1,299 research outputs found
Theoretical approaches to ribocell modeling
No full textThe so-called Ribocell (ribozymes-based cell) is a theoretical cellular model that has been proposed some years ago as a possible minimal cell prototype. It consists in a self-replicating minimum RNA genome coupled with a self-reproducing lipid vesicle compartment. This model assumes the existence of two hypothetical ribozymes one able to catalyze the conversion of molecular precursors into lipids and the second one able to replicate RNA strands. Therefore, in an environment rich both of lipid precursors and activated nucleotides, the ribocell can self-reproduce if the genome self-replication and the compartment self-reproduction mechanisms are somehow synchronized. The aim of this contribution is to explore the feasibility of this model with in silico simulations using kinetic parameters available in literature
Autopoietic self-Reproduction of chiral fatty acid vesicles
No full textThe self-reproduction of vesicles formed by (S)- and (R)-2-methyldodecanoic acid (4) was investigated in order to relate the autocatalytic increase of the vesicle concentration with enantioselectivity. 4(R) and 4(S) were synthesized with an enantiomeric excess greater than 98%. 4 forms vesicles in aqueous solution in the pH region between 8.8 and 7.5. Chiral properties of the vesicles were studied by differential scanning calorimetry (DSC) and circular dichroism (CD). For self-reproduction studies, the hydrolysis of the water-insoluble 2-methyldodecanoic anhydride (8) was investigated in a biphasic system consisting of an aqueous solution and 8. The reaction rates of 8(RR) and 8(SS) catalyzed by 4(R) or 4(S) vesicles were the same within experimental errors, indicating that the chiral vesicles cannot induce significant enantioselectivity. However, a clear effect was observed at 10 °C: racemic vesicles destabilized during hydrolysis, causing phase separation, whereas homochiral vesicles remained stable and continued to self-reproduce
Polymerase Chain Reaction in liposomes
No full textBackground: Compartmentalization of biochemical reactions within a spherically closed bilayer is an important step in the molecular evolution of cells. Liposomes are the most suitable structures to model this kind of chemistry. We have used the polymerase chain reaction (PCR) to demonstrate that complex biochemical reactions such as DNA replication can be carried out inside these compartments.
Results: We describe the first example of DNA amplification by the PCR occurring inside liposomes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), or of a mixture of POPC and phosphatidylserine. We show that these liposomes are stable even under the high temperature conditions used for PCR. Although only a very small fraction of liposomes contains all eight different reagents together, a significant amount of DNA is produced which can be observed by polyacrylamide gel electrophoresis.
Conclusions: This work shows that it is possible to carry out complex biochemical reactions within liposomes, which may be germane to the question of the origin of living cells. We have established the parameters and conditions that are critical for carrying out this complex reaction within the liposome compartment
Warum sind Enzyme Makromoleküle?
No full textThe paper is devoted to the question wheter the macromolecular structure is really essential for the chemical and biological qualities of enzymes. Recognizing that high molecular weight compounds are relevant both in nature and in modern technology, the question is first approached by considering whether there are some common and quite general properties intrinsic to long chains which may explain the overall success of a polymer. It is pointed out that in the process of building polymers from low molecular weight monomers so many structural parameters (copolymerisation, branching, cross-linking, stereoisomerism, grafting, compounding, etc.) can be utilized and regulated, that the physical and chemical properties of the final products can be modulated almost at will. For functional proteins, the two most important of such structural parameters are the copolymerization (from twenty different amino acid residues) and the resultant rigid folding in solution. One cannot, however, easily dispell the doubt that enzymes are oversized, particularly if one considers the principles by which enzymes develop in the evolutionary process. Indeed, the products synthesized by nature do not correspond to the optimal structural efficiency; on the contrary, structures are built at random without a prefixed finality, and they assume a function that depends on the circumstances. As a consequence of this “molecular tinkering” all enzymes may be mostly oversized, at least in principle.To analyze the question of whether the large structure of enzymes is really necessary or whether it is an unnecessary amount of molecular tinkering and fossil sequences, an enzyme can be ideally depicted as having three regions: the active site region, its overall folding, and the region in contact with its environment. Concerning the active site region, it is argued that the main structural feature is an ordered high atomic packing density (the meaning of the term order is also briefly discussed). This is a prerequisite for catalysis also in other types of chemistry, e.g. in inorganic catalysis by zeolites or other inorganic crystalline solids. In the case of enzymes the high molecular packing density is expressed in four main phenomenological properties: a good binding energy for the substrate, the stereochemical complementary of the active concavity, the obliged proximity of active amino acid residues, and the physical microenvironment of the site where the reaction has to take place. It is shown that a long chain fulfills in the best way the chemical pre-requisites for providing these four properties.After the active site region, the conformational properties of enzymes are discussed. It is argued that they are based on a compromise between two seemingly contadictory qualities, i.e. the conformational rigidity on the one hand, and the ability of undergoing conformational changes on the other. A few examples are discussed. It is shown that a long chain is the best and perhaps the only way to accomodate both conformational rigidity (via long series of intramolecular interactions) and flexibility upon ligand binding. This can give rise both to local conformational changes, very often quite important for catalysis, as well as to long-range channelled conformational changes, very often quite relevant for allostery and other biologically important mechanisms.Concerning the external surface of the enzyme, it is recognized that a long chain permits a best fitting with the environment (e. g. solubilizing, with the help of hydrophilic residues, a largely insoluble water-active site region). But the macromolecular chain is also valuable for permitting the enzyme to go from one environment to another by selective conformational changes: in this way the enzyme body behaves like an elastic buffer which imparts the enzyme’s chameleon-like properties. This can be put to use in biotechnology, and the particular case of enzymes solubilized in hydrocarbon solvents with the help of reverse micelles is used as illustration. Here enzymes like lysozyme or chymotrypsin undergo gross conformational changes, without loss of activity (which actually in some cases becomes even greater than in bulk water).It is therefore concluded, on the basis of the analysis of the active site, of the protein folding and of the protein surface, that a macromolecular chain is indeed necessary for the chemical properties of an enzyme. The question however, as to what extent does a long chain help in decreasing the activation energy in catalysis, could be answered with the present analysis only in an indirect form.Finally, some considerations are made as to the philosophical implications of the question “Why are Enzymes Macromolecules” and to its analysis. This is viewed within the general framework of molecular Darwinistic evolution which is dominated by strict reductionism, at least at the level of the molecular structures. The points of view of Jacob and Monod in this respect are cited, and also some conceptual difficulties perceived in the Darwinistic reductionism are presented
Quasi-cellular Systems: Stochastic Simulation Analysis at Nanoscale Range
No full textI complessi sistemi di reazioni biochimiche all’interno della cellula sono altamente compartimentalizzati, conseguenza di un importante fenomeno di macromolecolar crowding (sovraffollamento molecolare). E’ dunque importante determinare il comportamento e le proprietà di un sistema di reazioni in piccoli volumi. Sono stati riprodotti con successo diversi sistemi di semplici reazioni all’interno di vescicole lipidiche (liposomi) nell’ordine del micro/nanometro di diametro, osservando in molti casi una risposta cinetica diversa dalle reazioni in esame rispetto al comportamento in sistemi di grandi volumi. Questo fenomeno di divergenza tra piccoli e grandi volumi è in gran parte dipendente da fenomeni non completamente chiariti,
quali l’incapsulamento delle specie e il crowding molecolare, aspetti sempre più importanti man mano che l’attenzione si sposta verso i piccoli volumi. Recenti dati sperimentali dimostrano che il fenomeno dell'intrappolamento sembra non seguire un andamento casuale squisitamente probabilistico, ma un comportamento di tipo power-law (a legge di potenza), in cui solo pochissime vescicole intrappolano tante specie, mentre la maggior parte resta completamente vuota.
A tal proposito è stato intrapreso uno studio sui meccanismi generativi delle distribuzioni a legge di potenza calate nel contesto dell’incapsulamento (entrapment) delle specie all'interno di vescicole lipidiche.
Utilizzando un sistema cell-free di trascrizione/traduzione (PURESYSTEM™), volto alla produzione di EGFP all’interno di liposomi di POPC, è possibile monitorare la produzione di proteina fluorescente in liposomi di differente grandezza. Tuttavia, è molto difficile osservare la produzione di molecole fluorescenti in singole
vescicole di 100 nm di diametro; diventa così importante poter studiare in silico la di produzione di proteina in singole vescicole virtuali, utilizzando un modello formalmente valido del complesso sistema di reazioni del PURESYSTEM™.
QDC (Quick Direct-Method Controlled), è un software di simulazione stocastico precedentemente sviluppato in laboratorio, basato sull’algoritmo di simulazione SSA Direct-Method di Gillespie, tra i più usati in biologia computazionale/systems biology.
L’argomento della tesi riguarda l’uso di questo software nello studio delle oltre 100 reazioni biochimiche del PURESYSTEM™, comparando i risultati ottenuti in diverse condizioni (volume totale di reazione, concentrazioni delle specie, costanti cinetiche delle singole reazioni). Dopo aver affinato il modello in silico di Trascrizione/traduzione coupled (accoppiato), sono state effettuate delle simulazioni variando alcune variabili macroscopiche (concentrazioni delle specie e costanti cinetiche), mostrando un'importante dipendenza della traduzione dalla trascrizione, soprattutto considerando il
grande limite energetico di un sistema che non produce al suo interno nucleotidi trifosfato
OPEN QUESTIONS IN ORIGIN OF LIFE: EXPERIMENTAL STUDIES ON THE ORIGIN OF NUCLEIC ACIDS AND PROTEINS WITH SPECIFIC AND FUNCTIONAL SEQUENCES BY A CHEMICAL SYNTHETIC BIOLOGY APPROACH
No full textIn this mini-review we present some experimental approaches to the important issue in the origin of life, namely the origin of nucleic acids and proteins with specific and functional sequences. The formation of macromolecules on prebiotic Earth faces practical and conceptual difficulties. From the chemical viewpoint, macromolecules are formed by chemical pathways leading to the condensation of building blocks (amino acids, or nucleotides) in long-chain copolymers (proteins and nucleic acids, respectively). The second difficulty deals with a conceptual problem, namely with the emergence of specific sequences among a vast array of possible ones, the huge “sequence space”, leading to the question “why these macromolecules, and not the others?”
We have recently addressed these questions by using a chemical synthetic biology approach. In particular, we have tested the catalytic activity of small peptides, like Ser-His, with respect to peptide- and nucleotides-condensation, as a realistic model of primitive organocatalysis. We have also set up a strategy for exploring the sequence space of random proteins and RNAs (the so-called “never born biopolymer” project) with respect to the production of folded structures. Being still far from solved, the main aspects of these “open questions” are discussed here, by commenting on recent results obtained in our groups and by providing a unifying view on the problem and possible solutions. In particular, we propose a general scenario for macromolecule formation via fragment-condensation, as a scheme for the emergence of specific sequences based on molecular growth and selection
Silver sheaves; gathered through clouds and sunshine. In two parts. Part 1st: Civil and military life of the author. Part 2d: Miscellaneous collection of prose and poetry.
No full text2d ed.2 p.l., [9]-360 p
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