Recursiveness, Switching, and Fluctuations in a Replicating Catalytic Network

A protocell model consisting of mutually catalyzing molecules is studied in order to investigate how chemical compositions are transferred recursively through cell divisions under replication errors. Depending on the path rate, the numbers of molecules and species, three phases are found: fast switching state without recursive production, recursive production, and itinerancy between the above two states. The number distributions of the molecules in the recursive states are shown to be log-normal except for those species that form a core hypercycle, and are explained with the help of a heuristic argument.Comment: 4 pages (with 7 figures (6 color)), submitted to PR

<i>De novo</i> design of a four-fold symmetric TIM-barrel protein with atomic-level accuracy

Despite efforts for over 25 years, de novo protein design has not succeeded in achieving the TIM-barrel fold. Here we describe the computational design of 4-fold symmetrical (β/α)(8)-barrels guided by geometrical and chemical principles. Experimental characterization of 33 designs revealed the importance of sidechain-backbone hydrogen bonding for defining the strand register between repeat units. The X-ray crystal structure of a designed thermostable 184-residue protein is nearly identical with the designed TIM-barrel model. PSI-BLAST searches do not identify sequence similarities to known TIM-barrel proteins, and sensitive profile-profile searches indicate that the design sequence is distant from other naturally occurring TIM-barrel superfamilies, suggesting that Nature has only sampled a subset of the sequence space available to the TIM-barrel fold. The ability to de novo design TIM-barrels opens new possibilities for custom-made enzymes

Mimicking enzyme evolution by generating new (βα)(8)-barrels from (βα)(4)-half-barrels

Gene duplication and fusion events that multiply and link functional protein domains are crucial mechanisms of enzyme evolution. The analysis of amino acid sequences and three-dimensional structures suggested that the (βα)(8)-barrel, which is the most frequent fold among enzymes, has evolved by the duplication, fusion, and mixing of (βα)(4)-half-barrel domains. Here, we mimicked this evolutionary strategy by generating in vitro (βα)(8)-barrels from (βα)(4)-half-barrels that were deduced from the enzymes imidazole glycerol phosphate synthase (HisF) and N′[(5′-phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide-ribonucleotide isomerase (HisA). To this end, the gene for the C-terminal (βα)(4)-half-barrel (HisF-C) of HisF was duplicated and fused in tandem to yield HisF-CC, which is more stable than HisF-C. In the next step, by optimizing side-chain interactions within the center of the β-barrel of HisF-CC, the monomeric and compact (βα)(8)-barrel protein HisF-C*C was generated. Moreover, the genes for the N- and C-terminal (βα)(4)-half-barrels of HisF and HisA were fused crosswise to yield the chimeric proteins HisFA and HisAF. Whereas HisFA contains native secondary structure elements but adopts ill-defined association states, the (βα)(8)-barrel HisAF is a stable and compact monomer that reversibly unfolds with high cooperativity. The results obtained suggest a previously undescribed dimension for the diversification of enzymatic activities: new (βα)(8)-barrels with novel functions might have evolved by the exchange of (βα)(4)-half-barrel domains with distinct functional properties

Establishing wild-type levels of catalytic activity on natural and artificial (βα)8-barrel protein scaffolds

The generation of high levels of new catalytic activities on natural and artificial protein scaffolds is a major goal of enzyme engineering. Here, we used random mutagenesis and selection in vivo to establish a sugar isomerisation reaction on both a natural (βα)8-barrel enzyme and a catalytically inert chimeric (βα)8-barrel scaffold, which was generated by the recombination of 2 (βα)4-half barrels. The best evolved variants show turnover numbers and substrate affinities that are similar to those of wild-type enzymes catalyzing the same reaction. The determination of the crystal structure of the most proficient variant allowed us to model the substrate sugar in the novel active site and to elucidate the mechanistic basis of the newly established activity. The results demonstrate that natural and inert artificial protein scaffolds can be converted into highly proficient enzymes in the laboratory, and provide insights into the mechanisms of enzyme evolution

Modelling of Aeolian Vibrations of Single Conductors

This chapter deals with an analytical approach which may be used to investigate alternatives in the design or redesign process of a line. In particular, this section describes the energy balance principle (EBP) which is used to estimate an upper bound to the expected vibratory motions, gives examples of measured wind and conductor self-damping data used, and some comparisons with available field measurements. Important problems are tackled related to the reliability of available data, investigated through: 1. a critical analysis of the available data pertaining to wind power input; 2. an analysis of data on conductor self-damping; 3. comparison of analytical and experimental vibration measurements; 4. study of how uncertainties regarding wind power input and conductor self-damping are reflected in analytical predictions of vibration behaviour, obtained through the EBP (Energy Balance Principal). These results offer guidance for future research to improve the reliability of such predictions. The main conclusion is that through predictions of aeolian vibration level in operating lines, obtained using the EBP and the various available databases, it is possible to obtain a good reproduction of the frequency range and of the distribution of vibration amplitudes with frequency. If the wind power functions and self-damping models employed in the study are indicative of the range of uncertainty in these parameters, then the range of uncertainty in EBP predictions of vibration amplitude can be about ±50–60%, when steady-state conditions aren’t reached