced that oxygen permeation across the protein-rich mitochondrial membranes can be a rate-limiting step for oxygen consumption under hypoxic conditions in vivo.     

Stimulation of chloride transport by fatty acids in corneal epithelium and relation to changes in membrane fluidity.

The effect of altering cell membrane lipids on ion transport across isolated corneas was studied. Corneas mounted in Ussing-type chambers showed a rapid increase in short-circuit current following treatment with a variety of unsaturated fatty acids of varying chain length and unsaturation. Measurements of membrane fluidity which utilize immunofluorescence labelling of membrane proteins showed corneal epithelial cell membranes to be significantly more fluid following linoleic acid treatment. Uptake studies indicate rapid incorporation of [14C]linoleic acid into corneal cell membranes. Highly unsaturated fatty acids were found to have the greatest ability to stimulate chloride transport. Saturated fatty acids were tested and were found to have no effect on chloride transport at any concentration. It is proposed that unsaturated fatty acids activate chloride transport by increasing membrane lipid fluidity. The relationship of these parameters is discussed in terms of a mobile receptor model. We speculate that an increase in membrane lipid fluidity promotes lateral diffusion of membrane receptor proteins and enzymes, increasing protein-protein interactions within the membrane, ultimately resulting in the enhancement of cyclic AMP synthesis.     

A calorimetric investigation of a series of mixed-chain polyunsaturated phosphatidylcholines: effect of sn-2 chain length and degree of unsaturation.

Although mammalian tissues contain high levels of polyunsaturated fatty acids, our knowledge of the effects of the degree of unsaturation and double-bond location upon bilayer organization is limited. Therefore, a series of mixed-chain unsaturated phosphatidylcholines (PC) comprised of 18:0 at the sn-1 position and various unsaturates at the sn-2 position (18:1n9, 18:2n6, 18:3n6, 18:3n3, 20:2n6, 20:3n6, 20:4n6, 20:5n3, 22:4n6, 22:5n6, or 22:6n3) was studied with differential scanning calorimetry, and their gel to liquid-crystalline phase transitions yielded measurements of Tm, Tonset, delta H, and delta S. Minimal delta H values were obtained for the diene species, 1.7 and 2.9 kcal/mole for 18:2n6 and 20:2n6, respectively. These results are consistent with the dienes having an acyl chain conformation that results in perturbed chain packing. Increasing the degree of unsaturation to three or more double bonds resulted in higher delta H values, 3.7, 4.3, and 4.6 kcal/mole for 18:3n6, 20:3n6, and 20:4n6, respectively, consistent with the occurrence of a gel-state chain conformation(s), which is more tightly packed than the dienes. The 18:0,22:6n3-PC species yielded the highest delta H (6.1 kcal/mole) and delta S(22.7 cal/mol degree) of all the polyunsaturates studied. The distinctive packing properties of phospholipid bilayers containing 22:6n3 may underlie its essential role in the nervous system.     

Proton magnetic relaxation dispersion in solutions of the cuproprotein diamine oxidase.

Proton nuclear magnetic resonance relaxation measurements were made over the range 4.7--220 MHz for aqueous solutions of hog kidney diamine oxidase. The values of 1/T1 give rise in two distinct dispersions, at 16 and 75 MHz, whereas 1/T2 displays a minimum at 20 MHz. The temperature dependence of relaxation rates in all cases yield apparent activation energies less than 0.6 kcal/mol. These data indicate to us that the two Cu(II) ions of diamine oxidase are intrinsically different in terms of their electronic relaxation characteristics and hence, chemical environments. Low field limits of the two electronic relaxation times are 2 and 10 ns, with one of these correlation times being frequency dependent. The value of the frequency-dependent electronic relaxation time is governed by interactions that are modulated by a process having a correlation time of 5 ps.     

Electrostatic effects on the stability of discoidal high-density lipoproteins

High-density lipoproteins (HDL) remove cholesterol from peripheral tissues and thereby help to prevent atherosclerosis. Nascent HDL are discoidal complexes composed of a phospholipid bilayer surrounded by protein alpha-helices that are thought to form extensive stabilizing interhelical salt bridges. Earlier we showed that HDL stability, which is necessary for HDL functions, is modulated by kinetic barriers. Here we test the role of electrostatic interactions in the kinetic stability by analyzing the effects of salt, pH, and point mutations on model discoidal HDL reconstituted from human apolipoprotein C-1 (apoC-1) and dimyristoyl phosphatidylcholine (DMPC). Circular dichroism, Trp fluorescence, and light scattering data show that molar concentrations of NaCl or Na(2)SO(4) increase the apparent melting temperature of apoC-1:DMPC complexes by up to 20 degrees C and decelerate protein unfolding. Arrhenius analysis shows that 1 M NaCl stabilizes the disks by deltaDeltaG* approximately equal 3.5 kcal/mol at 37 degrees C and increases the activation energy of their denaturation and fusion by deltaE(a) approximately equal deltaDeltaH* approximately equal 13 kcal/mol, indicating that the salt-induced stabilization is enthalpy-driven. Denaturation studies in various solvent conditions (pH 5.7-8.2, 0-40% sucrose, 0-2 M trimethylamine N-oxide) suggest that the salt-induced disk stabilization results from ionic screening of unfavorable short-range Coulombic interactions. Thus, the dominant electrostatic interactions in apoC-1:DMPC disks are destabilizing. Comparison of the salt effects on the protein:lipid complexes of various composition reveals an inverse correlation between the lipoprotein stability and the salt-induced stabilization and suggests that short-range electrostatic interactions significantly contribute to lipoprotein stability: the better-optimized these interactions are, the more stable the complex is.     

Effects of various numbers and positions of cis double bonds in the sn-2 acyl chain of phosphatidylethanolamine on the chain-melting temperature

In an attempt to investigate systematically the effects of various single and multiple cis carbon-carbon double bonds in the sn-2 acyl chains of natural phospholipids on membrane properties, we have de novo synthesized unsaturated C20 fatty acids comprised of single or multiple methylene-interrupted cis double bonds. Subsequently, 15 molecular species of phosphatidylethanolamine (PE) with sn-1 C20-saturated and sn-2 C20-unsaturated acyl chains were semi-synthesized by acylation of C20-lysophosphatidylcholine with unsaturated C20 fatty acids followed by phospholipase D-catalyzed base-exchange reaction in the presence of excess ethanolamine. The gel-to-liquid crystalline phase transitions of these 15 mixed-chain PE, in excess H2O, were investigated by high resolution differential scanning calorimetry. In addition, the energy-minimized structures of these sn-1 C20-saturated/sn-2 C20-unsaturated PE were simulated by molecular mechanics calculations. It is shown that the successive introduction of cis double bonds into the sn-2 acyl chain of C(20):C(20)PE can affect the gel-to-liquid crystalline phase transition temperature, Tm, of the lipid bilayer in some characteristic ways; moreover, the effect depends critically on the position of cis double bonds in the sn-2 acyl chain. Specifically, we have constructed a novel Tm diagram for the 15 species of unsaturated PE, from which the effects of the number and the position of cis double bonds on Tm can be examined simultaneously in a simple, direct, and unifying manner. Interestingly, the characteristic Tm profiles exhibited by different series of mixed-chain PE with increasing degree of unsaturation can be interpreted in terms of structural changes associated with acyl chain unsaturation.