1,721,002 research outputs found
The production of de novo folded proteins by a stepwise chain elongation: A model for prebiotic chemical evolution of macromolecular sequences RID A-4573-2009
No full textWe describe an experimental procedure to mimic the formation of long (over 40 residues) co-oligopetide sequences in many identical copies which may have occurred in the prebiotic molecular evolution. The basic hypothesis is that chain formation is based on the stepwise fragment condensation of randomly generated short oligopeptides, whereby the elongation takes place under the contingent environmental constraints (solubility, pH, salinity), which eliminate most of the products, and thus determine the selection towards one particular small set of chains. The present work aims at verifying the validity of this scheme. In order to do so, we utilize a classic synthetic procedure based on the Merrifield solid-phase synthesis of peptides for the synthesis of randomly produced peptides as well as for their stepwise fragment condensation. Thus, starting from a library of peptides with n=10, the first condensation step produces a library of 16 peptides with 20 residues each (n=20), of which only four remain water-soluble and, therefore, capable to undergo the next fragment condensation step. This gives rise to 16 peptides with n = 30, out of which twelve precipitate out under the chosen pH and buffer conditions and are eliminated. Finally, a 44-residue-long water-soluble de novo protein is obtained. This has no homologies or similarities with extant proteins, and, based on circular dichroism (CD), it assumes a stable three-dimensional folding. In agreement with CD data, molecular-modelling simulations suggest an helical fold for the protein with poor, if any, structural homology with known proteins. The implication of this procedure as a general mechanism for the etiology of de novo macromolecular sequences and globular proteins in the origin of life is briefly discussed
Quasi-cellular systems: stochastic simulation analysis at nanoscale range
No full textBackground: The wet-lab synthesis of the simplest forms of life (minimal cells) is a challenging aspect in modern
synthetic biology. Quasi-cellular systems able to produce proteins directly from DNA can be obtained by
encapsulating the cell-free transcription/translation system PURESYSTEMTM(PS) in liposomes. It is possible to detect
the intra-vesicle protein production using DNA encoding for GFP and monitoring the fluorescence emission over
time. The entrapment of solutes in small-volume liposomes is a fundamental open problem. Stochastic simulation
is a valuable tool in the study of biochemical reaction at nanoscale range. QDC (Quick Direct-Method Controlled), a
stochastic simulation software based on the well-known Gillespie’s SSA algorithm, was used. A suitable model
formally describing the PS reactions network was developed, to predict, from inner species concentrations (very
difficult to measure in small-volumes), the resulting fluorescence signal (experimentally observable).
Results: Thanks to suitable features specific of QDC, we successfully formalized the dynamical coupling between
the transcription and translation processes that occurs in the real PS, thus bypassing the concurrent-only
environment of Gillespie’s algorithm. Simulations were firstly performed for large liposomes (2.67μm of diameter)
entrapping the PS to synthetize GFP. By varying the initial concentrations of the three main classes of molecules
involved in the PS (DNA, enzymes, consumables), we were able to stochastically simulate the time-course of GFPproduction.
The sigmoid fit of the GFP-production curves allowed us to extract three quantitative parameters
which are significantly dependent on the various initial states. Then we extended this study for small-volume
liposomes (575 nm of diameter), where it is more complex to infer the intra-vesicle composition, due to the
expected anomalous entrapment phenomena. We identified almost two extreme states that are forecasted to give
rise to significantly different experimental observables.
Conclusions: The present work is the first one describing in the detail the stochastic behavior of the PS. Thanks to our
results, an experimental approach is now possible, aimed at recording the GFP production kinetics in very small microemulsion
droplets or liposomes, and inferring, by using the simulation as a reverse-engineering procedure, the internal
solutes distribution, and shed light on the still unknown forces driving the entrapment phenomenon
Investigation of de novo totally random biosequences Part I A general method for in vitro selection of folded domains from a random polypeptide library displayed on phage
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