Nanosecond time-resolved fluorescence investigations of temperature-induced conformational changes in cytochrome oxidase in phosphatidylcholine vesicles and solubilized systems

Intrinsic and lipid phase transition-induced conformational changes in cytochrome oxidase in phosphatidylcholine vesicle and solubilized systems were examined by the fluorescence lifetime of N-(1-anilinonaphthyl-4)-maleimide conjugated with the enzyme. The time-dependent fluorescence intensity of N-(1-anilinonaphthyl-4)-maleimide attached to cytochrome oxidase was described as a triple exponential decay. Both the intrinsic and lipid phase transition-induced conformational changes were detectable in plots of the average lifetime against temperature. In most cases a peak occurred at the temperature of the conformational change. The time-dependent emission anisotropy showed that N-(1-anilinonaphthyl-4)-maleimide embedded in cytochrome oxidase in phosphatidylcholine vesicles underwent a rapid restricted wobbling within a cone. The half-angle of the cone was around 30 degrees for cytochrome oxidase in dimyristoyl phosphatidylcholine vesicles.     

Quantitative fluorescence analysis of the adsorption of lysozyme to phospholipid vesicles

Experiments directed to measure the interaction of lysozyme with liposomes consisting of phosphatidylcholine (PC) and phosphatidylserine (PS) have been conducted by monitoring both protein and lipid fluorescence and fluorescence anisotropy of the protein. The binding of lysozyme to the unilamellar vesicles was quantified using a novel method of analysis in which the fractional contribution at moderate binding conditions is determined from either total fluorescence decay or anisotropy decay curves of tryptophan at limiting binding conditions. In the energy transfer experiments PC and PS lipids labelled with two pyrene acyl chains served as energy acceptors of the excited tryptophan residues in lysozyme. The binding was strongly dependent on the molar fraction of negatively charged PS in neutral PC membranes and on the ionic strength. Changes in the tryptophan fluorescence decay characteristics were found to be connected with long correlation times, indicating conformational rearrangements induced by binding of the protein to these lipid membranes. The dynamics of membrane bound protein appeared to be dependent on the physical state of the membrane. Independent of protein fluorescence studies, formation of a protein-membrane complex can also be observed from the lipid properties of the system. The interaction of lysozyme with di-pyrenyl-labelled phosphatidylserine in anionic PS/PC membranes resulted in a substantial decrease of the intramolecular excimer formation, while the excimer formation of dipyrenyl-labelled phosphatidylcholine in neutral PC membranes barely changed in the presence of lysozyme.Abbreviations dipyr₄ sn-1,2-(pyrenylbutyl) - dipyr₁₀ sn-1,2-(pyrenyldecanoyl). - DMPC dimyristoyl-phosphatidylcholine - DOPC dioleoyl-phosphatidylcholine - DPPC dipalmitoyl-phosphatidylcholine - DPPC dipalmitoylphosphatidylcholine - PC phosphatidylcholine - PS phosphatidylserine Correspondence to: A. J. W. G. Visser     

Nitroxide spin-probe study of amphotericin B-ergosterol interaction in egg phosphatidylcholine multilayers

The interaction between the polyene macrolide antibiotic, amphotericin B, and ergosterol in egg phosphatidylcholine multilayers was investigated using head group and acyl chain nitroxide spin-labelled phosphatidylcholine as probes. At physiological concentrations of less than 15 mol% sterol in egg phosphatidylcholine multilayers amphotericin B accumulates near the head group region until an amphotericin B : ergosterol ratio of approximately 0.7 is achieved. As the proportion of amphotericin B is increased above this value, formation of an acyl chain disordering complex occurs which has an approximate antibiotic:sterol ratio of unity. Dicetyl phosphate was used to increase the solubility of ergosterol past its normal limit in pure egg phosphatidylcholine (approximately 15 mol%). At concentrations of ergosterol higher than 15 mol% a complex of two ergosterol molecules and one amphotericin B was postulated when there was insufficient antibiotic to form a 1:1 complex.     

Apomyoglobin forms a micellar complex with phospholipid at low pH

Apomyoglobin was found to break down phosphatidylcholine vesicles to form a micellar complex at pH 4. Size reduction during this process was observed by measuring light scattering at 400 nm and by passing the complex through a gel-filtration column. Micellar complex formation was confirmed by electron microscopy.     

Membrane-protein interaction and the molten globule state: Interaction of α-lactalbumin with membranes

The insertion of soluble proteins into membranes has been a topic of considerable interest. We have studied the insertion of bovineα-lactalbumin into single-bilayer vesicles prepared from egg phosphatidylcholine (PC). Fluoresence studies indicated rapid and tight binding of apo-α-lactalbumin (apo-α-LA) to PC vesicles as a function of pH. The binding was maximal at pH values which favor the formation of the molten globule state. As an increase of hydrophobic surface is observed in the molten globule state, this conformational state can provide a molecular basis for insertion of soluble proteins into membranes. The membrane-bound complex formed at low pH (3.0) could be isolated and was found to be stable at neutral pH. The structural characterization of the apo-α-LA-PC complex was studied by fluorescence quenching using iodide, acrylamide, and 9,10-dibromostearic acid. The results obtained indicated that some of the tryptophans of apo-α-LA were buried in the membrane interior and some were exposed on the outer side. Fluorescence quenching and CD studies indicated the membrane-bound conformation of apo-α-LA was some conformational state that is between the soluble, fully folded conformation and the molten globule state.     

Mechanism of 8-amino-7-oxononanoate synthase: spectroscopic, kinetic, and crystallographic studies

8-Amino-7-oxononanoate synthase (also known as 7-keto-8-aminopelargonate synthase, EC 2.3.1.47) is a pyridoxal 5'-phosphate-dependent enzyme which catalyzes the decarboxylative condensation of L-alanine with pimeloyl-CoA in a stereospecific manner to form 8(S)-amino-7-oxononanoate. This is the first committed step in biotin biosynthesis. The mechanism of Escherichia coli AONS has been investigated by spectroscopic, kinetic, and crystallographic techniques. The X-ray structure of the holoenzyme has been refined at a resolution of 1.7 A (R = 18.6%, R(free) = 21. 2%) and shows that the plane of the imine bond of the internal aldimine deviates from the pyridine plane. The structure of the enzyme-product external aldimine complex has been refined at a resolution of 2.0 A (R = 21.2%, R(free) = 27.8%) and shows a rotation of the pyridine ring with respect to that in the internal aldimine, together with a significant conformational change of the C-terminal domain and subtle rearrangement of the active site hydrogen bonding. The first step in the reaction, L-alanine external aldimine formation, is rapid (k(1) = 2 x 10(4) M(-)(1) s(-)(1)). Formation of an external aldimine with D-alanine, which is not a substrate, is significantly slower (k(1) = 125 M(-)(1) s(-)(1)). Binding of D-alanine to AONS is enhanced approximately 2-fold in the presence of pimeloyl-CoA. Significant substrate quinonoid formation only occurs upon addition of pimeloyl-CoA to the preformed L-alanine external aldimine complex and is preceded by a distinct lag phase ( approximately 30 ms) which suggests that binding of the pimeloyl-CoA causes a conformational transition of the enzyme external aldimine complex. This transition, which is inferred by modeling to require a rotation around the Calpha-N bond of the external aldimine complex, promotes abstraction of the Calpha proton by Lys236. These results have been combined to form a detailed mechanistic pathway for AONS catalysis which may be applied to the other members of the alpha-oxoamine synthase subfamily.     

Order-disorder transition in bilayers of diphytanoyl phosphatidylcholine

A comparative study on bilayers of diphytanoyl phosphatidylcholine (DPhPC) and bilayers of dimyristoyl phosphatidylcholine (DMPC) was made by X-ray lamellar diffraction as a function of temperature and the degree of hydration. An order-disorder phase transition of DPhPC reveals an interesting contrast to the standard model of DMPC. Electron density profiles allow us to deduce the conformational changes which occur in the headgroup-glycerol region and in the chain region. The important conclusion is that the lipid headgroup may have different conformational energetics in lipids of different chains. We explain why this is important to protein-membrane interactions.     

Lipid lateral organization in fluid interfaces controls the rate of colipase association.

Colipase, a cofactor of pancreatic triacylglycerol lipase, binds to surfaces of lipolysis reactants, like fatty acid and diacylglycerol, but not to the nonsubstrate phosphatidylcholine. The initial rate of colipase binding to fluid, single-phase lipid monolayers was used to characterize the interfacial requirements for its adsorption. Colipase adsorption rates to phosphatidylcholine/reactant mixed monolayers depended strongly on lipid composition and packing. Paradoxically, reactants lowered colipase adsorption rates only if phosphatidylcholine was present. This suggests that interactions between phosphatidylcholine and reactants create dynamic complexes that impede colipase adsorption. Complex formation was independently verified by physical measurements. Colipase binding rate depends nonlinearly on the two-dimensional concentration of phosphatidylcholine. This suggests that binding is initiated by a cluster of nonexcluded surface sites smaller than the area occupied by a bound colipase. Binding rates are mathematically consistent with this mechanism. Moreover, for each phosphatidylcholine-reactant pair, the complex area obtained from the analysis of binding rates agrees well with the independently measured collapse area of the complex. The dynamic complexes between phosphatidylcholine and lipids, like diacylglycerols, exist independently of the presence of colipase. Thus, our results suggest that lipid complexes may regulate the fluxes of other proteins to membranes during, for example, lipid-mediated signaling events in cells.     

Amphotericin B-sterol complex formation and competition with egg phosphatidylcholine: a monolayer study

The conditions of formation of amphotericin B-cholesterol or -ergosterol complexes in monolayers are investigated by the penetration into a monolayer of egg phosphatidylcholine/sterol of 14C-labelled N-fructosyl-amphotericin B dissolved in the aqueous subphase. An increase of both surface pressure and radioactivity as a function of concentration are observed simultaneously while a 'saturation' effect occurs only for the surface pressure. The experiments are not accurate enough to make conclusions about the number of actually penetrated amphotericin B molecules. Therefore, the existence of an amphotericin B-sterol complex was evidenced from a study of surface pressure area per molecule isotherm. The results indicate that a complex with a 2:1 stoichiometry is formed and that the amphotericin B-ergosterol interaction is larger than the amphotericin B-cholesterol interaction. The complex is dissociated by addition of egg phosphatidylcholine due to a competition between egg phosphatidylcholine and amphotericin B for sterol.     

Study of the physico-chemical properties of prostaglandin F2 alpha and its complexes

To study the intracellular action mechanisms of prostaglandin F2 alpha its interaction with lipid components of biological membranes was investigated. It has been found that prostaglandin forms a complex with phosphatidylcholine and cholesterol. Immobilization ability of phospholipids is changed in the course of complex formation. Ability of prostaglandin F2 alpha for complex formation with insulin was also observed. Combination of monolayer technique and electron microscopy made possible to discover molecular reconstructions when prostaglandin is incorporated into monolayer of biologically active compounds.     

Regulation of phospholipid-ATPase complex interaction by the adenine nucleotide carrier

(1) The effect of phospholipids on a preparation containing the ATPase complex and the adenine nucleotide carrier is studied in the presence of ligands known to affect the conformation of these components of the mitochondrial inner membrane. (2) When ATPase activity is abolished by phospholipid depletion, the reactivation induced by phosphatidylcholine is prevented by the simultaneous addition of ATP. ADP partially reproduces the ATP effect. AMP, GTP, UTP and P_i are ineffective. (3) The influence of ATP is associated with reduced phospholipid binding to the membrane fragments and is reversible. The ATP effect on reconstitution is not manifest when phosphatidylcholine is added together with negatively charged phospholipids. (4) Carboxyatractyloside does not modify the phospholipid-ATPase complex interaction but bongkrekic acid is as effective as ATP. In the presence of ADP, the influence of bongkrekic acid is considerably increased. (5) It is concluded that the binding of ATP to the adenine nucleotide carrier enables the complex to select between the charged and uncharged phospholipids. As a result of the carrier conformational change, the ATPase complex is induced to prefer a negatively charged phospholipid environment.     

Use of bromthymol blue to study the complex of methemoglobin with liposomes

Methemoglobin effect on the structure of phosphatidylcholine liposomes was studied using sulfophthalein dye bromthymol blue. It is shown that formation of the protein-lipid complex is accompanied by the change in conformation of phospholipid polar groups.     

A deactivating conformational change induced by reduced nicotinamide-adenine dinucleotide phosphate in a Neurospora glutamate dehydrogenase.

Stopped-flow fluorescence techniques have been used to observe the formation of the binary comples of E-NADPH. At pH 7.5 there is a protein conformational change after the formation of the binary complex. This conformational change can be detected by a decrease in the fluorescence intensity of the complex at 350 nm and by an increase in its fluorescence intensity at 450 nm.     

Characterization of discoidal complexes of phosphatidylcholine, apolipoprotein A-I and cholesterol by gradient gel electrophoresis

Complexes of egg yolk phosphatidylcholine and apolipoprotein A-I were prepared by a detergent (sodium cholate)-dialysis method and characterized by gradient gel electrophoresis, gel filtration, electron microscopy and chemical analysis. Multicomponent electrophoretic patterns were obtained indicating formation of at least eight classes of discoidal complexes. The relative contribution of the different classes to the electrophoretic pattern was a function of the molar ratio of phosphatidylcholine:apolipoprotein A-I in the interaction mixture. Molar ratios of phosphatidylcholine:apolipoprotein A-I in isolated complexes were strongly and positively correlated with disc diameter obtained by electron microscopy. Incorporation of unesterified cholesterol into phosphatidylcholine/apolipoprotein A-I interaction mixtures also resulted in formation of unique complexes but with considerably different particle size distributions relative to those observed in the absence of cholesterol. One common consequence of cholesterol incorporation into interaction mixtures of 87.5:1 and 150:1 molar ratio of phosphatidylcholine:apolipoprotein A-I was the disappearance of a major complex class with diameter of 10.8 nm and the appearance of a major component with diameter of approximately 8.8 nm. Electrophoretic patterns of cholesterol-containing complexes showed a strong similarity to patterns recently published for high density lipoproteins from plasma of lecithin:cholesterol acyltransferase-deficient subjects, suggesting that the complexes formed in vitro by the detergent-dialysis method may serve as appropriate models for investigation of the origins of the HDL particle size distribution.     

Effects of diacylglycerols on conformation of phosphatidylcholine headgroups in phosphatidylcholine/phosphatidylserine bilayers.

The effects of five diacylglycerols (DAGs), diolein, 1-stearoyl,2-arachidonoyl-sn-glycerol, dioctanoylglycerol, 1-oleoyl,2-sn-acetylglycerol, and dipalmitin (DP), on the structure of lipid bilayers composed of mixtures of phosphatidylcholine and phosphatidylserine (4:1 mol/mol) were examined by 2H nuclear magnetic resonance (NMR). Dipalmitoylphosphatidylcholine deuterated at the alpha- and beta-positions of the choline moiety was used to probe the surface region of the membranes. Addition of each DAG except DP caused a continuous decrease in the beta-deuteron quadrupole splittings and a concomitant increase in the alpha-deuteron splittings indicating that DAGs induce a conformational change in the phosphatidylcholine headgroup. Additional evidence of conformational change was found at high DAG concentrations (> or = 20 mol%) where the alpha-deuteron peaks became doublets indicating that the two alpha-deuterons were not equivalent. The changes induced by DP were consistent with the lateral phase separation of the bilayers into gel-like and fluid-like domains with the phosphatidylcholine headgroups in the latter phase being virtually unaffected by DP. The DAG-induced changes in alpha-deuteron splittings were found to correlate with DAG-enhanced protein kinase C (PK-C) activity, suggesting that the DAG-induced conformational changes of the phosphatidylcholine headgroups are either directly or indirectly related to a mechanism of PK-C activation. 2H NMR relaxation measurements showed significant increase of the spin-lattice relaxation times for the region of the phosphatidylcholine headgroups, induced by all DAGs except DP. However, this effect of DAGs did not correlate with the DAG-induced activation of PK-C.     

Differential interaction of equinatoxin II with model membranes in response to lipid composition

Equinatoxin II is a 179-amino-acid pore-forming protein isolated from the venom of the sea anemone Actinia equina. Large unilamellar vesicles and lipid monolayers of different lipid compositions have been used to study its interaction with membranes. The critical pressure for insertion is the same in monolayers made of phosphatidylcholine or sphingomyelin (approximately 26 mN m(-1)) and explains why the permeabilization of large unilamellar vesicles by equinatoxin II with these lipid compositions is null or moderate. In phosphatidylcholine-sphingomyelin (1:1) monolayers, the critical pressure is higher (approximately 33 mN m(-1)), thus permitting the insertion of equinatoxin II in large unilamellar vesicles, a process that is accompanied by major conformational changes. In the presence of vesicles made of phosphatidylcholine, a fraction of the protein molecules remains associated with the membranes. This interaction is fully reversible, does not involve major conformational changes, and is governed by the high affinity for membrane interfaces of the protein region comprising amino acids 101-120. We conclude that although the presence of sphingomyelin within the membrane creates conditions for irreversible insertion and pore formation, this lipid is not essential for the initial partitioning event, and its role as a specific receptor for the toxin is not so clear-cut.     

The interaction of various lanthanide ions and some anions with phosphatidylcholine vesicle membranes. A 31P NMR study of the surface potential effects

The interaction of various lanthanide ions with vesicles of phosphatidylcholine from egg yolk has been followed by 31P NMR at 30 degrees C. From known magnetic properties of these ions, separation of the paramagnetic shift into a pure contact and a pseudo-contact part was carried out. Binding curves for the contact contribution (F curves) were obtained from vesicles in solutions of sodium salts with monovalent anions over a wide concentration range. These curves should be insensitive to any conformational effects due to ion binding. Indication of a conformational change in the lipid head group at low ion binding was obtained by studying the ratio between the contact and the pseudo-contact contributions. Besides the adsorption of lanthanide ions, specific anion binding to the surface was introduced to account for the enhanced chemical shifts (Cl- < Br- < NO3-). The results were analyzed in terms of the theory for the diffuse double layer (Gouy-Chapman-Grahame) with equilibrium conditions for the adsorbing cations and anions. Simulations of the titration curves furnished parameters for the ion-lipid interactions. The synergism between the cations and anions follows from the potential effects. Comparison of results with lanthanide ions and Ca2+ indicates that the anion adsorption probably depends on the nature of the adsorbed cation. Lanthanide ion binding to L-glycerophosphorylcholine is not influenced by sodium salts. The binding constant for this complex is weaker than with phosphatidylcholine. The chemical shifts for the lanthanide ion complexes with these two phosphorus compounds seem to be about the same.     

Rapid formation of 4-hydroxy-2-nonenal, malondialdehyde, and phosphatidylcholine aldehyde from phospholipid hydroperoxide by hemoproteins

4-Hydroxy-2-nonenal (HNE) and malondialdehyde (MDA) are well-known toxic products of lipid peroxidation. Phosphatidylcholine aldehydes are also known as oxidation products of phosphatidylcholine. The mechanism of the formation of these compounds in vivo has been a long-standing question. We observed that the rapid reaction of hemoproteins (methemoglobin, metmyoglobin, and cytochrome c) with 1-palmitoyl-2-(13-hydroperoxy-cis-9, trans-11-octadecadienoyl) phosphatidylcholine (PLPC-OOH), having a hydroperoxylinoleoyl residue, generated HNE, MDA, and the phosphatidylcholine aldehyde 1-palmitoyl-2-(9-oxononanoyl) phosphatidylcholine. The efficiencies (mol% yield) of the formation of HNE and MDA from decomposed PLPC-OOH by methemoglobin, metmyoglobin, and cytochrome c after incubation for 10 min were 1.6, 1.0, and 1.0% for HNE and 1.2, 0.6, and 0.9% for MDA, respectively. When 1-palmitoyl-2-linoleoyl phosphatidylcholine was incubated with lipoxidase and methemoglobin, the formation of HNE and the phosphatidylcholine aldehyde 1-palmitoyl-2-(9-oxononanoyl) phosphatidylcholine was observed. When 1-palmitoyl-2-arachidonyl phosphatidylcholine was used instead of 1-palmitoyl-2-linoleoyl phosphatidylcholine, the phosphatidylcholine aldehyde 1-palmitoyl-2-oxovaleroyl phosphatidylcholine was obtained. These data suggest that HNE and phosphatidylcholine aldehydes might be rapidly formed from phosphatidylcholine by lipoxygenase and hemoproteins. Furthermore, hemichrome, converted from methemoglobin by deoxycholic acid and ursodeoxycholic acid, showed marked decomposition of HNE. These results suggest that hemoproteins are related to both the formation and the decomposition of HNE.     

Distortion of the catalytic domain of tissue-type plasminogen activator by plasminogen activator inhibitor-1 coincides with the formation of stable serpin-proteinase complexes

Plasminogen activator inhibitor-1 (PAI-1) is a typical member of the serpin family that kinetically traps its target proteinase as a covalent complex by distortion of the proteinase domain. Incorporation of the fluorescently silent 4-fluorotryptophan analog into PAI-1 permitted us to observe changes in the intrinsic tryptophan fluorescence of two-chain tissue-type plasminogen activator (tPA) and the proteinase domain of tPA during the inhibition reaction. We demonstrated three distinct conformational changes of the proteinase that occur during complex formation and distortion. A conformational change occurred during the initial formation of the non-covalent Michaelis complex followed by a large conformational change associated with the distortion of the proteinase catalytic domain that occurs concurrently with the formation of stable proteinase-inhibitor complexes. Following distortion, a very slow structural change occurs that may be involved in the stabilization or regulation of the trapped complex. Furthermore, by comparing the inhibition rates of two-chain tPA and the proteinase domain of tPA by PAI-1, we demonstrate that the accessory domains of tPA play a prominent role in the initial formation of the non-covalent Michaelis complex.     

A conformational change in E. coli DNA polymerase I (Klenow fragment) is induced in the presence of a dNTP complementary to the template base in the active site

It is well established that the insertion of a nucleotide into a growing DNA chain requires a conformational change in the structure of a DNA polymerase. These enzymes have been shown to bind a primer-template in the open conformation and then upon binding of a complementary dNTP undergo a conformational rearrangement to the closed ternary complex. This movement results in the positioning of the incoming nucleotide in the proper geometry for the nucleophilic attack by the 3'-hydroxyl of the primer. In this work, tryptic digestion experiments were performed to detect this conformational change in the structure of the exonuclease-deficient DNA polymerase I (Klenow fragment). Three distinct digestion patterns were observed: one for the polymerase alone, one for the binary complex with the primer-template, and one for the ternary polymerase-DNA-dNTP complex. The latter conformational change leads to a stable ternary closed complex formation only when the correct nucleotide is present in the reaction mixture. Positioning of nucleotides with incorrect geometry in the protein active site inhibits or eliminates formation of the closed complex. Similarly, this conformational change is inhibited when the primer terminus of the DNA molecule is altered by the presence of the 2'-hydroxyl.