Interfacial Enzymes: Membrane Binding, Orientation, Membrane Insertion, and Activity.

Most interfacial enzymes undergo activation upon membrane binding. Interfacial activation is determined not only by the binding strength but also by the specific mode of protein-membrane interactions, including the angular orientation and membrane insertion of the enzymes. This chapter describes biophysical techniques to quantitatively evaluate membrane binding, orientation, membrane insertion, and activity of secreted phospholipase A2 (PLA2) and lipoxygenase (LO) enzymes. Procedures for recombinant production and purification of human pancreatic PLA2 and human 5-lipoxygenase (5-LO) are also presented. Several methods for measurements of membrane binding of peripheral proteins are described, i.e., fluorescence resonance energy transfer (FRET) from tryptophan or tyrosine residues of the protein to a fluorescent lipid in vesicles, changes in fluorescence of an environment-sensitive fluorescent lipid upon binding of proteins to membranes, and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. These methods produce the apparent binding constant, the protein-to-lipid binding stoichiometry, and the Hill cooperativity coefficient. Experimental procedures for segmental isotope labeling of proteins and determination of the orientation of membrane-bound proteins by polarized ATR-FTIR spectroscopy are described. Furthermore, evaluation of membrane insertion of peripheral proteins by a fluorescence quenching technique is outlined. Combination of the orientation and membrane insertion provides a unique configuration of the protein-membrane complex and hence elucidates certain details of the enzyme function, such as the modes of acquisition of a membrane-residing substrate and product release. Finally, assays for determination of the activities of secreted PLA2, soybean LO, and human 5-LO are described.

Toward understanding interfacial activation of secretory phospholipase A2 (PLA2): membrane surface properties and membrane-induced structural changes in the enzyme contribute synergistically to PLA2 activation.

Phospholipase A2 (PLA2) hydrolyzes phospholipids to free fatty acids and lysolipids and thus initiates the biosynthesis of eicosanoids and platelet-activating factor, potent mediators of inflammation, allergy, apoptosis, and tumorigenesis. The relative contributions of the physical properties of membranes and the structural changes in PLA2 to the interfacial activation of PLA2, that is, a strong increase in the lipolytic activity upon binding to the surface of phospholipid membranes or micelles, are not well understood. The present results demonstrate that both binding of PLA2 to phospholipid bilayers and its activity are facilitated by membrane surface electrostatics. Higher PLA2 activity toward negatively charged membranes is shown to result from stronger membrane-enzyme electrostatic interactions rather than selective hydrolysis of the acidic lipid. Phospholipid hydrolysis by PLA2 is followed by preferential removal of the liberated lysolipid and accumulation of the fatty acid in the membrane that may predominantly modulate PLA2 activity by affecting membrane electrostatics and/or morphology. The previously described induction of a flexible helical structure in PLA2 during interfacial activation was more pronounced at higher negative charge densities of membranes. These findings identify a reciprocal relationship between the membrane surface properties, strength of membrane binding of PLA2, membrane-induced structural changes in PLA2, and the enzyme activation.

Secondary structure, orientation, oligomerization, and lipid interactions of the transmembrane domain of influenza hemagglutinin.

Influenza virus hemagglutinin (HA), the viral envelope glycoprotein that mediates fusion between the viral and cellular membranes, is a homotrimer of three subunits, each containing two disulfide-linked polypeptide chains, HA(1) and HA(2). Each HA(2) chain spans the viral membrane with a single putative transmembrane alpha-helix near its C-terminus. Fusion experiments with recombinant HAs suggest that this sequence is required for a late step of membrane fusion, as a glycosylphosphatidylinositol-anchored analogue of HA only mediates "hemifusion" of membranes, i.e., the merging of the proximal, but not distal, leaflets of the two juxtaposed lipid bilayers [Kemble et al. (1994) Cell 76, 383-391]. To find a structural explanation for the function of the transmembrane domain of HA(2) in membrane fusion, we have studied the secondary structure, orientation, oligomerization, and lipid interactions of a synthetic peptide representing the transmembrane segment of X:31 HA (TMX31) by circular dichroism and attenuated total reflection Fourier transform infrared spectroscopy and by gel electrophoresis. The peptide was predominantly alpha-helical in detergent micelles and in phospholipid bilayers. The helicity was increased in lipid bilayers composed of acidic lipids compared to pure phosphatidylcholine bilayers. In planar lipid bilayers, the helices were oriented close to the membrane normal. TMX31 aggregated into small heat-resistant oligomers composed of two to five subunits in SDS micelles. Amide hydrogen exchange experiments indicated that a large fraction of the helical residues were accessible to water, suggesting the possibility that TMX31 forms pores in lipid bilayers. Finally, the peptide increased the acyl chain order in lipid bilayers, which may be related to the preferential association of HA with lipid "rafts" in the cell surface and which may be an important prerequisite for complete membrane fusion.

Uncovering a calcium-regulated membrane-binding mechanism for soybean lipoxygenase-1.

Lipoxygenases catalyze the biosynthesis of leukotrienes, lipoxins, and other lipid-derived mediators that are involved in a wide variety of pathophysiological processes, including inflammation, allergy, and tumorigenesis. Mammalian lipoxygenases are activated by a calcium-mediated translocation to intracellular membranes upon cell stimulation, and cooperate with cytosolic phospholipase A2 at the membrane surface to generate eicosanoids. Although it has been documented that plant cell stimulation increases intracellular Ca2+ concentration and activates cytosolic phospholipase A2, followed by lipoxygenase-catalyzed conversion of the liberated linolenic acid to jasmonic acid, no evidence is available for Ca2+-regulated membrane binding and activity of plant lipoxygenases. Plant lipoxygenases, unlike their mammalian counterparts, are believed to function independently of calcium or membranes. Here we present spectroscopic evidence for a calcium-regulated membrane-binding mechanism of soybean lipoxygenase-1 (L-1). Both calcium and membrane binding affect the structure and the mode of action of L-1. Free L-1 in solution is less accessible to the polar solvent and converts linoleic acid to conjugated dienes, whereas surface binding increases solvent accessibility and stimulates conjugated ketodiene production. Calcium exerts a biphasic effect on the structure and activity of L-1. Our results uncover a new regulatory mechanism for plant lipoxygenases and delineate common features in animal and plant cell signaling pathways.

Structural dynamics of the Streptomyces lividans K+ channel (SKC1): secondary structure characterization from FTIR spectroscopy.

Fourier transform infrared (FTIR) spectroscopy was used to probe the secondary structure, orientation, and the kinetics of amide hydrogen-deuterium exchange (HX) of the small K+ channel from Streptomyces lividans. Frequency component analysis of the amide I band showed that SKC1 is composed of 44-46% alpha-helix, 21-24% beta-sheet, 10-12% turns and 18-20% unordered structures. The order parameter S of the helical component of SKC1 was between 0.60 and 0.69. Close to 80% of SKC1 amide protons exchange within approximately 3 h of D2O exposure, suggesting that the channel is largely accessible to solvent exchange. These results are consistent with a model of SKC1 in which helices slightly tilted from the membrane normal line the water-filled vestibules that flank the K+ selectivity filter.

Structural changes in a secretory phospholipase A2 induced by membrane binding: a clue to interfacial activation?

Activation of phospholipase A2 (PLA2) upon binding to phospholipid assemblies is poorly understood. X-ray crystallography revealed little structural change in the enzyme upon binding of monomeric substrate analogs, whereas small conformational changes in PLA2 complexed with substrate micelles and an inhibitor were found by NMR. The structure of PLA2 bound to phospholipid bilayers is not known. Here we uncover by FTIR spectroscopy a splitting in the alpha-helical region of the amide I absorbance band of PLA2 upon binding to lipid bilayers. We provide evidence that a higher frequency component, which is only observed in the membrane-bound enzyme, is a property of more flexible helices. Formation of flexible helices upon interaction with the membrane is likely to contribute to PLA2 activation.

Reversible pH-dependent conformational change of reconstituted influenza hemagglutinin.

Fusion between influenza virus and cell membranes is mediated by a major acid-induced conformational change of the spike glycoprotein of the viral envelope, hemagglutinin (HA). The conformational change of HA is commonly believed to be a kinetically controlled irreversible process, although the experimental evidence for this is controversial. Here we show by polarized infrared spectroscopy that the previously described acid-induced inclination of HA reconstituted in supported phospholipid bilayers is reversible in the absence, but irreversible in the presence, of bound target membranes. We also demonstrate reversible pH-dependent changes in the capability of reconstituted HA to bind target membranes. These results support a thermodynamically controlled mechanism of the conformational change of HA and provide new insight into the understanding of the energetics of influenza-mediated membrane fusion.

Effect of the N-terminal glycine on the secondary structure, orientation, and interaction of the influenza hemagglutinin fusion peptide with lipid bilayers.

The amino-terminal segment of the membrane-anchored subunit of influenza hemagglutinin (HA) plays a crucial role in membrane fusion and, hence, has been termed the fusion peptide. We have studied the secondary structure, orientation, and effects on the bilayer structure of synthetic peptides corresponding to the wild-type and several fusogenic and nonfusogenic mutants with altered N-termini of the influenza HA fusion peptide by fluorescence, circular dichroism, and Fourier transform infrared spectroscopy. All peptides contained segments of alpha-helical and beta-strand conformation. In the wild-type fusion peptide, 40% of all residues were in alpha-secondary and 30% in beta-secondary structures. By comparison, the nonfusogenic peptides exhibited larger beta/alpha secondary structure ratios. The order parameters of the helices and the amide carbonyl groups of the beta-strands of the wild-type fusion peptide were measured separately, based on the infrared dichroism of the respective absorption bands. Order parameters in the range 0.1-0.7 were found for both segments of the wild-type peptide, which indicates that they are most likely aligned at oblique angles to the membrane normal. The nonfusogenic but not the fusogenic peptides induced splitting of the infrared absorption band at 1735 cm(-1), which is assigned to stretching vibrations of the lipid ester carbonyl bond. This splitting, which reports on an alteration of the hydrogen bonds formed between the lipid ester carbonyls and water and/or hydrogen-donating groups of the fusion peptides, correlated with the beta/alpha ratio of the peptides, suggesting that unpaired beta-strands may replace water molecules and hydrogen-bond to the lipid ester carbonyl groups. The profound structural changes induced by single amino acid replacements at the extreme N-terminus of the fusion peptide further suggest that tertiary or quaternary structural interactions may be important when fusion peptides bind to lipid bilayers.

Influenza hemagglutinin assumes a tilted conformation during membrane fusion as determined by attenuated total reflection FTIR spectroscopy.

Fusion of influenza virus with target membranes is mediated by an acid-induced conformational change of the viral fusion protein hemagglutinin (HA) involving an extensive reorganization of the alpha-helices. A 'spring-loaded' displacement over at least 100 A provides a mechanism for the insertion of the fusion peptide into the target membrane, but does not explain how the two membranes are brought into fusion contact. Here we examine, by attenuated total reflection Fourier transform infrared spectroscopy, the secondary structure and orientation of HA reconstituted in planar membranes. At neutral pH, the orientation of the HA trimers in planar membranes is approximately perpendicular to the membrane. However, at the pH of fusion, the HA trimers are tilted 55-70 degrees from the membrane normal in the presence or absence of bound target membranes. In the absence of target membranes, the overall secondary structure of HA at the fusion pH is similar to that at neutral pH, but approximately 50-60 additional residues become alpha-helical upon the conformational change in the presence of bound target membranes. These results are discussed in terms of a structural model for the fusion intermediate of influenza HA.

Secondary structure and orientation of phospholamban reconstituted in supported bilayers from polarized attenuated total reflection FTIR spectroscopy.

We have studied the secondary structure of native phospholamban (PLB), a 52-residue integral membrane protein that regulates calcium uptake into the cardiac sarcoplasmic reticulum, as well as its 27-residue carboxy-terminal transmembrane segment (PLB26-52). The relative contents of alpha-helix, beta-strand, and random coil, as well as the spatial orientations of the alpha-helices of these molecules, reconstituted in dimyristoylphosphatidylcholine (DMPC) and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) bilayer membranes, were determined using polarized attenuated total reflection (ATR) Fourier transform infrared (FTIR) spectroscopy. The major component of the amide I' bands of PLB and PLB26-52 was centered at 1654-1657 cm-1 and was assigned to alpha-helix. The fraction of alpha-helix in native PLB was 64-67% (33-35 residues), and the transmembrane peptide PLB26-52 contained 73-82% alpha-helix (20-22 residues); small fractions of beta- and random structures were also identified. The orientational order parameter (S) of the alpha-helical component of PLB26-52 in DMPC was S = 0.86 +/- 0.09, indicating that the transmembrane helix was oriented approximately perpendicular to the membrane plane. Assuming the transmembrane domain of PLB resembles the peptide PLB26-52, the additional alpha-helical residues in PLB were assigned to the cytoplasmic helix and determined to have an order parameter S = -0.15 +/- 0.30. This may imply that the cytoplasmic helix was tilted from the membrane normal by an angle of 61 +/- 13 degrees or, alternatively, may indicate a wide angular distribution.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional reconstitution of recombinant phospholamban with rabbit skeletal Ca(2+)-ATPase.

Phospholamban (PLB) is a small, transmembrane protein that resides in the cardiac sarcoplasmic reticulum (SR) and regulates the activity of Ca(2+)-ATPase in response to beta-adrenergic stimulation. We have used the baculovirus expression system in Sf21 cells to express milligram quantities of wild-type PLB. After purification by antibody affinity chromatography, the function of this recombinant PLB was tested by reconstitution with Ca(2+)-ATPase purified from skeletal SR. The results obtained with recombinant PLB were indistinguishable from those obtained with purified, canine cardiac PLB. In particular, PLB reduced the apparent calcium affinity of Ca(2+)-ATPase but had no effect on Vmax. At pCa 6.8, PLB inhibited both calcium uptake and ATPase activity of Ca(2+)-ATPase by 50%. This inhibition was fully reversed by addition of a monoclonal antibody to PLB, which mimics the physiological effects of PLB phosphorylation. Maximal PLB regulatory effects occurred at a molar stoichiometry of approximately 3:1, PLB/Ca(2+)-ATPase. We also investigated peptides corresponding to the two main domains of PLB. The membrane-spanning domain, PLB26-52, appeared to uncouple ATPase hydrolysis from calcium transport, even though the permeability of the reconstituted vesicles was not altered. The cytoplasmic peptide, PLB1-31, had little effect, even at a 300:1 molar excess over Ca(2+)-ATPase.

Characterization of two membrane-bound forms of OmpA.

The insertion of the outer membrane protein A (OmpA) into lipid bilayers was studied by limited proteolysis, polarized Fourier transform infrared (FTIR) spectroscopy, and fluorescence spectroscopy. In the native state, OmpA is thought to form a barrel of eight antiparallel beta-strands. For the present study, it was isolated in an unfolded form, purified, and exposed to performed vesicles of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), and three phospholipids that were brominated in different positions of their sn-2 chains (4,5-BrPC, 9,10-BrPC, and 11,12-BrPC). Limited proteolysis revealed two membrane-bound forms of OmpA, namely an "adsorbed" (35 kDa) and an "inserted" (30 kDa) form [Surrey, T., & Jähnig, F. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 7457-7461]. Which form was found after membrane binding and refolding depended on the lipids used and on the temperature. Polarized attenuated total reflection (ATR)-FTIR spectra were recorded with OmpA bound to germanium-supported bilayers in both forms. The position of the amide I' band indicated quite large fractions of beta-structure of OmpA in both membrane-bound forms (35-45% in the adsorbed form and 45-55% in the inserted form). Measurements of the linear dichroism of the amide I' bands in the inserted form are consistent with an antiparallel beta-barrel in which the strands are inclined at about 36 degrees from the membrane normal. The average angle of the beta-strands to the bilayer normal is likely larger in the 35 kDa form than in the inserted form.(ABSTRACT TRUNCATED AT 250 WORDS)

Orientation of functional and nonfunctional PTS permease signal sequences in lipid bilayers. A polarized attenuated total reflection infrared study.

Synthetic peptides corresponding to the N-terminal 23 and 22 residues, respectively, of two integral plasma membrane proteins of Escherichia coli, namely the mannitol- and glucitol-specific permeases of the bacterial sugar phosphotransferase system, were incorporated into single planar phospholipid bilayers supported on germanium plates. Polarized attenuated total reflection infrared spectra were recorded, and order parameters were derived from the measured dichroic ratios. The order parameters of the two wild-type peptides which form amphiphilic alpha-helices in membranes were -0.4 to -0.5, indicating a preferential alignment of the alpha-helix long axis parallel to the membrane surface. Nonfunctional mutant peptides of the mannitol permease sequence in which serine-3 or aspartate-4 were substituted with prolines (S3P and D4P) or lysine (D4K), but which were still largely alpha-helical, exhibited peptide order parameters close to zero, indicating a high degree of disorder of these peptides in the lipid bilayers. The lipid was well ordered at low concentrations of peptides in the membranes but became disordered at high peptide concentrations. This effect of lipid disordering was more pronounced for the D4K mutant than for the wild-type mannitol peptide.

A neutron diffraction study of the influence of ions on phospholipid membrane interactions.

Neutron diffraction is used to examine the effects of Ca2+ and ClO4- ions on interactions and some structural features of dipalmitoylphosphatidylcholine membranes in both solid and fluid lamellar phases. The results are described within the framework of Derjaguin-Landau-Verwey-Overbeek (DLVO) theory with reference to electrostatic, van der Waals, and hydration components of disjoining pressure. The Hamaker constants are evaluated under equilibrium conditions. Addition of 100 mM CaCl2 to the aqueous phase substantially increases the lamellar repeat spacing (d), which is interpreted in terms of adsorption of Ca2+ ions to bilayers followed by electrostatic repulsion between membranes. The rise of NaClO4 concentration in the presence of 100 mM CaCl2 leads to gradual decrease in d, evidently resulted from the diminution of Ca(2+)-induced positive surface potential by both electrostatic screening and binding of ClO4- ions. In the absence of CaCl2, elevation of NaClO4 concentration to 100-300 mM drastically enhances the repeat spacing and then dramatically decreases d at about 1 M NaClO4. Estimation of the hydration coefficients showed that the pronounced decrease of the repeat spacing at high NaClO4 concentrations was resulted mainly from the (partial) disruption of the structure of intermembrane bound water by chaotropic ClO4- ions and subsequent decrease in hydration repulsive pressure. Moreover, in the case of solid membranes (20 degrees C) high concentrations of ClO4- induced formation of interdigitated phase paralleled with marked reduction in bilayer thickness and corresponding increase in the effective cross-sectional area per lipid molecule.

Effect of ion adsorption on the electrokinetic properties of erythrocytes.

The microelectrophoresis technique was used to determine the dependence of human erythrocyte surface potential on the concentration of various cations and anions. The interpretation of the results is based on the Gouy--Chapman--Stern theory. Values of pK, characterizing the binding of ions to the external surface of erythrocytes, as well as numbers of binding sites per unit area were determined. The affinities of ions for the red cell membrane were shown to decrease in the sequence: H+ greater than Ca2+ greater than Sr2+ greater than Mg2+ greater than Ba2+ greater than Li+ greater than Na+ congruent to congruent to K+ congruent to NH4+ and trinitrophenol greater than IO4- greater than CIO4- greater than salicylate congruent to I- greater than greater than SCN- greater than H2PO4- greater than Br- greater than Cl- greater than HPO4(2-). Changes in the ionic strength of the medium resulted in changes in numbers of exposed ion-binding sites. This phenomenon is interpreted in terms of ionic strength-dependent structural transformations of the cell surface coat.

Binding of alkaline-earth metal cations and some anions to phosphatidylcholine liposomes.

The dependence of electrophoretic mobility of multilamellar liposomes composed of egg phosphatidylcholine (PtdCho), dimyristoyl-glycerophosphocholine (Myr2Gro-P-Cho) and dipalmitoyl-glycerophosphocholine (Pam2-Gro-P-Cho) on the concentration of several cations and anions has been measured. Values of surface densities of binding sites and intrinsic binding constants of ions to liposome membranes were determined by processing the results in the framework of Gouy-Stern theory. Sharp reductions in the positive surface potential of Myr2Gro-P-Cho and Pam2Gro-P-Cho liposomes have been detected at the thermotropic transition of the lipids from the gel to liquid-crystalline phase. Similar alterations of liposome surface potential were revealed at the temperature of pretransition, as well as at about 50 degrees C, in the case of Pam2Gro-P-Cho. A model is suggested for ion binding to PtdCho membranes, according to which the ion-binding sites are considered as point defects (vacancies) in the structure of lipid head-groups arranged over a trigonal lattice.

[Electrokinetic and functional characteristics of natural killers in mice]. / Elektrokineticheskaia i funktsional'naia kharakteristiki estestvennykh killerov u myshei.

Using adsorption methods and elimination of Thy-1+ lymphocytes, the null cell fraction was obtained. The electrophoretic mobilities of unfractionated splenocytes and different fractions of lymphoid cells and null fractions (to which the natural killers belong) were measured with "Parmoquant-2" device. Simultaneously, the natural killer activity of unfractionated splenocytes and null cell fractions was examined.

Fluidity-dependence of membrane adhesiveness can be explained by thermotropic shifts in surface potential.

It is demonstrated that the transition of both dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) from the gel to liquid-crystalline phase is paralleled by a pronounced increase in the negative surface potential of liposomes composed of either lipid. The Derjaguin-Landau-Verwey-Overbeek (DLVO) theory is applied to show that this phenomenon can serve as a simple explanation of diverse adhesive properties of solid and fluid lipid bilayers.