Acyl chain composition determines cardiolipin clustering induced by mitochondrial creatine kinase binding to monolayers

It has been recently shown that mitochondrial creatine kinase (mtCK) organizes mitochondrial model membrane by modulating the state and fluidity of lipids and by promoting the formation of protein-cardiolipin clusters. This report shows, using Brewster angle microscopy, that such clustering is largely dependent on the acyl chain composition of phospholipids. Indeed, mtCK-cardiolipin domains were observed not only with unsaturated cardiolipins, but also with the cardiolipin precursor phosphatidylglycerol. On the other hand, in the case of saturated dimyristoylphosphatidylglycerol and tetramyristoylcardiolipin, mtCK was homogeneously distributed underneath the monolayer. However, an overall decrease in membrane fluidity was indicated by infrared spectroscopy as well as by extrinsic fluorescence spectroscopy using Laurdan as a fluorescent probe, both for tetramyristoylcardiolipin and bovine heart cardiolipin containing liposomes. The binding mechanism implicated the insertion of protein segments into monolayers, as evidenced from alternative current polarography, regardless of the chain unsaturation for the phosphatidylglycerols and cardiolipins tested.     

Apocytochrome c binding to negatively charged lipid dispersions studied by spin-label electron spin resonance

The interaction of apocytochrome c with aqueous dispersions of phosphatidylserine from bovine spinal cord and with other negatively charged phospholipids has been studied as a function of pH and salt concentration by using spin-label electron spin resonance (ESR) spectroscopy and chemical binding assays. The ESR spectra of phospholipids spin-labeled at different positions on the sn-2 chain indicate a generalized decrease in mobility of the lipids, while the characteristic flexibility gradient toward the terminal methyl end of the chain is maintained, on binding of apocytochrome c to phosphatidylserine dispersions. This perturbation of the bulk lipid mobility or ordering is considerably greater than that observed on binding of cytochrome c. In addition, a second, more motionally restricted, lipid component is observed with lipids labeled close to the terminal methyl ends of the chains. This second component is not observed on binding of cytochrome c and can be taken as direct evidence for penetration of apocytochrome c into the lipid bilayer. It is less strongly motionally restricted than similar spectral components observed with integral membrane proteins and displays a steep flexibility gradient. The proportion of this second component increases with increasing protein-to-lipid ratio, but the stoichiometry per protein bound decreases from 4.5 lipids per 12 000-dalton protein at low protein contents to 2 lipids per protein at saturating amounts of protein. Apocytochrome c binding to phosphatidylserine dispersions decreases with increasing salt concentration from a saturation value corresponding to approximately 5 lipids per protein in the absence of salt to practically zero at 0.4 M NaCl.(ABSTRACT TRUNCATED AT 250 WORDS)     

An electron spin resonance spin-label study of lipophilin in oriented phospholipid bilayers

Model membranes consisting of dimyristoyl phosphatidylcholine and a hydrophobic protein from bovine myelin, lipophilin, were studied using the cholesterol-resembling cholestane ESR spin label. Orientation of the membranes made it possible to deconvolute the spectra into two fractions, one of oriented spin labels reflecting phospholipid bilayer of high order, and one of isotropically tumbling spin labels ascribed to the lipid fraction surrounding the protein molecule (boundary lipid). This isotropic tumbling is different from the behavior of phospholipid molecules near the protein, which retain some degree of order, and indicates that the boundary lipid fraction in our model system forms a rather fluid environment for the protein. A nonlinear relation was found between protein concentration and amount of boundary spin labels. Addition of cholesterol decreases the amount of boundary spin labels. Both findings form evidence for a preferential binding of cholesterol by the membrane protein.     

Lipid-protein interactions with cardiac phospholamban studied by spin-label electron spin resonance

Phospholamban is a cardiac regulatory protein that, in its monomeric form, inhibits the Ca(2+)-ATPase. Lipid-protein interactions with a synthetic variant of phospholamban, in which all cysteine residues are replaced with alanine, have been studied by spin-label electron spin resonance (ESR) in different lipid host membranes. Both the stoichiometry and selectivity of lipid interactions were determined from the two-component ESR spectra of phospholipid species spin-labeled on the 14 C atom of the sn-2 chain. The lipid stoichiometry is determined by the oligomeric state of the protein and the selectivity by the membrane disposition of the positively charged residues in the N-terminal section of the protein. In dimyristoylphosphatidylcholine (DMPC) membranes, the stoichiometry (N(b)) is 7 lipids/monomer for the full-length protein and 4 for the transmembrane section (residues 26-52). These stoichiometries correspond to the dimeric and pentameric forms, respectively. In palmitoyloleoylphosphatidylcholine, N(b) = 4 for both the whole protein and the transmembrane peptide. In negatively charged membranes of dimyristoylphosphatidylglycerol (DMPG), the lipid stoichiometry is N(b) = 10-11 per monomer for both the full-length protein and the transmembrane peptide. This stoichiometry corresponds to monomeric dispersion of the protein in the negatively charged lipid. The sequence of lipid selectivity is as follows: stearic acid > phosphatidic acid > phosphatidylserine = phosphatidylglycerol = phosphatidylcholine > phosphatidylethanolamine for both the full-length protein and the transmembrane peptide in DMPC. Absolute selectivities are, however, lower for the transmembrane peptide. A similar pattern of lipid selectivity is obtained in DMPG, but the absolute selectivities are reduced considerably. The results are discussed in terms of the integration of the regulatory species in the lipid membrane.     

Specificity of the interaction of amino- and carboxy-terminal fragments of the mitochondrial precursor protein apocytochrome c with negatively charged phospholipids. A spin-label electron spin resonance study

The contribution of the various regions of the mitochondrial precursor protein apocytochrome c to the interaction of the protein with phosphatidylserine dispersions has been studied with chemically and enzymatically prepared fragments of horse heart apocytochrome c and phospholipids spin-labeled at different positions of the sn-2 chain. Three amino-terminal heme-less peptides, two heme-containing amino-terminal fragments, one central fragment, and three carboxy-terminal fragments were studied. The electron spin resonance spectra of phospholipids spin-labeled at the C5 position of the fatty acid chain indicate that both amino-terminal and carboxy-terminal fragments of the apocytochrome c molecule cause a restriction of motion of the lipids, whereas the heme-containing peptides and protein have less effect. In addition, a second motionally more restricted lipid component, which is observed for apocytochrome c interacting with phosphatidylserine dispersions containing lipids spin-labeled at the C12 or C14 position [G?rrissen, H., Marsh, D., Rietveld, A., & de Kruijff, B. (1986) Biochemistry 25, 2904-2910], was observed both on binding the carboxy-terminal fragments and on binding of the amino-terminal fragments of the precursor protein. Interestingly, even a small water-soluble peptide consisting of the 24 carboxy-terminal residues gave rise to a two-component spectrum, with an outer hyperfine splitting of the restricted lipid component of 59 G, indicating a considerable restriction of the chain motion. This suggests that both the carboxy- and amino-terminal parts of the protein penetrate into the center of the bilayer and cause a strong perturbation of the fatty acyl chain motion. The implications of these findings for the mechanism of apocytochrome c translocation across membranes are discussed.     

Solution of the nitroxide spin-label spectral overlap problem using pulse electron spin resonance.

Short-pulse saturation-recovery (SR) electron spin resonance (ESR) methods have been used to measure the lateral diffusion of a nitroxide-labeled cholesterol analogue (3-spiro-[2'-(N-oxyl-4',4'-dimethyloxazoladine)]-cholestane, CSL) in multilamellar liposomal dispersions. SR experiments were performed on samples containing 14NCSL:15NCSL pairs, and recovery signals were analyzed for initial conditions and multiexponential time constants by computer simulation. Rate equations describing the system were written and solved. The time constants contain combinations of electron spin lattice relaxation times Tle for both isotopes and the Heisenberg exchange rate constant Kx. We have investigated the complication that occurs from overlap of ESR spectral fragments from 14N and 15N moieties. The time constants of the multiexponential signals are independent of ESR line shape and position. From Kx, lateral diffusion constants of CSL in dimyristoylphosphatidylcholine (DMPC) were calculated (D = 1.7 x 10(-8) at 27 degrees C and 2.7 x 10(-8) cm2/s at 37 degrees C). It is shown that short-pulse saturation-recovery methods are able to overcome the ESR spectral overlap problem that is encountered in conventional ESR and continuous wave electron-electron double resonance (CW ELDOR) studies of spin-spin interactions. The present method can be extended to more complex situations involving spin labels in different environments with physical and chemical exchange.     

Dynamic molecular structure of DPPC-DLPC-cholesterol ternary lipid system by spin-label electron spin resonance

The hydrated ternary lamellar lipid mixture of dipalmitoyl-PC/dilauroyl-PC/cholesterol (DPPC/DLPC/Chol) has been studied by electron spin resonance (ESR) to reveal the dynamic structure on a molecular level of the different phases that exist and coexist over virtually the full range of composition. The spectra for more than 100 different compositions at room temperature were analyzed by nonlinear least-squares fitting to provide the rotational diffusion rates and order parameters of the end-chain labeled phospholipid 16-PC. The ESR spectra exhibit substantial variation as a function of composition, even though the respective phases generally differ rather modestly from each other. The Lalpha and Lbeta phases are clearly distinguished, with the former exhibiting substantially lower ordering and greater motional rates, whereas the well-defined Lo phase exhibits the greatest ordering and relatively fast motional rates. Typically, smaller variations occur within a given phase. The ESR spectral analysis also yields phase boundaries and coexistence regions which are found to be consistent with previous results from fluorescence methods, although new features are found. Phase coexistence regions were in some cases confirmed by observing the existence of isosbestic points in the absorption mode ESR spectra from the phases. The dynamic structural properties of the DPPC-rich Lbeta and DLPC-rich Lalpha phases, within their two-phase coexistence region do not change with composition along a tie-line, but the ratio of the two phases follows the lever rule in accordance with thermodynamic principles. The analysis shows that 16-PC spin-label partitions nearly equally between the Lalpha and Lbeta phases, making it a useful probe for studying such coexisting phases. Extensive study of two-phase coexistence regions requires the determination of tie-lines, which were approximated in this study. However, a method is suggested to accurately determine the tie-lines by ESR.     

Competitive ion binding to low density lipoproteins: an electron spin resonance study

The electron spin resonance (ESR) technique was used to evaluate binding constants for Ca(II) and Mg(II) in interaction with low density lipoprotein (LDL). The Ca(II) or Mg(II) ions competed with the paramagnetic Mn(II) ions for the same binding sites of two different classes on the LDL surface. For each ion competing with Mn(II), the solutions of eight non-linear competition equations were fit to the experimental titration curves, with two adjustable parameters, the two binding constants. The derived "intrinsic" values (the values corrected for the electrolyte-induced change of the surface potential) for "strong" binding sites for Ca(II) (170 +/- 85 M-1) and Mg(II) (60 +/- 30 M-1) differ significantly from the respective value for Mn(II) (760 M-1). The values for the "weak" binding sites (18 M-1, 15 M-1 and 10 M-1 for Mn(II), Ca(II) and Mg(II), respectively are in the range of the binding constants for these ions in interaction with model membranes.     

Exchange rates at the lipid-protein interface of myelin proteolipid protein studied by spin-label electron spin resonance

The electron spin resonance (ESR) spectra from spin-labeled phospholipids in recombinants of myelin proteolipid apoprotein with dimyristoylphosphatidylcholine have been simulated with the exchanged-coupled Bloch equations to obtain values for both the fraction of motionally restricted lipids and the exchange rate between the fluid and motionally restricted lipid populations. The rate of exchange between the two spin-labeled lipid components is found to lie in the slow exchange regime of nitroxide ESR spectroscopy. The values obtained for the fraction of motionally restricted component in the exchanged-coupled spectra are found to be in good agreement with those obtained previously by spectral subtraction for the same system [Brophy, P. J., Horváth, L. I., & Marsh, D. (1984) Biochemistry 23, 860-865]. The rate of lipid exchange off the protein is independent of lipid/protein ratio for a given spin-labeled phospholipid, as expected, and decreases with increasing selectivity of the various phospholipids for the protein. At 30 degrees C and for ionic strength 0.1 and pH 7.4, the off-rate constants are 4.6 X 10(6) s-1 for phosphatidic acid, 1.1 X 10(7) s-1 for phosphatidylserine, 1.6 X 10(7) s-1 for phosphatidylcholine, and 2.2 X 10(7) s-1 for phosphatidylethanolamine. These values are in the inverse ratio of the relative association constants of the various lipids for the protein (Brophy et al., 1984) and are appreciably slower than the rate of lipid lateral diffusion in dimyristoylphosphatidylcholine bilayers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transfer of stearic acids from albumin to polymer-grafted lipid containing membranes probed by spin-label electron spin resonance

Human serum albumin (HSA) has been spin-labelled with stearic acids having the nitroxide moiety attached to the hydrocarbon chain either at the 5th or at the 16th carbon atom (n-SASL, n = 5 and 16, respectively) with respect to the carboxyl groups. Its interaction with sterically stabilized liposomes (SSL) composed of dipalmitoylphosphatidylcholine (DPPC) mixed with submicellar content of poly(ethylene glycol:2000)-grafted dipalmitoyl phosphatidylethanolamine (PEG:2000-DPPE) has been studied by conventional electron spin resonance (ESR) spectroscopy. In the absence of bilayer membranes, the ESR spectra of nitroxide stearic acids non-covalently bound to HSA are single component powder patterns, indicative of spin labels undergoing temperature dependent anisotropic motion in the slow motional regime on the conventional ESR timescale. The adsorption of HSA to DPPC bilayers results in two component ESR spectra. Indeed, superimposed to an anisotropic protein-signal appears a more isotropic signal due to the labels in the lipid environment. This accounts for the transfer of fatty acids from the protein to DPPC bilayers. Two spectral components with different rotational mobility are also singled out in the spectra of n-SASL bound to HSA when DPPC/PEG:2000-DPPE mixtures are present in the dispersion medium. The fraction, f(L)(16-SASL), of spin labels transferred from the protein to lipid/polymer-lipid lamellar membranes has been quantified performing spectral subtraction. It is found that f(L)(16-SASL) decreases on increasing the content of the polymer-lipid mixed with DPPC. It is strongly reduced in the low-density mushroom regime and levels off in the high-density brush regime of the polymer-lipid content as a result of the steric stabilization exerted by the PEG-lipids. Moreover, the fraction of transferred fatty acids from HSA to SSL is dependent on the physical state of the lipid bilayers. It progressively increases with increasing the temperature from the gel to the liquid-crystalline lamellar phases of the mixed lipid/polymer-lipid membranes, although such a dependence is much weaker in the brush regime.     

Membrane lysis by the antibacterial peptides cecropins B1 and B3: A spin-label electron spin resonance study on phospholipid bilayers

Custom antibacterial peptides, cecropins B1 (CB1) and B3 (CB3), were synthesized. These peptides have particular sequence characteristics, with CB1 having two amphipathic alpha-helical segments and CB3 having two hydrophobic alpha-helical segments. These differences were exploited for a study of their efficacy in breaking up liposomes, which had different combinations of phosphatidic acid (PA) and phosphatidylcholine (PC), and a study of their lipid binding ability. Binding and nonbinding lysis actions of CB1 and CB3 on liposomes were examined further by electron spin resonance (ESR). The spin-labeled lipids 5'SL-PC, 7'SL-PC, 10'SL-PC, 12'SL-PC, and 16'SL-PC were used as probes. The ESR spectra revealed larger outer hyperfine splittings (2A(max)) for CB1 when the interactions of CB1 and CB3 with liposomes were compared. These observations indicate a larger restriction of the motion of the spin-labeled chains in the presence of CB1. Plots of the effective order parameter at the various probe positions (chain flexibility gradient) versus the peptide-lipid ratio further suggested that the lysis action of CB1 is related to its capacity to bind to the lipid bilayers. In contrast, there is no evidence of binding for CB3. To augment these findings, four spin-labeled peptides, C8SL-CB1, C32SL-CB1, C5SL-CB3, and C30SL-CB3, were also examined for their binding to and their state of aggregation within the lipid bilayers. Association isotherms of the peptides were measured for liposomes containing two molar fractions of PA (0.25 and 0.75). The membrane binding of the CB1 peptides exhibited a cooperative behavior, whereas the association isotherm of CB3 revealed binding to the lipid only for beta = 0.75 liposomes. To further identify the location of CB1 in the lipid bilayers, measurements of the collision rate with chromium oxalate in solution were conducted. Results from ESR power saturation measurements suggested that the NH(2)-terminal alpha-helix of CB1 is located on the surface of the lipid bilayers, whereas the COOH-terminal alpha-helix of CB1 is embedded below the surface of the lipid bilayers. These conclusions were further supported by the observed relationship between the partition distribution of peptides bound to liposomes at different PA/PC ratios and the amounts of free peptides. Based on the above observations, possible mechanisms of the bilayer lysis induced by CB1 and CB3 on liposomes of different composition are discussed.     

Diffusion of oxygen in water and hydrocarbons using an electron spin resonance spin-label technique.

The Smoluchowski equation for the bimolecular collision rate of dissolved oxygen molecules with spin labels yielded values for the diffusion constant of oxygen in water that are in agreement with the Stokes-Einstein equation (D infinity T/eta, where eta is the macroscopic viscosity) and with published values obtained by conventional methods. Heisenberg exchange at an interaction distance of 4.5 A occurs with a probability close to one for each encounter. In mixed hydrocarbons (olive oil, paraffin oils) and sec-butyl benzene, D infinity (T/eta)rho, where rho lies between 0.5 and 1. Oxygen diffuses in the hydrocarbons between 10 and 100 times more rapidly than predicted from the macroscopic viscosity. Similar results would be expected for diffusion of oxygen in model and biological membranes. Parallel measurements of rotational diffusion of the spin labels show little correlation with measurements of translational diffusion of oxygen. Dipolar interactions between spin labels and oxygen appear negligible except in the limit of highest viscosities.     

Head group and chain length dependence of phospholipid self-assembly studied by spin-label electron spin resonance

The critical micelle concentrations (cmc's) of a variety of spin-labeled phospholipids, 1-acyl-2-[4-(4,4-dimethyloxazolidine-N-oxyl)valeryl]-sn-glycero-3-pho sph o derivatives, have been determined by electron spin resonance (ESR) spectroscopy. The narrow, three-line ESR spectra of the rapidly tumbling monomers are clearly distinguished from the spin-spin broadened spectra of the micellar aggregates, allowing a direct determination of the concentrations of the two species. The influence of both the hydrocarbon chain length and the polar head group on the energetics of self-assembly has been studied. For phosphatidylcholine, 1n [cmc] decreases linearly with the length of the sn-1 chain. The gradient of this linear dependence corresponds to a free energy of transfer of the monomer from the aqueous phase to the micelle of delta Gtr = -1.1RT per CH2 group. The cmc's of the 1-lauroyl derivatives of both phosphatidylcholine and phosphatidylglycerol have relatively shallow, biphasic temperature dependences with a minimum at approximately 20 degrees C. Both of these properties are characteristic of the hydrophobic effect, with the free energy of transfer being slightly less than that for the solubility of n-hydrocarbons in water, corresponding to the reduced configurational entropy of the lipid chains in the micellar state. The cmc's of the 1-lauroyl derivatives of the phospholipids in 0.15 M NaCl, for their various charge states, are as follows: phosphatidic acid(2-), 0.77 mM; phosphatidic acid(1-), 0.13 mM; phosphatidylserine(1-), 0.24 mM; phosphatidylglycerol(1-), 0.17 mM; phosphatidylcholine, 0.10 mM; phosphatidylethanolamine, 0.05 mM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Domain formation in sphingomyelin/cholesterol mixed membranes studied by spin-label electron spin resonance spectroscopy

Interactions of palmitoylsphingomyelin with cholesterol in multilamellar vesicles have been studied over a wide range of compositions and temperatures in excess water by using electron spin resonance (ESR) spectroscopy. Spin labels bearing the nitroxide free radical group on the 5 or 14 C-atom in either the sn-2 stearoyl chain of phosphatidylcholine (predominantly 1-palmitoyl) or the N-stearoyl chain of sphingomyelin were used to determine the mobility and ordering of the lipids in the different phases. Two-component ESR spectra of the 14-position spin labels demonstrate the coexistence first of gel (L(beta)) and liquid-ordered (L(o)) phases and then of liquid-ordered and liquid-disordered (L(alpha)) phases, with progressively increasing temperature. These phase coexistences are detected over a limited range of cholesterol contents. ESR spectra of the 5-position spin labels register an abrupt increase in ordering at the L(alpha)-L(o) transition and a biphasic response at the L(beta)-L(o) transition. Differences in outer splitting between the C14-labeled sphingomyelin and phosphatidylcholine probes are attributed to partial interdigitation of the sphingomyelin N-acyl chains across the bilayer plane in the L(o) state. In the region where the two fluid phases, L(alpha) and L(o), coexist, the rate at which lipids exchange between phases (<7 x 10(7) s(-)(1)) is much slower than translational rates in the L(alpha) phase, which facilitates resolution of two-component spectra.     

The structure of mitochondrial creatine kinase and its membrane binding properties

The biochemical and biophysical characterization of the mitochondrial creatine kinase (Mi-CK) from chicken cardiac muscle is reviewed with emphasis on the structure of the octameric oligomer by electron microscopy and on its membrane binding properties. Information about shape, molecular symmetry and dimensions of the Mi-CK octamer, as obtained by different sample preparation techniques in combination with image processing methods, are compared. The organization of the four dimeric subunits into the Mi-CK complex as apparent in the end-on projections is discussed and the consistently observed high binding affinity of the four-fold symmetric end-on faces towards many support films and towards each other is outlined. A study on the oligomeric state of the enzyme in solution and in intact mitochondria, using chemical crosslinking reagents, is presented together with the results of a search for a possible linkage of Mi-CK with the adenine nucleotide translocator (ANT). The nature of Mi-CK binding to model membranes, demonstrating that rather the octameric than the dimeric subspecies is involved in lipid interaction and membrane contact formation, is resumed and put into relation to our structural observations. The findings are discussed in light of a possiblein vivo function of the Mi-CK octamer bridging the gap between outer and inner mitochondrial membranes at the contact sites.     

Bipolar tetraether lipids: chain flexibility and membrane polarity gradients from spin-label electron spin resonance

Membranes of thermophilic Archaea are composed of unique tetraether lipids in which C40, saturated, methyl-branched biphytanyl chains are linked at both ends to polar groups. In this paper, membranes composed of bipolar lipids P2 extracted from the acidothermophile archaeon Sulfolobus solfataricus are studied. The biophysical basis for the membrane formation and thermal stability is investigated by using electron spin resonance (ESR) of spin-labeled lipids. Spectral anisotropy and isotropic hyperfine couplings are used to determine the chain flexibility and polarity gradients, respectively. For comparison, similar measurements have been carried out on aqueous dispersions of diacyl reference lipid dipalmitoyl phosphatidylcholine and also of diphytanoyl phosphatidylcholine, which has methyl-branched chains. At a given temperature, the bolaform lipid chains are more ordered and less flexible than in normal bilayer membranes. Only at elevated temperatures (80 degrees C) does the flexibility of the chain environment in tetraether lipid assemblies approach that of fluid bilayer membranes. The height of the hydrophobic barrier formed by a monolayer of archaebacterial lipids is similar to that in conventional fluid bilayer membranes, and the permeability barrier width is comparable to that formed by a bilayer of C16 lipid chains. At a mole ratio of 1:2, the tetraether P2 lipids mix well with dipalmitoyl phosphatidylcholine lipids and stabilize conventional bilayer membranes. The biological as well as the biotechnological relevance of the results is discussed.     

Heterogeneity of mitochondrial creatine kinase

The heterogeneity of cardiac sarcomeric mitochondrial creatine kinase (creatine N-phosphotransferase, EC 2.7.3.2, sMi-CK), namely, brain ubiquitous Mi-CK (uMi-CK) and an atypical Mi-CK detected in the serum of a patient with ovarian cancer, was studied by isoelectric focusing. These Mi-CKs were found to be slightly different from each other with respect to their pIs under the examined conditions. The atypical Mi-CK was found to be an atypically oxidized form of uMi-CK. Results suggest that these heterogeneities of Mi-CK are caused by the genotypes, structures, biological functions and metabolism/dissimilation of Mi-CKs in the mitochondria and intravascular circulation.