Cell surface hydrophobicity and the orientation of certain bacteria at interfaces

Summary Individual cells of Flexibacter aurantiacus CW7 and Hyphomicrobium vulgare ZV580 orientate themselves perpendicularly to the interface in air-water, oil-water and solid-water systems. Electrostatic phenomena probably are not involved in this orientation, since no evidence was found of any localized distribution of positively-charged ionogenic groups on the bacterial surface. It is suggested that the orientation results from a relatively hydrophobic portion of each cell being rejected from the aqueous phase of the system. This property also may be related to the formation of rosettes by these bacteria. Electron micrographs of thin sections of cells sorbed to araldite blocks show that the cell proper is not in contact with the solid surface, but is anchored to it by extracellular adhesive material. The extracellular materials may be of a polysaccharide nature.     

A model of pancreatic lipase and the orientation of enzymes at interfaces

A model for the interfacial orientation and the mode of action of lipase is proposed. Lipase is oriented so that its active site is near the oil-water boundary. This orientation is achieved by oil-enzyme bonding at the “hydrophobic head” of the enzyme, a region free of electric charges and relatively resistant to unfolding. The measured K_M is a complex constant including the dissociation constant of this oil-enzyme “complex”. The interfacial orientation of lipase is further aided by hydrophilic negative charges on the “back” of the enzyme and by a hydrophilic carbohydrate “tail”.It is suggested that similar hydrophobic heads and hydrophilic tails and asymmetric charge distributions establish the orientation of many enzymes which act at interfaces. Many phospholipases, for instance, appear to be charge-oriented, and the carbohydrate residues of ribonucleases and many other glycoproteins may be hydrophilic tails.Lipase is probably a serine enzyme with a catalytic center similar to that of chymotrypsin, but more hindered, perhaps owing to the presence of a leucine residue, and there is no binding of substrate lipid chains in the “active complex”. The substrate molecule is fixated on the enzyme in a two-dimensional orientation, because its leaving alkoxy group must be received by the serine hydroxyl hydrogen which is directed towards the imidazol ring of the reactive histidine through a hydrogen bond. The high turnover rate of lipolysis, 5 × 10⁵/min, exceptional even for an enzyme, results from the extremely high substrate concentration near the active site, and from an almost complete extrusion of water because of the hydrophobicity of both the active site and the substrate. In addition, both substrate and enzyme, because of their polarity, are already so favorably positioned at the interface that the formation of the “active complex” requires only a proper two-dimensional alignment, perhaps with partial extraction of the substrate molecule from the lipid phase.     

Specific sequence combinations at parallel and antiparallel helix-helix interfaces

Orientation of helices at parallel and antiparallel helix-helix interfaces in proteins depends on interacting amino acids from both helices. Particularly important are amino acids at positions analogous to a and d in GCN4 leucine zipper nomenclature, which form hydrophobic core. In this work repeating sequence combinations at a and d positions characteristic for both parallel and antiparallel packing are shown. Layer packing of hydrophobic groups is compared for possible combinations of aliphatic amino acids at all four positions. Correlation between specific position of methyl groups and interhelical angle is found for parallel and antiparallel types of packing.     

Molecular dynamics of nanoparticle translocation at lipid interfaces

We report molecular dynamics simulations of bare and hydrophilic C60 nanoparticles at a dipalmitoylphosphatidylcholine–water interface representing a model lung surfactant layer. Bare C60 particles penetrate into the lipid layer from the vapour to sit just before the lipid head groups while hydrophilic nanoparticles penetrate into the head-group–water interface. The potential of mean force shows how the preferred position varies with the density of the lipid layer (in the physiological range) and with hydrophilicity. We conclude that C60 nanoparticles will not spontaneously diffuse across a surfactant monolayer but that functionalised nanoparticles may, depending on the membrane density, translocate the membrane to reach the water phase in 10–20 ns.     

Specific interaction and two-dimensional crystallization of histidine tagged yeast RNA polymerase I on nickel-chelating lipids.

Nickel-chelating lipid monolayers were used to generate two-dimensional crystals from yeast RNA polymerase I that was histidine-tagged on one of its subunits. The interaction of the enzyme with the spread lipid layers was found to be imidazole dependent, and the formation of two-dimensional crystals required small amounts of imidazole, probably to select the specific interaction of the engineered tag with the nickel. Two distinct preparations of RNA polymerase I tagged on different subunits yielded two different crystal forms, indicating that the position of the tag determines the crystallization process. The orientation of the enzyme in both crystal forms is correlated with the location of the tagged subunits in a three-dimensional model which shows that the tagged subunits are in contact with the lipid layer.     

Some properties of chlorophylla at hydrocarbon-water interfaces and in black lipid membranes

Summary Chlorophylla is very surface active in the system 2,2,4-trimethylpentanewater. The standard free energy of adsorption may be as high as 10.6 kcal/mole. However, chlorophyll adsorption at this interface is unable to stabilize black membranes. Black films formed from solutions of glyceryl monooleate and chlorophylla exhibit a weak fluorescence which indicates that a small amount of pigment, ca. 1 to 2% by area, may be contained in the membranes. Calculations based on adsorption data show that inclusion of somewhat more chlorophylla than this might be expected. However, interfacial tension data for mixed solutions do not support this expectation.Whether or not they are illuminated, black lipid membranes formed from mixed solutions of chlorophylla and glyceryl monooleate have electrical properties indistinguishable from those of films made in the absence of pigment.     

Simulation of two-dimensional streptavidin crystallization

We present lattice Monte Carlo simulations of the growth of streptavidin islands at a biotinylated lipid layer. The model employed takes into account attractive anisotropic lateral interactions between streptavidin tetramers. With a minimal set of interactions, we reproduce the formation of rectangular islands experimentally observed at pH > or = 9.0. Specifically, we analyze two scenarios of the island growth. First, if streptavidin is rapidly adsorbed at t = 0 (stepwise coverage change without ongoing adsorption), the average linear island size is found to grow according to the Lifshitz-Slyozov law, R proportional to t(1/3). Second, if the island growth occurs in parallel with streptavidin adsorption limited by diffusion in the solution, the Lifshitz-Slyozov law is also applicable, but only at the late stage, when the streptavidin coverage is appreciable.     

Conditions of two-dimensional crystallization of cholera toxin B-subunit on lipid films containing ganglioside GM1

A systematic study of the lipid-layer two-dimensional crystallization technique has been carried out on the system composed of cholera toxin B-subunit and monosialoganglioside GM1, by electron microscopy, image analysis, and lipid film surface pressure measurements. Concentrations of protein and lipid components required for two-dimensional crystallization of toxin-GM1 complexes have been determined. Crystals were only obtained in the presence of mixed lipid films, composed of GM1 and of unsaturated lipids, such as dioleoylphosphatidylcholine or dioleoylphosphatidylethanolamine, in agreement with a previous report [D. S. Ludwig et al., (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 8585–8588]. Crystals were obtained with cholera toxin B-subunit concentration as low as 5 μg/ml, as well as in the presence of protein contaminants. They were obtained over a wide range of concentrations of both GM1 and unsaturated lipids. The minimal lipid amount needed for crystallization corresponded to a lipid monolayer at, or near, the maximal spreading pressure (50 mN/m). The use of an excess of lipid resulted in a stabilization of lipid monolayers and in a higher reproducibility or crystallization experiments.     

Electrical interactions of membrane active peptides at lipid/water interfaces

Vital functions of biological membranes are frequently controlled by amphipathic peptides that are associated with the lipid bilayer. The extent of association is largely determined by influences encountered at the interface between the aqueous and lipid moieties, especially involving electrostatic interactions. A basic thermodynamic analysis is presented in terms of a partitioning equilibrium where the membrane is treated as a non-ideal solution of peptide molecules in a two-dimensional lipid solvent. This may then be employed to interpret experimental association isotherms (i.e. the ratio of associated peptide per lipid plotted versus the free aqueous peptide concentration) in the light of a molecular mechanism. Special emphasis is directed towards the evaluation of original titration data under most general circumstances when the association can be monitored using a suitable linear signal (preferentially an optical one). The experimental approaches as well as the merits regarding possible information about the underlying structural and functional features are discussed with pertinent practical examples.     

Impact of hapten presentation on antibody binding at lipid membrane interfaces

We report the effects of ligand presentation on the binding of aqueous proteins to solid supported lipid bilayers. Specifically, we show that the equilibrium dissociation constant can be strongly affected by ligand lipophilicity and linker length/structure. The apparent equilibrium dissociation constants (K_D) were compared for two model systems, biotin/anti-biotin and 2,4-dinitrophenyl (DNP)/anti-DNP, in bulk solution and at model membrane surfaces. The binding constants in solution were obtained from fluorescence anisotropy measurements. The surface binding constants were determined by microfluidic techniques in conjunction with total internal reflection fluorescence microscopy. The results showed that the bulk solution equilibrium dissociation constants for anti-biotin and anti-DNP were almost identical, K_D(bulk) = 1.7 ± 0.2 nM vs. 2.9 ± 0.1 nM. By contrast, the dissociation constant for anti-biotin antibody was three orders of magnitude tighter than for anti-DNP at a lipid membrane interface, K_D = 3.6 ± 1.1 nM vs. 2.0 ± 0.2 μM. We postulate that the pronounced difference in surface binding constants for these two similar antibodies is due to differences in the ligands’ relative lipophilicity, i.e., the more hydrophobic DNP molecules had a stronger interaction with the lipid bilayers, rendering them less available to incoming anti-DNP antibodies compared with the biotin/anti-biotin system. However, when membrane-bound biotin ligands were well screened by a poly(ethylene glycol) (PEG) polymer brush, the K_D value for the anti-biotin antibody could also be weakened by three orders of magnitude, 2.4 ± 1.1 μM. On the other hand, the dissociation constant for anti-DNP antibodies at a lipid interface could be significantly enhanced when DNP haptens were tethered to the end of very long hydrophilic PEG lipopolymers (K_D = 21 ± 10 nM) rather than presented on short lipid-conjugated tethers. These results demonstrate that ligand presentation strongly influences protein interactions with membrane-bound ligands.     

Bombyx mori silk fibroin liquid crystallinity and crystallization at aqueous fibroin-organic solvent interfaces.

A banded morphology has been observed for Bombyx mori silk fibroin films obtained from an aqueous hexane interface; the period of the banding is approximately 1 microm. Morphology and diffraction from different regions of the banded structure suggest that it is a free surface formed by a cholesteric liquid crystal. Truncated hexagonal lamellar crystallites of B. mori silk fibroin have been observed in films formed in the surface excess layer of fibroin at the interface between aqueous fibroin and hexane or chloroform. Based on initial crystallographic evidence, a three-fold helical conformation has been ascribed to the fibroin chains within the crystals. The chain conformation and crystalline habit appear to be similar to the silk III structure previously observed at the air-water interface (Valluzzi R, Gido SP. Biopolymers 1997;42:705-717; Valluzzi R, Gido S, Zhang W, Muller W, Kaplan D. Macromolecules 1996;29:8606-8614) but the crystalline packing is different. Diffraction data obtained for the crystallites are similar to diffraction behavior for a collagen-like model peptide. Diffraction patterns obtained from crystallized regions of the banded morphology can be indexed using the same unit cell as the hexagonal lamellar crystallites. Surfactancy of fibroin and subsequent aggregation and mesophase formation may help to explain the liquid crystallinity reported for silk, which is long suspected to play a role in the biological silk spinning process (Valluzzi R, Gido SP. Biopolymers 1997;42:705-717; Willcox, P. J.; Gido, SP, Muller W, Kaplan DL. Macromolecules 1996:29:5106-5110; Magoshi J, Magoshi Y, Nakamura S. In: Kaplan D, Adams W, Farmer B, Viney C, editors, Mechanism of Fiber Formation of Silkworm. Washington, DC: American Chemical Society 1994:292-310; Magoshi J, Magoshi Y, Nakamura S. J Appl Polym Sci Appl Polym Symp 1985;41:187-204; Magoshi J, Magoshi Y, Nakamura S. Polym Commun 1985;26:309.).     

Formation of stable polypeptide monolayers at interfaces: controlling molecular conformation and orientation.

The molecular self-organization and structural properties of peptide assemblies at different interfaces, using either amphipathic or hydrophobic polypeptide helices, is described. The two peptides under investigation form stable monolayers on the water surface under the conservation of their molecular conformation, as studied by circular dichroism and polarization-modulation Fourier transform infrared (FTIR) spectroscopy. Using surface plasmon resonance and reflection-absorption FTIR, we show that such molecular layers can be transferred unaltered to solid substrates. Most importantly, the molecular orientation of the hydrophobic helices on solid supports such as gold can be controlled by choosing a particular procedure for the layer formation. The helices were oriented parallel to the interface in Langmuir-Blodgett monolayers, and perpendicular to the interface in self-assembled monolayers. Our reflection-absorption FTIR measurements have delivered for the first time direct experimental evidence for the molecular conformation and orientation of pure peptide monolayers. Suitable reference spectra of polypeptides with defined conformation and orientation are necessary to use this technique for the determination of the molecular orientation of peptides in monomolecular films. We have solved the problem for alpha-helical polypeptides by using bacteriorhodopsin as a reference in combination with synthetic alpha-helices of defined interfacial orientation. The present study shows the possibility of constructing immobilized peptide monolayers with predefined macroscopic properties and molecular structure by choosing the proper polypeptide amino acid sequence, the technique used for layer formation, and the supporting surface properties.     

X-ray and neutron surface scattering for studying lipid/polymer assemblies at the air-liquid and solid-liquid interfaces

Simple mono- and bilayers, built of amphiphilic molecules and prepared at air-liquid or solid-liquid interfaces, can be used as models to study such effects as water penetration, hydrocarbon chain packing, and structural changes due to head group modification. In the paper, we will discuss neutron and X-ray reflectometry and grazing incidence X-ray diffraction techniques used to explore structures of such ultra-thin organic films in different environments. We will illustrate the use of these methods to characterize the morphologies of the following systems: (i) polyethylene glycol-modified distearoylphosphatidylethanolamine monolayers at air-liquid and solid-liquid interfaces; and (ii) assemblies of branched polyethyleneimine polymer and dimyristoylphophatidylcholine lipid at solid-liquid interfaces.     

Intrinsic molecules in lipid membranes change the lipid-domain interfacial area: cholesterol at domain interfaces

A theoretical analysis of the effects of intrinsic molecules on the lateral density fluctuations in lipid bilayer membranes is carried out by means of computer simulations on a microscopic interaction model of the gel-to-fluid chain-melting phase transition. The inhomogeneous equilibrium structures of gel and fluid domains, which in previous work (Cruzeiro-Hansson, L. and Mouritsen, O.G. (1988) Biochim. Biophys. Acta 944, 63-72) were shown to characterize the transition region of pure lipid membranes, are here shown to be enhanced by intrinsic molecules such as cholesterol. Cholesterol is found to increase the interfacial area and to accumulate in the interfaces. The interfacial area, the average cluster size, the lateral compressibility, and the membrane area are calculated as functions of temperature and cholesterol concentration. It is shown that the enhancement by cholesterol of the lateral density fluctuations and the lipid-domain interfacial area is most pronounced away from the transition temperature. The implications of the results are discussed in relation to passive ion permeability and function of interfacially active enzymes such as phospholipase.     

Effect of monolayer lipid charges on the structure and orientation of protein VAMP1 at the air-water interface

SNARE proteins are implicated in membrane fusion during neurotransmission and peptide hormone secretion. Relatively little is known about the molecular interactions of their trans- and juxtamembrane domains with lipid membranes. Here, we report the structure and the assembling behavior of one of the SNARE proteins, VAMP1/synaptobrevin1 incorporated in a lipid monolayer at an air-water interface which mimics the membrane environment. Our results show that the protein is extremely sensitive to surface pressure as well as the lipid composition. Monolayers of proteins alone or in the presence of the neutral phospholipid DMPC underwent structural transition from α-helix to β-sheet upon surface compression. In contrast, the anionic phospholipid DMPG inhibited this transition in a concentration-dependent manner. Moreover, the orientation of the proteins was highly sensitive to the charge density of the lipid layers. Thus, the structure of VAMP1 is clearly controlled by protein-lipid interactions.