A study of the surface-active properties of the Mg2+-activated ATPase from cytoplasmic membranes of Streptococcus faecalis

The surface activity and enzymic properties of the factor F₁, the catalytic moiety of Streptococcus faecalis H⁺-ATPase, has been studied at the air-water and phospholipid-water interfaces. F₁ does not interact with the monolayer phospholipids, hence its adsorption on a biological membrane must be due mainly to its recognition of proteins of the hydrophobic complex. The dimensions of the F₁ molecule at the air-water interface have been estimated. In the presence of Mg²⁺, base area is $\text{S = 1.8 · 10}{}^4\text{A}\text{?}{}^2$, height $\text{h = 27}\text{A}\text{?}$. Bearing in mind the size of a globular subunit, it follows from the measurements that the major F₁ subunits should all lie in the same plane. The ATPase activity of F₁ at the interface is inversely proportional to the monolayer density. With low density monolayer, the specific ATPase activity is higher at the interface than in the bulk of the solution.Adsorption of F₁ at the interface shifts the isoelectric point of the protein, apparently due to changes in its conformation. The findings are discussed relative to the proton-active transport mechanism.     

Two-dimensional crystallization of a membrane protein on a detergent-resistant lipid monolayer

Two-dimensional crystals of a membrane protein, the proton ATPase from plant plasma membranes, have been obtained by a new strategy based on the use of functionalized, fluorinated lipids spread at the air-water interface. Monolayers of the fluorinated lipids are stable even in the presence of high concentrations of various detergents as was established by ellipsometry measurements. A nickel functionalized fluorinated lipid was spread into a monolayer at the air-water interface. The overexpressed His-tagged ATPase solubilized by detergents was added to the subphase. 2D crystals of the membrane protein, embedded in a lipid bilayer, formed as the detergent was removed by adsorption. Electron microscopy indicated that the 2D crystals were single layers with dimensions of 10 microm or more. Image processing yielded a projection map at 9 A resolution, showing three well-separated domains of the membrane-embedded proton ATPase.     

Interaction of the GM2-activator protein with phospholipid-ganglioside bilayer membranes and with monolayers at the air-water interface.

Differential scanning calorimetry (DSC) and film balance measurements were performed to study the interactions of the GalNAcbeta1-->4(NeuAcalpha2-->3)Galbeta1-->4Glc1 -->1'Cer (GM2)-activator protein with phospholipid/ganglioside vesicles and monolayers. The nonglycosylated form of the GM2-activator protein, added to unilamellar lipid vesicles of different composition, causes differential effects on the gel to liquid-crystalline phase transition peaks. The phase transition temperature (Tm) of pure dimyristoylglycerophosphocholine (DMPC) bilayer is slightly decreased. When lipids which specifically bind the GM2-activator protein are incorporated into the vesicles (e.g. a sulfatide or gangliosides) a shoulder in the thermograms at higher temperatures is observed, indicating an increase of the stability of the gel phase in relation to the liquid-crystalline phase. We also studied the surface activity of a glycosylated and a nonglycosylated GM2-activator protein at the air-water interface. The glycosylated form showed a slightly lower surface activity than the GM2-activator protein without oligosaccharide moiety. When the GM2-activator protein is added to the sub-phase of a surface covered with a lipid monolayer, it can only insert into the monolayer and reach the air-water interface below a monolayer pressure of 25 mN.m-1, depending on the lipid composition, and not when the monolayers are at the bilayer equivalence pressure of 30-35 mN.m-1. Particularly for Galbeta1-->3GalNAcbeta1-->4(NeuAcalpha2-->3)Galbeta 1-->4Glc1-->1'Cer (GM1) and GM2 containing films, the critical pressures (picrit) when no additional increase in surface pressure is observed after addition of the protein into the subphase, are much lower. This leads to the conclusion that binding of the GM2 activator protein to the ganglioside headgroups prevents the protein from reaching the air-water interface. The protein is then located preferentially at the lipid-water interface and cannot penetrate into the chain region.     

Thermodynamic and dynamic characteristics of hydroxypropylmethylcellulose adsorbed films at the air-water interface

Surface pressure isotherms and structural and surface dilatational properties of three hydroxypropylmethycelluloses (HPMCs, called E4M, E50LV, and F4M) adsorbed films at the air-water interface were determined. In this work we present evidence that HPMC molecules are able to diffuse and saturate the air-water interface at very low concentrations in the bulk phase. As bulk concentration increased, structural changes at a molecular level occurred at the interface. These changes corresponded to transition from an expanded structure (structure I) to a condensed one (structure II). When the surface concentration of HPMC was high enough, the collapse of the monolayer was observed. The three HPMCs formed very elastic films at the air-water interface, even at low surface pressures. E4M showed features that make it unique. For instance it showed the highest surface activity, mainly at low bulk concentrations (<10(-4) wt %). The differences observed in surface activity may be attributed to differences in the hydroxypropyl molar substitution and molecular weight of HPMC. All three HPMCs formed films of similar viscoelasticity and elastic dilatational modulus, which can be accounted for by their similar degree of methyl substitution.     

Physical Properties of Lipid Monolayers on Alkylated Planar Glass Surfaces

A method for transferring a lipid monolayer from an air-water interface to an alkylated glass slide is described. Specific antibodies bind tightly to lipid haptens contained in these monolayers on the glass slides. We conclude that the polar head groups of the lipids face the aqueous phase. A monolayer containing a fluorescent lipid was used to show that the monolayer is homogeneous as observed with an epifluorescence microscope. A periodic pattern photobleaching technique was used to measure the lateral diffusion of this fluorescent lipid probe in monolayers composed of dipalmitoyl phosphatidylcholine and dimyristoyl phosphatidylcholine. Different regions of the pressure-area isotherms of the monolayers at the air-water interface can be correlated with the diffusion of the fluorescent probe molecules on the monolayer-coated glass slide. Monolayers derived from the so-called “solid-condensed” state of a monolayer at the air-water interface showed a very low probe diffusion coefficient in this monolayer when placed on a glass slide, D ≤ 10^-10 cm²/s. Monolayers derived from the “liquid condensed/liquid expanded” (LC/LE) region of the monolayer isotherms at the air-water interface showed rapid diffusion (D > 10^-8 cm²/s) when these same monolayers were observed on an alkylated glass slide. The monolayers attached to the glass slide appear to be homogeneous when derived from monolayers in the LC/LE region of monolayers at the air-water interface. There is no major variation of the diffusion coefficient of a fluorescent lipid probe when this diffusion is measured on a lipid monolayer on a glass slide, for monolayers derived from various regions of the LC/LE monolayers at the air-water interface. This is consistent with the view that the LC/LE region is most likely a single fluid phase. Monolayers supported on a planar glass substrate are of much potential interest for biophysical and biochemical studies of the interactions between model membranes and cellular membranes, and for physical chemical studies relating the properties of lipid monolayers to the properties of lipid bilayers.     

Identification and properties of an ATPase in vacuolar membranes of Neurospora crassa

Using a vacuolar preparation virtually free of contamination by other organelles, we isolated vacuolar membranes and demonstrated that they contain an ATPase. Sucrose density gradient profiles of vacuolar membranes show a single peak of ATPase activity at a density of 1.11 g/cm3. Comparison of this enzyme with the two well-studied proton-pumping ATPases of Neurospora plasma membranes and mitochondria shows that it is clearly distinct. The vacuolar membrane ATPase is insensitive to the inhibitors oligomycin, azide, and vanadate, but sensitive to N,N'-dicyclohexylcarbodiimide (Ki = 2 microM). It has a pH optimum of 7.5, requires a divalent cation (Mg2+ or Mn2+) for activity, and is remarkably unaffected (+/- 20%) by a number of monovalent cations, anions, and buffers. In its substrate affinity (Km for ATP = 0.2 mM), substrate preference (ATP greater than GTP, ITP greater than UTP greater than CTP), and loss of activity with repeated 1 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid washes, the vacuolar membrane ATPase resembles the F1F0 type of ATPase found in mitochondria and differs from the integral membrane type of ATPase in plasma membranes.     

Exchange and interactions between lipid layers at the surface of a liposome solution.

Lipid organization and lipid transport processes occurring at the air-water interface of a liposome (lipid vesicle) solution are studied by conventional surface pressure-area measurements and interpreted by an adequate theory. At the interface of a dioleoyl phosphatidylcholine vesicle solution, used for demonstration, a well defined two layer structure selfassembles: vesicles disintegrate at the interface forming a surface-adsorbed lipid monolayer, which prevents further disintegration beyond about 1 dyne/cm surface pressure. A layer of vesicles now assembles in close association with the monolayer. This layer is in vesicle diffusion exchange with the solution and in lipid exchange with the monolayer. The lipid exchange occurs exclusively between the monolayer and the outer lipid layer of the vesicles; it is absent between outer and inner vesicle layers. Equilibration of the lipid density in the monolayer with that in the vesicle outer layer provides a coherent and quantitative explanation of the observed hysteresis effects and equilibrium states. The correspondence between monolayer and vesicle outer layer is traced down to equilibrium constants and rate constants and their dependences on surface pressure, vesicle size and concentration. Other alternate realizations of surface structure and exchange, including induced lipid flip-flop within vesicles or vesicle monolayer adhesion or fusion are potential applications of the proposed analysis.     

Exchange and interactions between lipid layers at the surface of a liposome solution

Lipid organization and lipid transport processes occurring at the air-water interface of a liposome (lipid vesicle) solution are studied by conventional surface pressure-area measurements and interpreted by an adequate theory. At the interface of a dioleoyl phosphatidylcholine vesicle solution, used for demonstration, a well defined two layer structure selfassembles: vesicles disintegrate at the interface forming a surface-adsorbed lipid monolayer, which prevents further disintegration beyond about 1 dyne/cm surface pressure. A layer of vesicles now assembles in close association with the monolayer. This layer is in vesicle diffusion exchange with the solution and in lipid exchange with the monolayer. The lipid exchange occurs exclusively between the monolayer and the outer lipid layer of the vesicles; it is absent between outer and inner vesicle layers. Equilibration of the lipid density in the monolayer with that in the vesicle outer layer provides a coherent and quantitative explanation of the observed hysteresis effects and equilibrium states. The correspondence between monolayer and vesicle outer layer is traced down to equilibrium constants and rate constants and their dependences on surface pressure, vesicle size and concentration. p] Other alternate realizations of surface structure and exchange, including induced lipid flip-flop within vesicles or vesicle monolayer adhesion or fusion are potential applications of the proposed analysis.     

Monitoring of phospholipid monolayer hydrolysis by phospholipase A2 by use of polarization-modulated Fourier transform infrared spectroscopy

Polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS) was used to follow the hydrolysis of phospholipid monolayers at the air-water interface by phospholipase A2 (PLA2). The decrease in the intensity of the nuC=O ester band of dipalmitoylphosphatidylcholine at 1733 cm(-1) and the appearance of two new infrared bands in the 1530-1580 cm(-1) region allowed to monitor phospholipid hydrolysis by PLA2. Indeed, the decrease in the intensity of the band at 1733 cm(-1) was attributed to the enzymatic hydrolysis of the acyl ester linkage of the sn-2 fatty acid on the glycerol backbone whereas the doublet appearing at 1537 and 1575 cm(-1) was attributed to the nu(a) COO- vibration of the newly formed calcium-palmitate. The presence of this band as a doublet indicates the formation of a crystalline-like calcium-palmitate monolayer. This observation supports our previously postulated mechanism for the formation of PLA2 domains at the air-water interface. Definitive assignment of the infrared bands has been possible by measuring PM-IRRAS spectra of the individual hydrolysis products (palmitic acid and lysopalmitoylphosphatidylcholine) as well as of 1-caproyl-2-palmitoyl-phosphatidylcholine and 1-palmitoyl-2-caproylphosphatidylcholine monolayers before and after hydrolysis by PLA2.     

Control of the action of phospholipases A by "vertical compression" of the substrate monolayer

Monolayers of rac-1,2-didodecanoyl-sn-glycero-3-phosphoglycerol at an air-water interface were "vertically compressed" by substituting an alkylated glass plate for air while maintaining a constant surface pressure of 15 mN m-1. At this surface pressure the overlaying of the lipid film by the alkylated surface resulted in an average increase of 16 A2/molecule in the mean molecular area of those phospholipid molecules residing at the interface between water and the alkylated glass. Subsequently, the activities of phospholipases A1 and A2 toward the monolayers were measured both in the presence and in the absence of the support. While phospholipase A1 activity was increased 4-fold by the support, the activity of phospholipase A2 was reduced to 15% of the activity measured in the absence of the alkylated surface. These findings indicate that such a "vertical compression" of the monolayer is likely to induce a conformational change in the phospholipid molecules, which in turn would cause the above reciprocal changes in the activities of phospholipases A1 and A2. A molecular model accounting to these findings is presented.     

Incorporation of beta-lactoglobulin in a lipid/porphyrin monolayer at the air--water interface

A catanionic lipid/porphyrin monolayer was formed at the air-water interface by the tetra-anionic porphyrin, tetra-sodium-meso-tetra(4-sulfonatophenyl)porphine (TSPP), mixed with the cationic lipid dioctadecyldimethylammonium bromide (DODAB) in a 1:4 molar ratio. This binary mixture (TSPP/4DODAB) was used as the incorporation matrix of beta-lactoglobulin (betaLG). Binary and ternary systems (TSPP/4DODAB/zbetaLG, where z stands for the number of protein residues per TSPP) were characterized by surface pressure versus area (pi-A) measurements and by Brewster angle microscopy (BAM) observation at the air-water interface. Pi-A measurements and BAM images show that protein is incorporated in the expanded regime of the monolayer and is gradually expelled upon compression at high surface pressures. The successive compression-expansion cycles indicate that the protein under adsorbed to the floating film is reincorporated after the expansion of the monolayer. At low subphase pH, TSPP tends to aggregate decreasing the interaction with DODAB molecules. Electrostatic and hydrophobic interactions are responsible for the presence of betaLG at the interfacial film.     

Structural characteristics of hydrolysates of proteins from extracted sunflower flour at the air-water interface

The structural and topographical characteristics of a sunflower protein isolate (SPI) and its hydrolysates at different degrees of hydrolysis (DH = 5.62%, 23.5%, and 46.3%) spread at the air-water interface at pH 7 and 20 degrees C were determined from pi-A isotherms coupled with Brewster angle microscopy (BAM). The structural characteristics of SP hydrolysate spread monolayers depend on the degree of hydrolysis. We observed a significant shift of the pi-A(APPARENT) isotherms toward lower molecular areas as the degree of hydrolysis (DH) increased. This phenomenon was attributed to spreading of the protein at the interface, especially at DH 46.3%. A change in the monolayer structure was observed at a surface pressure of 12-15 mN/m. At a microscopic level, the heterogeneous monolayer structures visualized near the monolayer collapse and during the monolayer expansion proved the existence of large regions of protein aggregates. Reflectivity increased with surface pressure and was a maximum at the monolayer collapse. The monolayer thickness decreased as the degree of hydrolysis increased. These phenomena explain the poor functional properties for the formation and stabilization of a dispersion (emulsion or foam) of protein hydrolysates at high degrees of hydrolysis.     

Monolayer film behavior of lipopolysaccharide from Pseudomonas aeruginosa at the air-water interface

Lipopolysaccharide (LPS) is an essential biomacromolecule making up approximately 50% of the outer membrane of gram-negative bacteria. LPS chemistry facilitates cellular barrier and permeability functions and mediates interactions between the cell and its environment. To better understand the local interactions within LPS membranes, the monolayer film behavior of LPS extracted from Pseudomonas aeruginosa, an opportunistic pathogen of medical importance, was investigated by Langmuir film balance. LPS formed stable monolayers at the air-water interface and the measured lateral stresses and modulus (rigidity) of the LPS film in the compressed monolayer region were found to be appreciable. Scaling theories for two-dimensional (2D) polymer chain conformations were used to describe the pi-A profile, in particular, the high lateral stress region suggested that the polysaccharide segments reside at the 2D air-water interface. Although the addition of monovalent and divalent salts caused LPS molecules to adopt a compact conformation at the air-water interface, they did not appear to have any influence on the modulus (rigidity) of the LPS monolayer film under biologically relevant stressed conditions. With increasing divalent salt (CaCl2) content in the subphase, however, there is a progressive reduction of the LPS monolayer's collapse pressure, signifying that, at high concentrations, divalent salts weaken the ability of the membrane to withstand elevated stress. Finally, based on the measured viscoelastic response of the LPS films, we hypothesize that this property of LPS-rich outer membranes of bacteria permits the deformation of the membrane and may consequently protect bacteria from catastrophic structural failure when under mechanical-stress.     

Physical and surface properties of insect apolipophorin III

Apolipophorin III (apoLp-III) from Manduca sexta has a molecular weight of 18,100. Based on its hydrodynamic properties (sedimentation and diffusion coefficients, frictional ratio, intrinsic viscosity) and its behavior during gel permeation chromatography, we concluded that apoLp-III is a prolate ellipsoid with an axial ratio of about 3. The circular dichroic spectrum of apoLp-III suggests that the protein contains approximately 50% alpha-helix. At the air-water interface, apoLp-III forms a monolayer which is gaseous at surface pressures less than or equal to 1 dyne/cm. The isotherm of this phase yields an excluded molecular area of 3800 A2/molecule (23 A2/amino acid). At a surface pressure of 22.1 dynes/cm, the monolayer undergoes a phase transition reminiscent of a first-order phase transition of pure lipids. The monolayer can be compressed in this surface pressure range to an area per molecule of 480 A2 (2.9 A2/amino acid). Since a globular protein of molecular weight 18,100 could occupy an area of only about 2000 A2 when bound to a surface, it is suggested that in the expanded state, apoLp-III must unfold on the surface, whereas in the compressed state, the molecule is oriented with its minor axis parallel to the water surface. ApoLp-III binds with high affinity (Kd = 1.9 X 10(-7)M) to both phosphatidylcholine- and diacylglycerol-coated polystyrene beads. All of these results are consistent with the proposal that apoLp-III plays a key role in increasing the capacity of the insect lipoprotein, lipophorin, to transport diacylglycerol by stabilizing the increment of lipid-water interface that results from diacylglycerol uptake.     

Molecular interaction between organophosphorus acid anhydrolase and diisopropylfluorophosphate

Organophosphorus acid anhydrolases (OPAA; E.C.3.1.8.2) are a class of enzymes that hydrolyze a variety of toxic acetylcholinesterase-inhibiting organophosphorus (OP) compounds, including pesticides and fluorine-containing chemical nerve agents. In this paper, subphase conditions have been optimized to obtain stable OPAA Langmuir films, and the diisopropylfluorophosphate (DFP) hydrolysis reaction catalyzed by OPAA in aqueous solution and at the air-water interface was studied. OPAA-DFP interactions were investigated utilizing different spectroscopic techniques, that is, circular dichroism and fluorescence in aqueous solution and infrared reflection absorption spectroscopies at the air-water interface. The characterization of OPAA and its secondary structure in aqueous solution and as a monolayer at the air-water interface in the absence and in the presence of DFP dissolved in aqueous solution or in the aqueous subphase demonstrated significantly distinctive features. The research described herein demonstrated that OPAA can be used in an enzyme-based biosensor for DFP detection.     

A long-lived amphiphilic fluorescent probe studied in POPC air-water monolayer and solution bilayer systems.

A novel amphiphilic fluorescent probe (Fluorazophore-L) with a strongly dipolar, nonionic azoalkane as headgroup and a palmitoyl tail has been synthesized and characterized. Pure Fluorazophore-L was found to be sufficiently amphiphilic to form stable air-water monolayers. An analysis of the surface pressure versus area suggests an area per molecule of about 34+/-2 A(2) at 29 mN m(-1). The partitioning into a lipid membrane model was quantified at the air-water interface by spreading 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) monolayers. Measurements with different molar fractions of Fluorazophore-L revealed a small but significant reduction of the mean area in the mixed monolayer. The excess free energy of mixing (-0.5+/-0.1 kT) indicated a weakly attractive interaction slightly above thermal energy, suggesting a good miscibility of the fluorescent probe within the lipid monolayer without major structural modifications. Spectroscopic measurements confirmed the incorporation of Fluorazophore-L into POPC vesicles. The fluorescence lifetime was very long (125+/-5 ns under air) with monoexponential fluorescence decays.     

Two-dimensional recognition pattern of lipid-anchored Fab'' fragments.

A two-dimensional pattern of oriented antibody fragments was formed at the air-water interface and transferred onto a solid support. The Fab'-fragments of a monoclonal antibody against the hapten dinitrophenyl (DNP) were covalently linked via a hydrophilic spacer to phospholipid vesicles. A monomolecular lipid-protein layer at equilibrium with these vesicles was allowed to form at the air-water interface. The monolayer was separated from the vesicle phase and transferred to a Langmuir-Blodgett trough. By cooling and compressing, the previously homogeneous lipid-protein film was driven into a two-dimensional phase separation resulting in protein-rich domains and a second phase consisting mainly of lipid. This film was transferred onto a solid support in a way that preserved the protein-lipid pattern. The specificity as well as the contrast in the binding activity of the two different separated phases were then quantified using microfluorometry. DNP conjugated to fluorescein-labeled bovine serum albumin (BSA) showed virtually no binding to the lipid regions, but gave a ratio of bound DNP-BSA to Fab'-lipid of greater than 50% in the protein-rich domains proving that the Fab'-moiety retained its biological activity. This demonstrates that the technique presented here is well suited to modify different solid surfaces with a pattern of a given biological function. The optional control of lateral packing and orientation of the components in the monolayer makes it a general tool for the reconstitution of supported lipid-protein membranes and might also open new ways for the two-dimensional crystallization of proteins at membranes.     

Interfacial properties and structural analysis of the antimicrobial peptide NK-2.

The structure of the antimicrobial peptide NK-2 has been studied at the air-water interface and in different solutions using spectroscopic methods such as circular dichroism (CD) and infrared reflection absorption spectroscopy (IRRAS) as well as specular X-ray reflectivity (XR). NK-2 adopts an unordered structure in water, buffer, and in the presence of monomeric cationic and noncharged amphiphiles. However, it forms a stable alpha-helix in 2,2,2-trifluoroethanol (TFE) and in micellar solutions of anionic, cationic as well as nonionic amphiphiles, whereas only in sodium dodecyl sulfonate solutions the alpha-helical structure can also be found below the critical micellar concentration (cmc). The amphiphilic molecule NK-2 is surface active and forms a Gibbs monolayer at the air-buffer interface. In contrast, no adsorption was observed if NK-2 is dissolved in water. During the adsorption process in buffer solutions, NK-2 undergoes a conformational transition from random coil in bulk to alpha-helix at the interface. This change of the peptide's secondary structure is known to be associated with its antimicrobial activity. A comparison of the experimental IRRA spectra with the simulated spectra indicates that the adsorbed NK-2 alpha-helix lies flat at the interface. This is confirmed by XR measurements which show that the thickness of the NK-2 layer is approximately 17 A, which is the average diameter of a alpha-helix, indicating that only a monomolecular adsorption layer is formed.     

Effect of hydrophobic surfactant peptides SP-B and SP-C on binary phospholipid monolayers. I. Fluorescence and dark-field microscopy.

The influence of the hydrophobic proteins SP-B and SP-C, isolated from pulmonary surfactant, on the morphology of binary monomolecular lipid films containing phosphocholine and phosphoglycerol (DPPC and DPPG) at the air-water interface has been studied using epifluorescence and dark-field microscopy. In contrast to previously published studies, the monolayer experiments used the entire hydrophobic surfactant protein fraction (containing both the SP-B and SP-C peptides) at physiologically relevant concentrations (approximately 1 wt %). Even at such low levels, the SP-B/C peptides induce the formation of a new phase in the surface monolayer that is of lower intrinsic order than the liquid condensed (LC) phase that forms in the pure lipid mixture. This presumably leads to a higher structural flexibility of the surface monolayer at high lateral pressure. Variation of the subphase pH indicates that electrostatic interaction dominates the association of the SP-B/C peptides with the lipid monolayer. As evidenced from dark-field microscopy, monolayer material is excluded from the DPPC/DPPG surface film on compression and forms three-dimensional, surface-associated structures of micron dimensions. Such exclusion bodies formed only with SP-B/C peptides. This observation provides the first direct optical evidence for the squeeze-out of pulmonary surfactant material in situ at the air-water interface upon increasing monolayer surface pressures.     

Hisactophilin-mediated binding of actin to lipid lamellae: a neutron reflectivity study of protein membrane coupling.

The neutron reflectivity technique is applied to determine the adsorptive interaction of the 13.5-kDa actin-binding protein hisactophilin from Dictyostelium discoideum with lipid monolayers at a lateral pressure of 21 mN/m < or = pi < or = 25 mN/m at the air-water interface. We compare binding of natural hisactophilin exhibiting a myristic acid chain membrane anchor at the N-terminus (DIC-HIS) and a fatty acid-deficient genetic product expressed in Escherichia coli (EC-HIS). It is demonstrated that only the natural hisactophilin DIC-HIS is capable of mediating the strong binding of monomeric actin to the monolayer, where it forms a layer of about 40 A thickness corresponding to the average diameter of actin monomers. Monolayers composed of pure dimyristoyl phosphatidylcholine with fully deuterated hydrocarbon tails and headgroup (DMPC-d67) and 1:1 mixtures of this lipid with chain deuterated dimyristoyl phosphatidylglycerol (DMPG-d54) are studied on subphases consisting either of fully deuterated buffer (D2O) or of a 9:1 H2O/D2O buffer that matches the scattering length density of air (CMA buffer). The reflectivity data are analyzed in terms of layer models, consisting of one to three layers, depending on the contrast of the buffer and the system. We show that both protein species bind tightly to negatively charged 1:1 DMPC-d67/DMPG-d54 monolayers, thereby forming a thin and most probably monomolecular protein layer of 12-15 A thickness. We find that the natural protein (DIC-HIS) partially penetrates into the lipid monolayer, in contrast to chain-deficient species (EC-HIS), which forms only an adsorbed layer. The coverage of the monolayer with DIC-HIS strongly depends on the presence of anionic DMPG in the monolayer. At a bulk protein concentration of 1.5 micrograms/ml, the molar ratio of bound protein to lipid is about 1:45 for the 1:1 lipid mixture but only 1:420 for the pure DMPC.