Fabio Mavelli

Enzymatic reactions in confined environments

Within each biological cell, surface- and volume-confined enzymes control a highly complex network of chemical reactions. These reactions are efficient, timely, and spatially defined. Efforts to transfer such appealing features to in vitro systems have led to several successful examples of chemical reactions catalysed by isolated and immobilized enzymes. In most cases, these enzymes are either bound or adsorbed to an insoluble support, physically trapped in a macromolecular network, or encapsulated within compartments. Advanced applications of enzymatic cascade reactions with immobilized enzymes include enzymatic fuel cells and enzymatic nanoreactors, both for in vitro and possible in vivo applications. In this Review, we discuss some of the general principles of enzymatic reactions confined on surfaces, at interfaces, and inside small volumes. We also highlight the similarities and differences between the in vivo and in vitro cases and attempt to critically evaluate some of the necessary future steps to improve our fundamental understanding of these systems.

A Possible Route to Prebiotic Vesicle Reproduction

Spherical bounded structures such as those formed by surfactant aggregates (mostly micelles and vesicles), with an inside that is chemically and physically different from the outside medium, can be seen as primitive cell models. As such, they are fundamental structures for the theory of autopoiesis as originally formulated by Varela and Maturana. In particular, since self-reproduction is a very important feature of minimal cellular life, the study of self-reproduction of micelles and vesicles represents a quite challenging bio-mimetic approach. Our laboratory has put much effort in recent years into implementing self-reproduction of vesicles as models for self-reproduction of cellular bounded structures, and this article is a further contribution in this direction. In particular, we deal with the so-called matrix effect of vesicles, related to the fact that when fresh surfactant is added to an aqueous solution containing preformed vesicles of a very narrow size distribution, the newly formed vesicles (instead of being polydisperse, as is usually the case) have dimensions very close to those of the preformed ones. In practice, this corresponds to a mechanism of reproduction of vesicles of the same size. In this article, the matrix effect is re-elaborated in the perspective of the origin of life, and in particular in terms of the prebiotic mechanisms that might permit the growth and reproduction of vesicles. The data are analyzed by dynamic light scattering with a new program that permits the calculation of the number-weighted size distribution. It is shown that, on adding a stoichiometric amount of oleate micelles to preformed oleate vesicles extruded at 50 and 100 nm, the final distribution contains about twice the initial number of particles, centered around 50 and 100 nm. The same holds when oleate is added to preformed phospholipid liposomes. By contrast, when the same amount of oleate is added to an aqueous solution (as a control experiment), a very broad distribution ranging between 20 and 1000 nm is obtained. The data can then be seen as a kind of reproduction of the same size vesicles, and the argument is advanced that this may correspond to a simple prebiotic mechanism of vesicle multiplication in prebiotic times, when only physical forces might be responsible for the basic mechanisms of early protocell growth and division. Preliminary data also show that repeated addition of oleate maintains the same basic initial features, and that surfactants other than oleate also respect the reproductive mode of the matrix effect.

Stochastic simulations of minimal self-reproducing cellular systems

This paper is a theoretical attempt to gain insight into the problem of how self-assembling vesicles (closed bilayer structures) could progressively turn into minimal self-producing and self-reproducing cells, i.e. into interesting candidates for (proto)biological systems. With this aim, we make use of a recently developed object-oriented platform to carry out stochastic simulations of chemical reaction networks that take place in dynamic cellular compartments. We apply this new tool to study the behaviour of different minimal cell models, making realistic assumptions about the physico-chemical processes and conditions involved (e.g. thermodynamic equilibrium/non-equilibrium, variable volume-to-surface relationship, osmotic pressure, solute diffusion across the membrane due to concentration gradients, buffering effect). The new programming platform has been designed to analyse not only how a single protometabolic cell could maintain itself, grow or divide, but also how a collection of these cells could ‘evolve’ as a result of their mutual interactions in a common environment.

Structure and Enzymatic Properties of Molecular Dendronized Polymer-Enzyme Conjugates and Their Entrapment inside Giant Vesicles

Macromolecular hybrid structures were prepared in which two types of enzymes, horseradish peroxidase (HRP) and bovine erythrocytes Cu,Zn-superoxide dismutase (SOD), were linked to a fluorescently labeled, polycationic, dendronized polymer (denpol). Two homologous denpols of first and second generation were used and compared, and the activities of HRP and SOD of the conjugates were measured in aqueous solution separately and in combination. In the latter case the efficiency of the two enzymes in catalyzing a two-step cascade reaction was evaluated. Both enzymes in the two types of conjugates were highly active and comparable to free enzymes, although the efficiency of the enzymes bound to the second-generation denpol was significantly lower (up to a factor of 2) than the efficiency of HRP and SOD linked to the first-generation denpol. Both conjugates were analyzed by atomic force microscopy (AFM), confirming the expected increase in object size compared to free denpols and demonstrating the presence of enzyme molecules localized along the denpol chains. Finally, giant phospholipid vesicles with diameters of up to about 20 μm containing in their aqueous interior pool a first-generation denpol-HRP conjugate were prepared. The HRP of the entrapped conjugate was shown to remain active toward externally added, membrane-permeable substrates, an important prerequisite for the development of vesicular multienzyme reaction systems.

Matrix effect in oleate micelles-vesicles transformation.

It is accepted by many authors that the formation of closed molecular structures is a key step in the evolution of life. Oleate vesicles represent a good model system in this framework due to the fact that they self-assemble spontaneously and that fatty acids are considered as possible prebiotic structures. In this contribution, we will focus the attention on the transition from oleate micelles to oleic acid/oleate vesicles induced by a pH change. This transformation is strongly influenced by the presence of pre-formed vesicles. We called this phenomenon the matrix effect. The influence of pre-added POPC liposomes (POPC = 1-palmitoyl-2-oleoyl- {sn}-glycerol–3-phosphocholine) and oleic acido leate vesicles on the process rate and on the final size distribution will be discussed elucidating the main differences between these two systems.

Reverse micellar systems: self organised assembly as effective route for the synthesis of colloidal semiconductor nanocrystals

Nanoparticles can be obtained by using self-assembly molecules as a template, exploiting confined growth inside the surfactant film of microemulsion system. In this work, the feasibility of using quaternary water-in-oil microemulsion (CTAB/hexane/pentanol/water) to synthesise nanometer-sized structures of semiconductor material (CdS) with desired size distribution is described. Stochastic kinetic simulations have been utilised to account for the observed template effect of the microemulsion aggregate size on the nanocluster growth.

Recent Biophysical Issues About the Preparation of Solute-Filled Lipid Vesicles

Here we report some recent biophysical issues on the preparation of solute-filled lipid vesicles and their relevance to the construction of “synthetic cells.” First, we introduce the “semi-synthetic minimal cells” as the liposome-based cell-like systems, which contain a minimal number of biomolecules required to display simple and complex biological functions. Next, we focus on recent aspects related to the construction of synthetic cells. Emphasis is given to the interplay between the methods of synthetic cell preparation and the physics of solute encapsulation. We briefly introduce the notion of structural and compositional “diversity” in synthetic cell populations.

Highly oriented photosynthetic reaction centers generate a proton gradient in synthetic protocells

Significance The photosynthetic reaction center (RC), an integral membrane protein at the core of bioenergetics of all autotrophic organisms, has been reconstituted in the membrane of giant unilamellar vesicles (RC@GUV) by retaining the physiological orientation at a very high percentage (90 ± 1%). Owing to this uniform orientation, it has been possible to demonstrate that, under red-light illumination, photosynthetic RCs operate as nanoscopic machines that convert light energy into chemical energy, in the form of a proton gradient across the vesicle membrane. This result is of great relevance in the field of synthetic cell construction, proving that such systems can easily transduce light energy into chemical energy eventually exploitable for the synthesis of ATP. Photosynthesis is responsible for the photochemical conversion of light into the chemical energy that fuels the planet Earth. The photochemical core of this process in all photosynthetic organisms is a transmembrane protein called the reaction center. In purple photosynthetic bacteria a simple version of this photoenzyme catalyzes the reduction of a quinone molecule, accompanied by the uptake of two protons from the cytoplasm. This results in the establishment of a proton concentration gradient across the lipid membrane, which can be ultimately harnessed to synthesize ATP. Herein we show that synthetic protocells, based on giant lipid vesicles embedding an oriented population of reaction centers, are capable of generating a photoinduced proton gradient across the membrane. Under continuous illumination, the protocells generate a gradient of 0.061 pH units per min, equivalent to a proton motive force of 3.6 mV⋅min−1. Remarkably, the facile reconstitution of the photosynthetic reaction center in the artificial lipid membrane, obtained by the droplet transfer method, paves the way for the construction of novel and more functional protocells for synthetic biology.

Stochastic simulations of micellization kinetics

The Monte Carlo method introduced by Gillespie (cf. D. T. Gillespie, J. Phys. Chem. 81, 2340 1977), was applied to the kinetics of micelle formation according to a mechanism that allows associations and dissociations among ennamers of whatever aggregation number. A careful choice of thermodynamic and dissociation kinetic constants made it possible to reproduce both equilibrium and kinetic properties of hypothetical surfactant solutions. The results obtained by stochastic simulations are shown and compared to the accepted theory and to the experimental evidence available in literature.

Modelling lipid competition dynamics in heterogeneous protocell populations

Recent experimental work in the field of synthetic protocell biology has shown that prebiotic vesicles are able to ‘steal’ lipids from each other. This phenomenon is driven purely by asymmetries in the physical state or composition of the vesicle membranes, and, when lipid resource is limited, translates directly into competition amongst the vesicles. Such a scenario is interesting from an origins of life perspective because a rudimentary form of cell-level selection emerges. To sharpen intuition about possible mechanisms underlying this behaviour, experimental work must be complemented with theoretical modelling. The aim of this paper is to provide a coarse-grain mathematical model of protocell lipid competition. Our model is capable of reproducing, often quantitatively, results from core experimental papers that reported distinct types vesicle competition. Additionally, we make some predictions untested in the lab, and develop a general numerical method for quickly solving the equilibrium point of a model vesicle population.