Structural study of poly(beta-benzyl-L-aspartate) monolayers at air-liquid interfaces.

As normally studied, in the solid state or in solution, poly(beta-benzyl-L-aspartate) (PBLA) differs from the other helical polyamino acids in that its alpha-helical conformation is most stable in the left-handed rather than in the right-handed form. The slightly lower energy per residue for the left-handed form in PBLA is easily perturbed, however. The helical screw sense can be inverted in a polar environment and, upon heating above 100 degrees C, a distorted left-handed helix or omega-helix is irreversibly formed. From external reflectance Fourier transform infrared measurements at the air-water interface, the conformation of PBLA in the monolayer state is directly established for the first time. The infrared frequencies of the amide bands suggest that right-handed alpha-helices are formed on the surface of water immediately after spreading the monolayers and independently of the polypeptide conformational distribution in the spreading solution. The right-handed helical form prevails throughout the slow compression of the Langmuir monolayers to collapsed films. The helical screw sense can be reversed by lowering the polarity of the aqueous phase. In addition, an alternate conformation similar to the omega-helix forms on addition of small amounts of isopropanol to the aqueous subphase, and appears to be an intermediate in the helix-helix transition.     

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.     

Unique side-chain conformation encoding for chirality and azimuthal orientation in the molecular packing of skin collagen.

We used molecular mechanics to study the role of gly X-Y+ sequences, where X- was Asp or Glu and Y+ was Lys or Arg, in the molecular packing of type I collagen. In the minimal energy conformation of a triply stranded molecule having a coiled-coil configuration, the side-chains of these sequences segregated into two oppositely charged groupings of the forms X-Y+X- and Y+X-Y+. Groupings having the same net charge were clustered along two complementary azimuthal edges of the molecule. Intermolecular interactions, through these oppositely charged edges, align the molecules appropriately for the formation of the HHL crosslink of skin. This alignment also can account for the axial periodicity and chiral appearance of skin collagen fibrils.     

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 orientation and two-dimensional crystallization of the proteasome at metal-chelating lipid interfaces

The potential of a protein-engineered His tag to immobilize macromolecules in a predictable orientation at metal-chelating lipid interfaces was investigated using recombinant 20 S proteasomes His-tagged in various positions. Electron micrographs demonstrated that the orientation of proteasomes bound to chelating lipid films could be controlled via the location of their His tags: proteasomes His-tagged at their sides displayed exclusively side-on views, while proteasomes His-tagged at their ends displayed exclusively end-on views. The activity of proteasomes immobilized at chelating lipid interfaces was well preserved. In solution, His-tagged proteasomes hydrolyzed casein at rates comparable with wild-type proteasomes, unless the His tags were located in the vicinity of the N termini of alpha-subunits. The N termini of alpha-subunits might partly occlude the entrance channel in alpha-rings through which substrates enter the proteasome for subsequent degradation. A combination of electron micrographs and atomic force microscope topographs revealed a propensity of vertically oriented proteasomes to crystallize in two dimensions on fluid lipid films. The oriented immobilization of His-tagged proteins at biocompatible lipid interfaces will assist structural studies as well as the investigation of biomolecular interaction via a wide variety of surface-sensitive techniques including single-molecule analysis.     

Kinetics of adsorption of proteins at interfaces: role of protein conformation in diffusional adsorption

To elucidate the role of protein conformation in the kinetics of adsorption at interfaces, seven structural intermediates of bovine serum albumin were prepared and their adsorption at the air/water interface was studied. Molecular area calculations indicated two distinct molecular processes, the first being the creation of an area, delta A1, for anchoring the molecule during the initial phase of adsorption and the second being the delta A2 cleared during subsequent reorientation and rearrangement of adsorbed molecules at the interface. The delta A1 values for all the albumin intermediates were the same, indicating that the initial work pi delta A1 needed to anchor the molecule at the interface was independent of solution conformation of the protein. Unlike delta A1, delta A2 exhibited a bell-shaped relationship with the extent of refolded state of the intermediates. Calculation of diffusion coefficients indicated that greater the unfolded state of the albumin intermediate, the greater was the diffusion coefficient. It is shown that the simple diffusion theory is inadequate to explain quantitatively the kinetics of protein adsorption. Specific, conformation-dependent, solute-solvent and solute-interface interactions also seem to influence the kinetics of adsorption of proteins.     

Sensitive force technique to probe molecular adhesion and structural linkages at biological interfaces.

Adhesion and cytoskeletal structure are intimately related in biological cell function. Even with the vast amount of biological and biochemical data that exist, little is known at the molecular level about physical mechanisms involved in attachments between cells or about consequences of adhesion on the material structure. To expose physical actions at soft biological interfaces, we have combined an ultrasensitive transducer and reflection interference microscopy to image submicroscopic displacements of probe contact with a test surface under minuscule forces. The transducer is a cell-size membrane capsule pressurized by micropipette suction where displacement normal to the membrane under tension is proportional to the applied force. Pressure control of the tension tunes the sensitivity in operation over four orders of magnitude through a range of force from 0.01 pN up to the strength of covalent bonds (approximately 1000 pN)! As the surface probe, a microscopic bead is biochemically glued to the transducer with a densely-bound ligand that is indifferent to the test surface. Movements of the probe under applied force are resolved down to an accuracy of approximately 5 nm from the interference fringe pattern created by light reflected from the bead. With this arrangement, we show that local mechanical compliance of a cell surface can be measured at a displacement resolution set by structural fluctuations. When desired, a second ligand is bound sparsely to the probe for focal adhesion to specific receptors in the test surface. We demonstrate that monitoring fluctuations in probe position at low transducer stiffness enhances detection of molecular adhesion and activation of cytoskeletal structure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spontaneous formation of DPPC monolayers at aqueous/vapor interfaces and the impact of charged surfactants

Surface tensiometry and vibrational sum-frequency spectroscopy were used to examine the structure and organization in phospholipid monolayers at the aqueous/vapor interface in the absence and in the presence of simple, charged surfactants. 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was the phospholipid employed in these studies and surfactants included sodium dodecyl sulfate (SDS) and dodecyl trimethyl ammonium bromide (DTAB). DPPC spontaneously spreads on a pure water (pH = 5.5) surface to form monolayers as evidenced by an equilibrium spreading pressure (ESP) of 7.9 ± 2.3 mN/m and a clearly resolved vibrational spectrum. Low concentrations of surfactants inhibit the spreading of DPPC and result in significantly lower ESP values. Anionic and cationic surfactants at higher concentrations have opposite effects on monolayer organization; SDS creates well-organized monolayers while DTAB leads to poor organization of lipid molecules. Surface-specific vibrational spectra showed that high concentrations of charged surfactants (≥ 100 µM) lead to accumulation of net surface charges as evidenced by destructive and constructive interferences. Selectively deuterating surfactants results in changes in vibrational band intensities and phases enabling assignment of relative orientations of equivalent functional groups belonging to the lipid and surfactant.     

Activation of Candida rugosa lipase at alkane-aqueous interfaces: a molecular dynamics study

The effect of solvent hydrophobicity on activation of Candida rugosa lipase (CRL) was investigated by performing molecular dynamics simulations for four nano seconds (ns). The closed/inactive conformer of CRL (PDB code 1TRH) was solvated in three alkane-aqueous environments. The alkanes aggregated in a predominantly aqueous environment and by 1 ns a stable spherical alkane-aqueous interface had formed. This led to the interfacial activation of CRL. On analyzing the simulated conformers with the closed conformer of CRL, the flap was found to have opened from a closed state by 7.7 A, 10.2 A, 13.1 A at hexane-aqueous, octane-aqueous, and decane-aqueous interfaces. Further, essential dynamics analysis revealed that major anharmonic fluctuations were confined to residues 64-81, the flap of CRL.     

Structure and orientation of lung surfactant SP-C and L-alpha-dipalmitoylphosphatidylcholine in aqueous monolayers.

SP-C, a pulmonary surfactant-specific protein, aids the spreading of the main surfactant phospholipid L-alpha-dipalmitoylphosphatidylcholine (DPPC) across air/water interfaces, a process that has possible implications for in vivo function. To understand the molecular mechanism of this process, we have used external infrared reflection-absorption spectroscopy (IRRAS) to determine DPPC acyl chain conformation and orientation as well as SP-C secondary structure and helix tilt angle in mixed DPPC/SP-C monolayers in situ at the air/water interface. The SP-C helix tilt angle changed from approximately 24 degrees to the interface normal in lipid bilayers to approximately 70 degrees in the mixed monolayer films, whereas the acyl chain tilt angle of DPPC decreased from approximately 26 degrees in pure lipid monolayers (comparable to bilayers) to approximately 10 degrees in the mixed monolayer films. The protein acts as a "hydrophobic lever" by maximizing its interactions with the lipid acyl chains while simultaneously permitting the lipids to remain conformationally ordered. In addition to providing a reasonable molecular mechanism for protein-aided spreading of ordered lipids, these measurements constitute the first quantitative determination of SP-C orientation in Langmuir films, a paradigm widely used to simulate processes at the air/alveolar interface.     

Interactions between lipopolysaccharide and phosphatidylethanolamine in molecular monolayers.

Lipopolysaccharide and phosphatidylethanolamine are the two major lipid constituents of the membrane of Salmonella typhimurium. Interactions between the purified lipopolysaccharide and phosphatidylethanolamine were studied in molecular monolayers at air-water interfaces. The equilibrium surface pressures of mixed films of lipopolysaccharide and phosphatidylethanolamine were determined as a function of the film composition. The plot of the equilibrium surface pressrue vs. the area occupied by phosphatidylethanolamine molecules exhibited two distinct regions. Below a phosphatidylethanolamine surface concentration at which 55% of the surface was occupied by phosphatidylethanolamine molecules, the equilibrium pressure was invariant and had the value of a pure lipopolysaccharide monolayer at maximum compression. At phosphatidylethanolamine surface concentrations in excess of 55% surface area occupation (phosphatidylethanolamine/lipopolysaccharide (mol/mol) greater than 16), the equilibrium surface pressure was a function of the surface concentration of phosphatidylethanolamine. The results suggest a simple model in which lipopolysaccharide and phosphatidylethanolamine form a complex in which each lipopolysaccharide molecule is surrounded ("lipidated") by a shell of approx. 16 phosphatidylethanolamine molecules.     

Trypsin digestion of arginase: evidence for a stable conformation manganese directed.

1. Controlled tryptic digestion of native arginase from rat liver suggests that Mn2+ promotes a stable conformation as shown by the following features. 2. An 18-fold increase in the half-life of arginase activity in the presence of Mn2+ is produced. 3. The stability of subunit B of arginase is increased in the presence of Mn2+ as revealed by SDS-PAGE during the time-course of trypsin cleavage. 4. The different digestion products of arginase with and without Mn2+ appearing during the time-course of tryptic treatment. 5. Different activity/bands protein ratio at any time of the tryptic digestion in the incubation mixtures, with and without Mn2+, are apparent.     

Cavities and packing at protein interfaces.

An analysis of internal packing defects or "cavities" (both empty and water-containing) within protein structures has been undertaken and includes 3 cavity classes: within domains, between domains, and between protein subunits. We confirm several basic features common to all cavity types but also find a number of new characteristics, including those that distinguish the classes. The total cavity volume remains only a small fraction of the total protein volume and yet increases with protein size. Water-filled "cavities" possess a more polar surface and are typically larger. Their constituent waters are necessary to satisfy the local hydrogen bonding potential. Cavity-surrounding atoms are observed to be, on average, less flexible than their environments. Intersubunit and interdomain cavities are on average larger than the intradomain cavities, occupy a larger fraction of their resident surfaces, and are more frequently water-filled. We observe increased cavity volume at domain-domain interfaces involved with shear type domain motions. The significance of interfacial cavities upon subunit and domain shape complementarity and the protein docking problem, as well as in their structural and functional role in oligomeric proteins, will be discussed. The results concerning cavity size, polarity, solvation, general abundance, and residue type constituency should provide useful guidelines for protein modeling and design.