Mis-translation of a computationally designed protein yields an exceptionally stable homodimer: implications for protein engineering and evolution

We recently used computational protein design to create an extremely stable, globular protein, Top7, with a sequence and fold not observed previously in nature. Since Top7 was created in the absence of genetic selection, it provides a rare opportunity to investigate aspects of the cellular protein production and surveillance machinery that are subject to natural selection. Here we show that a portion of the Top7 protein corresponding to the final 49 C-terminal residues is efficiently mis-translated and accumulates at high levels in Escherichia coli. We used circular dichroism, size-exclusion chromatography, small-angle X-ray scattering, analytical ultra-centrifugation, and NMR spectroscopy to show that the resulting C-terminal fragment (CFr) protein adopts a compact, extremely stable, homo-dimeric structure. Based on the solution structure, we engineered an even more stable variant of CFr by disulfide-induced covalent circularisation that should be an excellent platform for design of novel functions. The accumulation of high levels of CFr exposes the high error rate of the protein translation machinery. The rarity of correspondingly stable fragments in natural proteins coupled with the observation that high quality ribosome binding sites are found to occur within E. coli protein-coding regions significantly less often than expected by random chance implies a stringent evolutionary pressure against protein sub-fragments that can independently fold into stable structures. The symmetric self-association between two identical mis-translated CFr sub-domains to generate an extremely stable structure parallels a mechanism for natural protein-fold evolution by modular recombination of protein sub-structures.     

Rational Design of Selective Antimetabolites of DNA-Thymine Biosynthesis: 5-Propynylpyrimidine Nucleoside Derivatives

Abstract

A novel series of 5-propynylpyrimidine nucleosides are proposed as potential antimetabolites of DNA-thymine biosynthesis. This proposal is based on the results of detailed mechanistic analyses of the molecular interactions between dTMP synthase and its inhibitors. It is proposed that a propynyl side-chain at the 5-position of dUMP, bearing an appropriate leaving group, would cause irreversible inactivation of dTMP synthase, which would not require the presence of the cofactor, CH₂H₄folate.     

The relationship between proteome size,structural disorder and organism complexity

Background  

Sequencing the genomes of the first few eukaryotes created the impression that gene number shows no correlation with organism complexity, often referred to as the G-value paradox. Several attempts have previously been made to resolve this paradox, citing multifunctionality of proteins, alternative splicing, microRNAs or non-coding DNA. As intrinsic protein disorder has been linked with complex responses to environmental stimuli and communication between cells, an additional possibility is that structural disorder may effectively increase the complexity of species.     

Analysis by <Emphasis Type="Italic">siRNA_profile</Emphasis> program displays novel thermodynamic characteristics of highly functional siRNA molecules

Objective  

Here we report the improved results of a new siRNA design program and analysis tool called siRNA_profile that reveals an additional criterion for bioinformatic search of highly functional siRNA sequences.     

The anatomy and functional morphology of the large hermaphroditic duct of three species of Aplysia, with special reference to the atrial gland

The anatomy and functional morphology of the large hermaphroditic duct of three species of gastropod mollusc (Aplysia californica, A. dactylomela, and A. brasiliana) were examined. Each duct is composed of two parallel compartments, the red hemiduct (RHD) and the white hemiduct (WHD), which are distinguishable from the outside of the duct. Four secretory regions, all exocrine in morphology, are recognizable: the RHD secretory epithelium, the atrial gland (or atrial gland-like epithelium), the WHD secretory epithelium, and the accessory gland of the copulatory duct (AGCD). Of these regions, only the atrial gland (or atrial gland-like epithelium) contains egglaying activity and only the atrial gland (or atrial gland-like epithelium) is immunocytochemically labeled by serum antibodies generated against low molecular weight A. californica atrial gland peptides. The RHD is the functional oviduct: the egg cordon passes through a channel lined by the RHD secretory epithelium and bordered by the atrial gland (or atrial gland-like epithelium); the eggs are separated from both the WHD secretory epithelium and the AGCD by internal folds of the duct. The WHD is the functional copulatory duct: the penis, exogenous sperm, and endogenous sperm pass directly by the AGCD and in close proximity to the WHD secretory epithelium; they are separated from both the RHD secretory epithelium and the atrial gland (or atrial gland-like epithelium) by internal folds. The atrial gland (or atrial gland-like epithelium) is thus not likely to have a prostatic function or to be directly stimulated by the penis during copulation; it may play a role in oviductal function.     

<Superscript>1</Superscript>H, <Superscript>15</Superscript>N, <Superscript>13</Superscript>C resonance assignment of plant dehydrin early response to dehydration 10 (ERD10)

Early response to dehydration 10 protein (ERD10) is an intrinsically disordered protein from Arabidopsis thaliana. The protein is upregulated during stress however its mechanism of action at atomic level is not well understood. In the present work multidimensional NMR methodologies are used in order to facilitate the process of chemical shift assignment. The information provided here supports further NMR spectroscopy experiments aimed at elucidation of ERD10 behaviour during molecular recognition events with other proteins.     

A new genus and species of mesoparasitic ergasilid (Copepoda: Cyclopoida) from brackish water pufferfishes collected in northern Australian waters

A new mesoparasitic ergasilid copepod, Majalincola buthi n. g., n. sp., is described based on material collected from the gills of Marilyna darwinii (Castelnau) and M. meraukensis (de Beaufort) (Tetraodontidae) captured in brackish waters in northern Australia. The new genus is characterised by the presence in the fully-transformed adult female of: four tagmata (antennary, neck, postantennary cephalothoracic region and trunk region); a 5-segmented antennule; a trimerous leg 1 endopod; and a free exopod segment armed with two setae on the fifth leg. The establishment of the new genus is supported by the results of a cladistic analysis of Majalincola and members of its sister taxon.     

Fuzzy complexes: polymorphism and structural disorder in protein-protein interactions

The notion that all protein functions are determined through macromolecular interactions is the driving force behind current efforts that aim to solve the structures of all cellular complexes. Recent findings, however, demonstrate a significant amount of structural disorder or polymorphism in protein complexes, a phenomenon that has been largely overlooked thus far. It is our view that such disorder can be classified into four mechanistic categories, covering a continuous spectrum of structural states from static to dynamic disorder and from segmental to full disorder. To emphasize its generality and importance, we suggest a generic term, 'fuzziness', for this phenomenon. Given the crucial role of protein disorder in protein-protein interactions and in regulatory processes, we envision that fuzziness will become integral to understanding the interactome.