Characterization of the emergent properties of a synthetic quasi-cellular system.

BACKGROUND: The process of solutes entrapment during liposomes formation is interesting for the investigation of the relationship between the formation of compartments and the distribution of molecules inside them; a relevant issue in the studies of the origin of life. Theoretically, when no interactions are supposed among the chemical species to be entrapped, the entrapment is described by a standard Poisson process. But very recent experimental findings show that, for small liposomes (100 nm diameter), the distribution of entrapped molecules is best described by a power-law function. This is of a great importance, as the two random processes give rise to two completely different scenarios. Here we present an in silico stochastic simulation of the encapsulation of a cell-free molecular translation system (the PURE system), obtained following two different entrapment models: a pure Poisson process, and a power-law. The protein synthesis inside the liposomes has been studied in both cases, with the aim to highlight experimental observables that could be measured to assess which model gives a better representation of the real process. RESULTS: Firstly, a minimal model for in vitro protein synthesis, based on the PURE system molecular composition, has been formalized. Then, we have designed a reliable experimental simulation where stochastic factors affect the reaction course inside the compartment. To this end, 24 solutes, which represent the PURE system components, have been stochastically distributed among vesicles by following either a Poisson or a power-law distribution. The course of the protein synthesis within each vesicle has been consequently calculated, as a function of vesicle size. Our study can predict translation yield in a population of small liposomes down to the attoliter (10(-18) L) range. Our results show that the efficiency of protein synthesis peaks at approximately 3 · 10(-16) L (840 nm diam.) with a Poisson distribution of solutes, while a relative optimum is found at around 10(-17) L (275 nm diam.) for the power-law statistics. CONCLUSIONS: Our simulation clearly shows that the wet-lab measurement of an effective protein synthesis at smaller volumes than 10(-17) L would rule out, according to our models, a Poisson distribution of solutes.

Multilamellar liposomes formed by phosphatidyl nucleosides: an NMR-HR-MAS characterization.

We present an NMR investigation of multilamellar vesicles (MLVs) obtained from phosphatidyl nucleosides, 5'-(1-palmitoyl-2-oleoyl-sn-glycero(3)phospho)cytidine (1), 5'-(1-palmitoyl-2-oleoyl-sn-glycero(3)phospho)inosine (2), and their mixtures. Because of the lower stability of liposomes obtained from 2, studies have been preferentially performed in this case with mixed liposomes 2/POPC (4:1). The investigation is conducted mostly via the HR-MAS technique and the general observation is that the resolution achieved in this way is superior to that obtained in the past with small unilamellar vesicles (SUVs). A full assignment is now possible, which includes the spectral region of the ribose ring and part of the glycerol moiety. Also in the case of MLVs, both for 1 and 2, a stacking between the aromatic bases of the same liposome layer seems to be ruled out, although in both cases the nucleobases appear to be exposed to the aqueous phase. The splitting of both aromatic H-5cyt and H-6cyt is ascribed to the presence of two aggregate populations that may correspond to the two syn and anti conformations observed for cytidine monophosphate in aqueous solution. On the basis of NOESY cross-peaks, it is not always possible to discriminate between inter- and intramolecular interactions; however, the distances found for 1 appear to be compatible with the intramolecular contacts in the anti conformation of the cytidine and also with intermolecular interactions between neighboring molecules of 1. We also find that the glycerol moiety does not seem to interact with the cytidine; however, part of the ribose ring seems to be close to the glycerol moiety. More generally, the interaction of one base with the sugar moiety of a neighboring base, previously observed for SUVs, still appears to be true for MLVs. Studies have been performed also for mixed liposomes obtained from the mixture of 1 and 2, where it is observed that the HR-MAS spectra of the corresponding MLVs are not simply the sum of the spectra of the two isolated components. In particular, there is the presence of a NOESY cross-peak between the aromatic protons H-6cyt and H-2ino, and this permits us to rule out large patchwork domains containing only one nucleoside components in the mixed liposomes. Finally, a study is performed on the time evolution of the system obtained by mixing the previously prepared liposomes of 1 and 2. No interaction is obtained in this case, i.e., the spectra are constitutive, which is consistent with the general picture of liposomes as kinetic traps that are not fusing with each other.