Evolution of Complexity. Molecular Aspects of Preassembly.

An extension of neo-Darwinism, termed preassembly, states that genetic material required for many complex traits, such as echolocation, was present long before emergence of the traits. Assembly of genes and gene segments had occurred over protracted time-periods within large libraries of non-coding genes. Epigenetic factors ultimately promoted transfers from noncoding to coding genes, leading to abrupt formation of the trait via de novo genes. This preassembly model explains many observations that to this present day still puzzle biologists: formation of super-complexity in the absence of multiple fossil precursors, as with bat echolocation and flowering plants; major genetic and physical alterations occurring in just a few thousand years, as with housecat evolution; lack of precursors preceding lush periods of species expansion, as in the Cambrian explosion; and evolution of costly traits that exceed their need during evolutionary times, as with human intelligence. What follows in this paper is a mechanism that is not meant to supplant neo-Darwinism; instead, preassembly aims to supplement current ideas when complexity issues leave them struggling.

Coordination among Bond Formation/Cleavage in a Bifunctional-Catalyzed Fast Amide Hydrolysis: Evidence for an Optimized Intramolecular N-Protonation Event.

A density functional theory (DFT) computational analysis, using the ωB97X-D functional, of a rapid amide cleavage in 2-carboxyphthalanilic acid (2CPA), where the amide group is flanked by two catalytic carboxyls, reveals key mechanistic information: (a) General base catalysis by a carboxylate coupled to general acid catalysis by a carboxyl is not operative. (b) Nucleophilic attack by a carboxylate on the amide carbonyl coupled to general acid catalysis at the amide oxygen can also be ruled out. (c) A mechanistic pathway that remains viable involves general acid proton delivery to the amide nitrogen by a carboxyl, while the other carboxylate engages in nucleophilic attack upon the amide carbonyl; a substantially unchanged amide carbonyl in the transition state; two concurrent bond-forming events; and a spatiotemporal-base rate acceleration. This mechanism is supported by molecular dynamic simulations which confirm a persistent key intramolecular hydrogen bonding. These theoretical conclusions, although not easily verified by experiment, are consistent with a bell-shaped pH/rate profile but are at odds with hydrolysis mechanisms in the classic literature.

An Alternative Molecular View of Evolution: How DNA was Altered over Geological Time.

Four natural phenomena are cited for their defiance of conventional neo-Darwinian analysis: human intelligence; cat domesticity; the Cambrian explosion; and convergent evolution. 1. Humans are now far more intelligent than needed in their hunting-gathering days >10,000 years ago. 2. Domestic cats evolved from wildcats via major genetic and physical changes, all occurring in less than 12,000 years. 3. The Cambrian explosion refers to the remarkable expansion of species that mystifies evolutionists, as there is a total lack of fossil evidence for precursors of this abundant new life. 4. Convergent evolution often involves formation of complex, multigene traits in two or more species that have no common ancestor. These four evolutionary riddles are discussed in terms of a proposed "preassembly" mechanism in which genes and gene precursors are collected silently and randomly over extensive time periods within huge non-coding sections of DNA. This is followed by epigenetic release of the genes, when the environment so allows, and by natural selection. In neo-Darwinism, macroevolution of complex traits involves multiple mutation/selections, with each of the resulting intermediates being more favorable to the species than the previous one. Preassembly, in contrast, invokes natural selection only after a partially or fully formed trait is already in place. Preassembly does not supplant neo-Darwinism but, instead, supplements neo-Darwinism in those important instances where the classical theory is wanting.

Transforming a Stable Amide into a Highly Reactive One: Capturing the Essence of Enzymatic Catalysis.

Aspartic proteinases, which include HIV-1 proteinase, function with two aspartate carboxy groups at the active site. This relationship has been modeled in a system possessing an otherwise unactivated amide positioned between two carboxy groups. The model amide is cleaved at an enzyme-like rate that renders the amide nonisolable at 35 °C and pH 4 owing to the joint presence of carboxy and carboxylate groups. A currently advanced theory attributing almost the entire catalytic power of enzymes to electrostatic reorganization is shown to be superfluous when suitable interatomic interactions are present. Our kinetic results are consistent with spatiotemporal concepts where embedding the amide group between two carboxylic moieties in proper geometries, at distances less than the diameter of water, leads to enzyme-like rate enhancements. Space and time are the essence of enzyme catalysis.

Lipid Segregation in Membranes of Anionic Liposomes Adsorbed onto Polycationic Brushes.

Two-phased: Complexation of liposomes to spherical polycationic brushes induces lipid segregation in the liposomal membrane. The greater the initial anionic lipid content in the membrane, the more the electroneutral lipid dilutes the induced anionic clusters.

Composition and properties of complexes between spherical polycationic brushes and anionic liposomes.

A spherical polycationic brush (SPB) is made by graft-polymerizing a cationic monomer onto the surface of a 100 nm polystyrene bead. It is possible to adsorb anionic liposomes (40-60 nm diameter) onto the SPBs while maintaining the liposome integrity. The liposomes were constructed with phosphatidyl choline (PC) admixed with 0.05-0.4 mol fraction of an dianionic lipid, cardiolipin (CL(2-)). As shown by electrophoretic mobility measurements, SPB-to-liposome complexation leads to a conversion from the initial positive charge of the copolymer to a negative charge. The higher the CL(2-) content of the liposomes, the lower the concentration needed for charge neutralization. Dynamic light scattering (DLS) revealed that multicomplex aggregates are formed with a maximum size at the SPB/liposome charge-equivalence point. Experiments with fluorescent-labeled liposomes show that at low CL(2-) content about 80 liposomes are adsorbed per SPB. As the mole fraction of CL(2-) increases from 0.05 to 0.4, fewer liposomes adsorb owing to electrostatic repulsion among neighboring liposomes. The effect of added NaCl also depends upon the CL(2-) content. With 0.05 mol fraction CL(2-), the SPB/liposome complex dissociates into its components at 0.15 M NaCl. With a mole fraction of >0.1, complexes fail to dissociate even at 1.2 M NaCl. Additional information about the SPB/liposome morphology was obtained from cryo-TEM. For example, cryo-TEM data confirm liposome integrity upon complexation, a behavior that contrasts with the liposome destruction as found with adsorption to many other types of surfaces.

Liposome fusion rates depend upon the conformation of polycation catalysts.

Cryo-TEM and NaCl-leakage experiments demonstrated that the cationic polymer polylysine induces fusion of anionic liposomes but that the cationic polymer poly(N-ethyl-4-vinylpyridinium bromide) (PEVP) does not, although both polymers bind strongly to the liposomes. The difference was traced to the thickness of the coatings at constant charge coverage. Polylysine is believed to form planar ß-sheets that are sufficiently thin to allow membrane fusion. In contrast, looping and disorganization among adsorbed PEVP molecules physically prevent fusion. A similar effect is likely to be applicable to important polycation-induced fusion of cell membranes.

Remembrances of self-assemblies past.

Research on four types of self-assemblies (micelles, coacervates, gels, and vesicles) is discussed via a particular investigative methodology (in order of appearance): kinetics, dynamic NMR, PGSE-NMR, double-(13)C labeling, molecular dynamics computations, phase diagrams, cryo-HRSEM, rheology, light/electron microscopy, electrophoretic mobility, electroformation, confocal microscopy, and calorimetry. The emphasis here is on how a given method, each in its own special way, illuminates a complex system.

Relationship between surface tension and surface coverage.

Surfactant action is caused in part by a dramatic reduction in surface tension. Using surface excess measurements from a radioactive surfactant, it was possible to show that (a) the surface tension declines only slightly when the occupancy of the air/water interface increases from 0 to 60% of the maximum and (b) the steep drop in surface tension in region B (Figure 1 ), frequently observed to be linear, begins at about 80% occupancy. Surfactant continues to enter the interface cooperatively up to and past the critical micelle concentration. Linearity in region B is not indicative of surface saturation despite a seemingly constant surface excess throughout the region. The disparity between interfacial areas determined by surface tension and by other methods is discussed in terms of these results.

Complexation of anionic liposomes with spherical polycationic brushes.

Spherical polycationic brushes, consisting of polystyrene particles with linear cationic macromolecules grafted onto their surfaces, were electrostatically complexed with small unilamellar anionic liposomes. Complexation was monitored using a multimethod approach that included laser electrophoresis, dynamic light scattering, fluorescence, cryogenic transmission electron microscopy, and conductivity. Liposomes adsorb onto the outer edges of the brushes rather than penetrate into their dense polycationic layer. The integrity of the liposomes remains unaltered when the liposomes reside on the polycationic brushes. The resulting complexes (roughly 40 liposomes per brush) do not dissociate into their components upon exposure to physiological solutions. The system is potentially useful in that liposomes are gathered into well-defined clusters with a high encapsulating potential. Multicomponent constructs can be easily prepared if polycationic brushes are allowed to bind to a mixture of liposomes that encapsulate different guests. This work provides an example of "systems chemistry" whereby as many as eight components, each with its own particular location and function (i.e., polystyrene core, polycationic graft, egg lecithin, cardiolipin, two fluorescent dyes, water, and buffer), collectively self-assemble.

Liposomes remain intact when complexed with polycationic brushes.

Anionic liposomes adsorb onto the surface of spherical polymer particles bearing grafted linear cationic macromolecules. The size, shape, and encapsulation ability of the liposomes remain unchanged upon adsorption, thus providing immobilized self-organizing containers that have potential applications in the biomedical field.

A singularity model for chemical reactivity.

This article proposes a model for chemical reactivity that involves singularities ("catastrophes") in the timing of bond-making and bond-breaking events. The common stapler is a good mechanical analogy: As hand-pressure is increased on the machine, the staple hardly changes its configuration until the staple suddenly bends. This is viewed as a singularity or catastrophe, defined classically as an abrupt change resulting from a smooth increase or decrease in external conditions (pressure in the case of a stapler, distance in the case of reactivity). Although experimental observations are provided to support the singularity effect, the model remains a heterodox notion at the present time.

Additional support for a revised Gibbs analysis.

Gibbs-determined areas of >60 A(2)/molecule for many common surfactants cause rather small surface tension reductions when measured on a Langmuir film balance. This is inconsistent with an air/water interface being saturated throughout the steep linear decline in plots of surface tension versus ln [surfactant]. In support for a gradually populating interface in the linear region, we have found that sodium docosanyl sulfate lowers the surface tension by only 7 mN/m when compressed to 50 A(2)/molecule. When docosanyltrimethylmmonium bromide is compressed to 65 A(2)/molecule, the surface tension is lowered about 15 mN/m as compared to a 30-40 mN/m drop occurring within the range of typical Gibbs analysis. Saturation of the interface is often obscured by competitive micelle formation that levels the surface tension versus ln [surfactant] plot before saturation has a chance to do so.

Electrostatic binding among equilibrating 2-D and 3-D self-assemblies.

Six organic additives, each bearing a different number of anionic charges, were added to a large excess of cationic surfactant (dodecyltrimethylammonium bromide, DTAB). The surface-tension vs log [DTAB] plot for solutions containing DTAB/trianion = 15:1 showed an abrupt break (routinely taken as the critical micelle concentration, CMC) at 2.9 mM. This constitutes a 5-fold decrease compared with a CMC of 15 mM for pure aqueous DTAB. There is a 10-fold decrease in the break-point concentration caused by a mere 3 mol % of hexanion. Corresponding CMC values from DTAB/trianion mixtures, measured by both conductivity and diffusion NMR, gave normal values of 14 mM. The unusual discrepancy between the CMC based on surface tension and on the two "bulk" methods was attributed to saturation of the air/water interface by a DTAB/trianion complex far below the concentration at which the micelles form. Thus, the sharp break seen in surface-tension "CMC plots" need not in fact attest to actual micelle formation as is almost universally assumed in colloid chemistry.

Re-evaluating the Gibbs analysis of surface tension at the air/water interface.

Molecular areas of soluble films at the air/water interface have traditionally been calculated by applying the Gibbs equation to the steep linear decline in surface tension as the bulk concentration increases. This approach presupposes that the interface is saturated in the "Gibbs region," thereby allowing a single unique area to be calculated. We show that the areas derived by the Gibbs equation (typically 50-60 A(2)/molecule) are much too large to account for the abrupt surface tension decline. Moreover, a surface tension/concentration plot was observed for a system where micelle formation does not interfere with the Gibbs region. Nonetheless, the surface tension plot leveled off, ostensibly owing to saturation, when the Gibbs approach predicted a continued linear decline, proving that the interface in the Gibbs region is not saturated as generally assumed. This conclusion means that the hundreds of published molecular areas obtained by the Gibbs approach should be reconsidered.

Biomembrane sensitivity to structural changes in bound polymers.

Anionic liposomes containing a 4:1 molar ratio of neutral to anionic phospholipids were treated with an excess of five zwitterionic polymers differing only in the spacer length separating their cationic and anionic moieties. Although the polymers do not disrupt the structural integrity of the liposomes, they can induce spacer-dependent molecular rearrangements within the liposomes. Thus, the following were observed: spacer length = 1, no binding to the liposomes; spacer length = 2, adsorption to the liposomes, but no molecular rearrangement; spacer length = 3, lateral lipid segregation but little or no flip-flop; spacer length = 4 or 5, lateral lipid segregation and flip-flop. These diverse behaviors are relevant to the use of biomedical formulations where polyelectrolytes play a role.

Polymer migration among phospholipid liposomes.

Complexation of phospholipid lipsomes with a cationic polymer, poly(N-ethyl-4-vinylpyridinium bromide) (PEVP), and subsequent interliposomal migration of the adsorbed macromolecules, have been investigated. Liposomes of two different charge types were examined: (a) a liposomal system, with an overall charge near zero, consisting of zwitterionic phosphatidylcholine (egg lecithin, EL) with added doubly anionic phospholipid, cardiolipin (CL(2-)), and cationic dihexadecyldimethylammonium bromide (HMAB(+)), in a CL(2-)/HMAB(+) charge-to-charge ratio of 1:1; (b) an anionic liposomal system composed of an EL/CL(2-) mixture plus polyoxyethylene monocetyl ether (Brij 58). Both three-component systems were designed specifically to preclude liposomal aggregation upon electrostatic association with the PEVP, a phenomenon that had complicated analysis of data from several two-component liposomes. PEVP macromolecules were found from fluorescence experiments to migrate among the charge-neutral EL/CL(2-)/HMAB(+) liposomes. In the case of anionic EL/CL(2-)/Brij liposomes, a combination of fluorescence and laser microelectrophoresis methods showed that PEVP macromolecules travel from liposome to liposome while being electrostatically associated with anionic lipids.

Interaction vs Preorganization in Enzyme Catalysis. A Dispute That Calls for Resolution.

This essay focuses on the debate between Warshel et al. (proponents of preorganization) and Menger and Nome (proponents of spatiotemporal effects) over the source of fast enzyme catalysis. The Warshel model proposes that the main function of enzymes is to push the solvent coordinate toward the transition state. Other physical-organic factors (e.g., desolvation, entropic effects, ground state destabilization, etc.) do not, ostensibly, contribute substantially to the rate. Indeed, physical organic chemistry in its entirety was claimed to be "irrelevant to an enzyme's active site". Preorganization had been applied by Warshel to his "flagship" enzyme, ketosteroid isomerase, but we discuss troubling issues with their ensuing analysis. For example, the concepts of "general acid" and "general base", known to play a role in this enzyme's mechanism, are ignored in the text. In contrast, the spatiotemporal theory postulates that enzyme-like rates (i.e., accelerations >108) occur when two functionalities are held rigidly at contact distances less than ca. 3 Å. Numerous diverse organic systems are shown to bear this out experimentally. Many of these are intramolecular systems where distances between functionalities are known. Among them are fast intramolecular systems where strain is actually generated during the reaction, thereby excluding steric compression as a source of the observed enzyme-like rates. Finally, the account ends with structural data from four active sites of enzymes, obtained by others, all showing contact distances between substrate analogues and enzyme. To our knowledge, contact distances less than the diameter of water are found universally among enzymes, and it is to this fact that we attribute their extremely fast rates given the assumption that enzymes, whatever their particular mechanism, obey elementary chemical principles.

Hydrogen-bond-induced hysteresis in the compression/relaxation of monolayer films.

Two stereoisomers of surfactants were synthesized in which a diketopiperazine ring was inserted between a hydrocarbon chain (of variable length) and an anionic headgroup. It was found (by HPLC, conductivity, surface tension, and diffusion NMR) that these compounds have low solubilities in water, remarkably high Krafft temperatures, and low critical micelle concentrations. Of particular interest were the pressure/area isotherms of insoluble monolayers of nonionic analogues of the amphiphiles. These isotherms showed an unprecedented hysteresis but only during the first compression/relaxation cycle. Additional cycles were normal in that they lacked the original hysteresis. These results were attributed to diketopiperazine rings that initially lie flat on the water surface at the air/water interface. Compression flips the diketopiperazine rings so that they now reside roughly perpendicular to the interface in a stable hydrogen-bonded orientation where the rings occupy far less space.

NMR studies of aggregation and hydration of surfactants containing amide bonds.

The consequences of including amide bonds into the structure of short-chain nonionic surfactants have been studied. Of particular interest were the possible effects of the hydrogen bonding ability of the amide group on the micellar shape. The aggregate structure and hydration of two different amide-containing surfactants, C7H15CO-NH-(CH2CH2O)4H and C7H15CO-(NH-C3H6-CO)2N(CH3)2, were investigated using NMR diffusometry (pulsed gradient spin echo NMR) as the main technique. Data from experiments on the surfactants, the hydrophobic probe molecule hexamethyldisilane (HMDS), and water were interpreted to gain information about the solution structures, and the results were compared to those on a previously studied alcohol ethoxylate surfactant of similar size, C8E4. Both of the amide-containing surfactants form small micelles within the whole investigated concentration range. At the critical micelle concentration, the aggregates are most probably spherical, and with increasing surfactant concentration there are indications of either a minor aggregate growth or agglomeration of the micelles. In addition, it was found that the presence of amide groups in the surfactant inhibits the intermicellar transport of HMDS, which occurs in the C8E4 system. From measurements on water diffusion in the three surfactant systems, it could be concluded that the surfactant hydration is higher when amide bonds are present.