On the Structural Regularity in Nucleobases and Amino Acids and Relationship to the Origin and Evolution of the Genetic Code

To explore how chemical structures of both nucleobases and amino acids may have played a role in shaping the genetic code, numbers of sp² hybrid nitrogen atoms in nucleobases were taken as a determinative measure for empirical stereo-electronic property to analyze the genetic code. Results revealed that amino acid hydropathy correlates strongly with the sp² nitrogen atom numbers in nucleobases rather than with the overall electronic property such as redox potentials of the bases, reflecting that stereo-electronic property of bases may play a role. In the rearranged code, five simple but stereo-structurally distinctive amino acids (Gly, Pro, Val, Thr and Ala) and their codon quartets form a crossed intersection “core”. Secondly, a re-categorization of the amino acids according to their β-carbon stereochemistry, verified by charge density (at β-carbon) calculation, results in five groups of stereo-structurally distinctive amino acids, the group leaders of which are Gly, Pro, Val, Thr and Ala, remarkably overlapping the above “core”. These two lines of independent observations provide empirical arguments for a contention that a seemingly “frozen” “core” could have formed at a certain evolutionary stage. The possible existence of this codon “core” is in conformity with a previous evolutionary model whereby stereochemical interactions may have shaped the code. Moreover, the genetic code listed in UCGA succession together with this codon “core” has recently facilitated an identification of the unprecedented icosikaioctagon symmetry and bi-pyramidal nature of the genetic code.     

Circumscription of Malvaceae (Malvales) as determined by a preliminary cladistic analysis of morphological, anatomical, palynological, and chemical characters

We report a phylogenetic analysis of “core” Malvales (Tiliaceae, Sterculiaceae, Bombacaceae, and Malvaceae) based on morphological, anatomical, palynological, and chemical features. The results of the analyses lead to the conclusion that Tiliaceae, Sterculiaceae, and Bombacaceae, as variously delimited, are paraphyletic; only the Malvaceae are likely monophyletic. The genera of “core” Malvales form a well-defined clade. Genera of “Tiliaceae” constitute the basal complex within “core” Malvales. The “Sterculiaceae” (most genera)+ “Bombacaceae” + Malvaceae form a clade on the basis of a monadelphous androecium; “Bombacaceae”+ Malvaceae also form a clade, which is diagnosable on the basis of monoloculate anthers. It is clear that the traditional classification, with its arbitrarily delimited evolutionary grades, is unsatisfactory, especially if one seeks to reflect phylogeny accurately. Thus, Malvaceae is redefined to refer to the most recent common ancestor of plants previously considered to be “Tiliaceae,” “Sterculiaceae,” “Bombacaceae,” and Malvaceae, and all of the descendants of that ancestor. This broadly circumscribed Malvaceae can be diagnosed by several presumed synapomorphies, but we draw special attention to the unusual floral nectaries that are composed of densely packed, multicellular, glandular hairs on the sepals (or less commonly on the petals or androgynophore).     

Question 7: Construction of a Semi-Synthetic Minimal Cell: A Model for Early Living Cells

Using a Synthetic Biology approach we are building a semi-synthetic minimal cell. This represents an exercise to shape a minimal-cell model system recalling the simplicity of early living cells in early evolution. We have recently introduced into liposome compartments a minimal set of enzymes named “Puresystem” (PS) synthesizing EGFP proteins. To establish reproduction of the shell compartment with a minimal set of genes we have cloned the genes for the Fatty Acid Synthase (FAS) type I enzymes. These FAS genes introduced into liposomes, translated into FAS enzymes by PS and in the presence of precursors produce fatty acids. The resulting release of fatty acid molecules within liposome vesicles should promote vesicle growth and reproduction. The core reproduction of a minimal cell corresponding to the replication of the minimal genome will require a few genes for the DNA replication and the PS, and a minimum set of genes for the synthesis of t-RNAs. In future the reconstruction of a minimal ribosome will bring the number of genes for ribosomal proteins from 54 of an existing minimal genome down to 30–20 genes. A Synthetic Biology approach could bring the number of essential genes for a minimal cell down to 100 or less. International School of Complexity–4th Course: Basic Questions on the Origins of Life; “Ettore Majorana” Foundation and Centre for Scientific Culture, Erice, Italy, 1–6 October 2006.     

Non-erythroid beta spectrin interacting proteins and their effects on spectrin tetramerization

With yeast two-hybrid methods, we used a C-terminal fragment (residues 1697–2145) of non-erythroid beta spectrin (βII-C), including the region involved in the association with alpha spectrin to form tetramers, as the bait to screen a human brain cDNA library to identify proteins interacting with βII-C. We applied stringent selection steps to eliminate false positives and identified 17 proteins that interacted with βII-C (IP_βII-C s). The proteins include a fragment (residues 38–284) of “THAP domain containing, apoptosis associated protein 3, isoform CRA g”, “glioma tumor suppressor candidate region gene 2” (residues 1-478), a fragment (residues 74–442) of septin 8 isoform c, a fragment (residues 704–953) of “coatomer protein complex, subunit beta 1, a fragment (residues 146–614) of zinc-finger protein 251, and a fragment (residues 284–435) of syntaxin binding protein 1. We used yeast three-hybrid system to determine the effects of these βII-C interacting proteins as well as of 7 proteins previously identified to interact with the tetramerization region of non-erythroid alpha spectrin (IP_αII-N s) [1] on spectrin tetramer formation. The results showed that 3 IP_βII-C s were able to bind βII-C even in the presence of αII-N, and 4 IP_αII-N s were able to bind αII-N in the presence of βII-C. We also found that the syntaxin binding protein 1 fragment abolished αII-N and βII-C interaction, suggesting that this protein may inhibit or regulate non-erythroid spectrin tetramer formation.     

Spectroscopic and metal-binding properties of DF3: an artificial protein able to accommodate different metal ions

The design, synthesis, and metal-binding properties of DF3, a new de novo designed di-iron protein model are described (“DF” represents due ferri, Italian for “two iron,” “di-iron”). DF3 is the latest member of the DF family of synthetic proteins. They consist of helix–loop–helix hairpins, designed to dimerize and form an antiparallel four-helix bundle that encompasses a metal-binding site similar to those of non-heme carboxylate-bridged di-iron proteins. Unlike previous DF proteins, DF3 is highly soluble in water (up to 3 mM) and forms stable complexes with several metal ions (Zn, Co, and Mn), with the desired secondary structure and the expected stoichiometry of two ions per protein. UV–vis studies of Co(II) and Fe(III) complexes confirm a metal-binding environment similar to previous di-Co(II)- and di-Fe(III)-DF proteins, including the presence of a μ-oxo-di-Fe(III) unit. Interestingly, UV–vis, EPR, and resonance Raman studies suggest the interaction of a tyrosine adjacent to the di-Fe(III) center. The design of DF3 was aimed at increasing the accessibility of small molecules to the active site of the four-helix bundle. Indeed, binding of azide to the di-Fe(III) site demonstrates a more accessible metal site compared with previous DFs. In fact, fitting of the binding curve to the Hill equation allows us to quantify a 150% accessibility enhancement, with respect to DF2. All these results represent a significant step towards the development of a functional synthetic DF metalloprotein.     

Molecular Cloning and Comparison of Avian Preproghrelin Genes

We report cDNA sequences for the preproghrelin gene from goose, duck, and emu. This gene is involved in stimulating the release of growth hormone in mammals and may play a similar role in avian species. The complete coding sequence of avian preproghrelin encodes a 116 amino acid (aa) protein, which is organized into three parts: the N-terminal hydrophobic signal peptide, a 26 aa peptide for mature ghrelin, and a long C-terminal polypeptide. Domain/motif structures of preproghrelin protein are highly conserved among avian species. Although the avian and mammalian homologs are not highly similar for the whole 116 aa sequence, the identity of the highly conserved “active core” sequence and the n-octanoyl modification of the serine 3 residue avian ghrelin protein with its mammalian homologs implies conserved function of ghrelin protein during evolution. Information provided in this study will be useful in further studies to determine the role the preproghrelin gene plays in the regulation of growth hormone release and body weight gain in avian species. Jing Yuan and Jianjun Zhou contributed equally to this work     

A comment on Warburg’s early understanding of biocatalysis

The early history of biochemistry was characterized by changing moods. The discovery of cell free fermentation (1897) led to the optimistic belief that all life processes were carried out by intracellular enzymes, being definite proteins with special catalytic properties. But, the persistent failure to isolate pure enzymes raised doubts. When Otto Warburg found cell respiration to be a membrane-bound iron catalysis (1914), he renewed the old position that biocatalysis was caused by surface forces and ferments could be heavy metal ions adsorbed on colloidal membrane carriers. This alternative view became popular when Warburg started his research in photosynthesis and explained his peculiar “photolyte” model. Neither the suggestion of surface-active colloidal ferments, nor the idea of “photolyte” stood the test of time and have now been rejected, but their history is of importance to how concepts evolve and die.     

Structural and functional studies on ?29 DNA polymerase

TheBacillus subtilis phage ?29 DNA polymerase, involved in protein-primed viral DNA replication, contains several amino acid consensus sequences common to other eukaryotic-type DNA polymerases. Using site-directed mutagenesis, we have studied the functional significance of a C-terminal conserved region, represented by the Lys-X-Tyr (“K-Y”) motif. Single point mutants have been constructed and the corresponding proteins have been overproduced and characterized. Measurements of the activity of the mutant proteins indicated that the invariant Lys and Tyr residues play a critical role in DNA polymerization. Interestingly, substitution of the invariant Lys either by Arg or Thr, produced enzymes with an increased or a largely reduced, respectively, capability to use a protein as primer, an intrinsic property of TP-priming DNA polymerases. On the other hand, the viral protein p6, which stimulates initiation of ?29 DNA replication by formation of a nucleoprotein complex at both DNA replication origins, increased (about 5-fold) the insertion fidelity of ?29 DNA polymerase during the formation of the TP-dAMP initiation complex. We propose a model in which the special strategy to maintain the integrity of the ?29 DNA ends, by means of a “sliding-back” mechanism, could also contribute to increase the fidelity of ?29 DNA replication.     

Question 7: Biosynthesis of Phosphatidic Acid in Liposome Compartments – Toward the Self-Reproduction of Minimal Cells

Self-reproduction is one of main properties that define living cells. In order to explore the self-reproduction process for the study of early cells, and to develop a research line somehow connected to the origin of life, we have built up a constructive ‘synthetic cells (minimal cells)’ approach. The minimal cells approach consists in the investigation of the minimal number of elements to accomplish simple cell-like processes – like self-reproduction. Such approach belongs to the field of synthetic biology. The minimal cells are reconstructed from a totally reconstituted cell-free protein synthesis system (PURESYSTEM) and liposome compartments as containers. Based on this approach, we synthesized two membrane proteins (enzymes), GPAT and LPAAT, which are involved in the phosphatidic acid biosynthesis in bacteria. Both membrane proteins were successfully synthesized by PURESYSTEM encapsulated inside POPC liposomes. Additionally, the enzymatic activity of GPAT was restored by mixing the expressed enzyme with lipid and by forming liposomes in situ. Through these experimental evidences, here we present a possible model to achieve self-reproduction in minimal cells. Our results would contribute to the idea that early cells could have been built by an extremely small number of genes. Presented at the International School of Complexity – 4th Course: Basic Questions on the Origins of Life; “Ettore Majorana” Foundation and Centre for Scientific Culture, Erice, Italy, 1–6 October 2006.     

Proteasome system of protein degradation and processing

In eukaryotic cells, degradation of most intracellular proteins is realized by proteasomes. The substrates for proteolysis are selected by the fact that the gate to the proteolytic chamber of the proteasome is usually closed, and only proteins carrying a special “label” can get into it. A polyubiquitin chain plays the role of the “label”: degradation affects proteins conjugated with a ubiquitin (Ub) chain that consists at minimum of four molecules. Upon entering the proteasome channel, the polypeptide chain of the protein unfolds and stretches along it, being hydrolyzed to short peptides. Ubiquitin per se does not get into the proteasome, but, after destruction of the “labeled” molecule, it is released and labels another molecule. This process has been named “Ub-dependent protein degradation”. In this review we systematize current data on the Ub-proteasome system, describe in detail proteasome structure, the ubiquitination system, and the classical ATP/Ub-dependent mechanism of protein degradation, as well as try to focus readers’ attention on the existence of alternative mechanisms of proteasomal degradation and processing of proteins. Data on damages of the proteasome system that lead to the development of different diseases are given separately.     

Microbial community analysis using high-throughput sequencing technology: a beginner’s guide for microbiologists

Microbial communities present in diverse environments from deep seas to human body niches play significant roles in the complex ecosystem and human health. Characterizing their structural and functional diversities is indispensable, and many approaches, such as microscopic observation, DNA fingerprinting, and PCR-based marker gene analysis, have been successfully applied to identify microorganisms. Since the revolutionary improvement of DNA sequencing technologies, direct and high-throughput analysis of genomic DNA from a whole environmental community without prior cultivation has become the mainstream approach, overcoming the constraints of the classical approaches. Here, we first briefly review the history of environmental DNA analysis applications with a focus on profiling the taxonomic composition and functional potentials of microbial communities. To this end, we aim to introduce the shotgun metagenomic sequencing (SMS) approach, which is used for the untargeted (“shotgun”) sequencing of all (“meta”) microbial genomes (“genomic”) present in a sample. SMS data analyses are performed in silico using various software programs; however, in silico analysis is typically regarded as a burden on wet-lab experimental microbiologists. Therefore, in this review, we present microbiologists who are unfamiliar with in silico analyses with a basic and practical SMS data analysis protocol. This protocol covers all the bioinformatics processes of the SMS analysis in terms of data preprocessing, taxonomic profiling, functional annotation, and visualization.     

Bacterial metallothioneins: past,present, and questions for the future

Bacterial metallothioneins (MTs) have been known since the mid-1980s. The only family known until recently was the BmtA family, exemplified by the zinc- and cadmium-binding SmtA from the cyanobacterium Synechococcus PCC 7942, for which a structure was determined in 2001. Only in 2008 was a second type of bacterial MT identified in mycobacteria, and the copper-binding gene product was called MymT. Many of the features of SmtA either have been unexpected or are otherwise “unusual”, for example the presence of a zinc finger fold and the kinetic inertness of one of the four zinc ions bound to the protein. The unpredictability of molecular properties of this protein exemplified the need for continued biophysical studies of novel proteins. Homologues for SmtA have been identified in a limited number of bacterial genomes from cyanobacteria, pseudomonads, alphaproteobacteria, gammaproteobacteria, and firmicutes. Except for the residues defining the zinc finger fold, these homologous protein sequences display an intriguing variety, especially in terms of metal ligand position and identity. The increased number of homologues has allowed use of hidden Markov models to look for more remote relatives of SmtA, leading to the identification of a novel family of putative hybrid LIM domain MTs. However, database searches based on sequence similarity are of limited use for mining for further “overlooked” bacterial MTs, as so far undiscovered bacterial MTs may be too diverse from any other known MTs, and other approaches are required.     

Engrained experience—a comparison of microclimate perception schemata and microclimate measurements in Dutch urban squares

Acceptance of public spaces is often guided by perceptual schemata. Such schemata also seem to play a role in thermal comfort and microclimate experience. For climate-responsive design with a focus on thermal comfort it is important to acquire knowledge about these schemata. For this purpose, perceived and “real” microclimate situations were compared for three Dutch urban squares. People were asked about their long-term microclimate perceptions, which resulted in “cognitive microclimate maps”. These were compared with mapped microclimate data from measurements representing the common microclimate when people stay outdoors. The comparison revealed some unexpected low matches; people clearly overestimated the influence of the wind. Therefore, a second assumption was developed: that it is the more salient wind situations that become engrained in people’s memory. A comparison using measurement data from windy days shows better matches. This suggests that these more salient situations play a role in the microclimate schemata that people develop about urban places. The consequences from this study for urban design are twofold. Firstly, urban design should address not only the “real” problems, but, more prominently, the “perceived” problems. Secondly, microclimate simulations addressing thermal comfort issues in urban spaces should focus on these perceived, salient situations.     

Protein design from in silico dynamic information: the emergence of the 'turn-dock-lock' motif

A protein design methodology based on ab initio folding simulations is described and illustrated. First, the time evolution of the chain topology is generated to identify a collapse-triggering nucleus. Then, a minimal spliced sequence of nuclear residues is created and systematically mutated in silico until it can sustain a stable conformation retaining the original nucleus topology. The mutations introduce a structural compensation for the deletions and eventually lead to the recovery of the native fold motif beyond topological identity. For ubiquitin, the systematically modified sequence is predicted to be a resilient folder, since it is 92% homologous to the hyperthermophile variant of B1-domain in streptococcal protein G. The methodology enabling us to identify the nucleus is independently validated vis-á-vis site-directed mutagenesis experiments on chymotrypsin inhibitor (CI2).     

Periodicities in protein sequences

The correlation between various amino acid residues (either same or different), along the polypeptide chain have been studied using a large data base. A table of preference values for pairs having strong correlations has been constructed, which can be used to study any sequence and by calculating the weight of these sequences based on these preference values, a rough distinction between a “natural” and a “random” sequence can be made, One can further comment on the evolutionary status of proteins based on these weights.     

Function2Form Bridge—Toward synthetic protein holistic performance prediction

Protein engineering and synthetic biology stand to benefit immensely from recent advances in silico tools for structural and functional analyses of proteins. In the context of designing novel proteins, current in silico tools inform the user on individual parameters of a query protein, with output scores/metrics unique to each parameter. In reality, proteins feature multiple “parts”/functions and modification of a protein aimed at altering a given part, typically has collateral impact on other protein parts. A system for prediction of the combined effect of design parameters on the overall performance of the final protein does not exist. Function2Form Bridge (F2F-Bridge) attempts to address this by combining the scores of different design parameters pertaining to the protein being analyzed into a single easily interpreted output describing overall performance. The strategy comprises of (a) a mathematical strategy combining data from a myriad of in silico tools into an OP-score (a singular score informing on a user-defined overall performance) and (b) the F2F Plot, a graphical means of informing the wetlab biologist holistically on designed construct suitability in the context of multiple parameters, highlighting scope for improvement. F2F predictive output was compared with wetlab data from a range of synthetic proteins designed, built, and tested for this study. Statistical/machine learning approaches for predicting overall performance, for use alongside the F2F plot, were also examined. Comparisons between wetlab performance and F2F predictions demonstrated close and reliable correlations. This user-friendly strategy represents a pivotal enabler in increasing the accessibility of synthetic protein building and de novo protein design.     

Fuzzy oil drop model to interpret the structure of antifreeze proteins and their mutants

Mutations in proteins introduce structural changes and influence biological activity: the specific effects depend on the location of the mutation. The simple method proposed in the present paper is based on a two-step model of in silico protein folding. The structure of the first intermediate is assumed to be determined solely by backbone conformation. The structure of the second one is assumed to be determined by the presence of a hydrophobic center. The comparable structural analysis of the set of mutants is performed to identify the mutant-induced structural changes. The changes of the hydrophobic core organization measured by the divergence entropy allows quantitative comparison estimating the relative structural changes upon mutation. The set of antifreeze proteins, which appeared to represent the hydrophobic core structure accordant with “fuzzy oil drop” model was selected for analysis.     

Halophilic β-lactamase as a new solubility- and folding-enhancing tag protein: production of native human interleukin 1α and human neutrophil α-defensin

The amino acid composition of halophilic enzymes is characterized by an abundant content of acidic amino acid, which confers to the halophilic enzymes extensive negative charges at neutral pH and high aqueous solubility. This negative charge prevents protein aggregation when denatured and thereby leads to highly efficient protein refolding. β-Lactamase from periplasmic space of moderate halophile (BLA), a typical halophilic enzyme, can be readily expressed as a native, active form in Escherichia coli cytoplasm. Similar to other halophilic enzymes, BLA is soluble upon denaturation by heat or urea treatments and, hence, can be efficiently refolded. Such high solubility and refolding efficiency make BLA a potential fusion partner for expression of aggregation-prone heterologous proteins to be expressed in E. coli. Here, we succeeded in the soluble expression of several “difficult-to-express” proteins as a BLA fusion protein and verified biological activities of human interleukin 1α and human neutrophil α-defensin, HNP-1.