A single aromatic core mutation converts a designed “primitive” protein from halophile to mesophile folding

The halophile environment has a number of compelling aspects with regard to the origin of structured polypeptides (i.e., proteogenesis) and, instead of a curious niche that living systems adapted into, the halophile environment is emerging as a candidate “cradle” for proteogenesis. In this viewpoint, a subsequent halophile‐to‐mesophile transition was a key step in early evolution. Several lines of evidence indicate that aromatic amino acids were a late addition to the codon table and not part of the original “prebiotic” set comprising the earliest polypeptides. We test the hypothesis that the availability of aromatic amino acids could facilitate a halophile‐to‐mesophile transition by hydrophobic core‐packing enhancement. The effects of aromatic amino acid substitutions were evaluated in the core of a “primitive” designed protein enriched for the 10 prebiotic amino acids (A,D,E,G,I,L,P,S,T,V)—having an exclusively prebiotic core and requiring halophilic conditions for folding. The results indicate that a single aromatic amino acid substitution is capable of eliminating the requirement of halophile conditions for folding of a “primitive” polypeptide. Thus, the availability of aromatic amino acids could have facilitated a critical halophile‐to‐mesophile protein folding adaptation—identifying a selective advantage for the incorporation of aromatic amino acids into the codon table.     

Toward a “Structural BLAST”: Using structural relationships to infer function

We outline a set of strategies to infer protein function from structure. The overall approach depends on extensive use of homology modeling, the exploitation of a wide range of global and local geometric relationships between protein structures and the use of machine learning techniques. The combination of modeling with broad searches of protein structure space defines a “structural BLAST” approach to infer function with high genomic coverage. Applications are described to the prediction of protein–protein and protein–ligand interactions. In the context of protein–protein interactions, our structure‐based prediction algorithm, PrePPI, has comparable accuracy to high‐throughput experiments. An essential feature of PrePPI involves the use of Bayesian methods to combine structure‐derived information with non‐structural evidence (e.g. co‐expression) to assign a likelihood for each predicted interaction. This, combined with a structural BLAST approach significantly expands the range of applications of protein structure in the annotation of protein function, including systems level biological applications where it has previously played little role.     

Creating a completely “cell‐free” system for protein synthesis

Cell‐free protein synthesis is a promising tool to take biotechnology outside of the cell. A cell‐free approach provides distinct advantages over in vivo systems including open access to the reaction environment and direct control over all chemical components for facile optimization and synthetic biology integration. Promising applications of cell‐free systems include portable diagnostics, biotherapeutics expression, rational protein engineering, and biocatalyst production. The highest yielding and most economical cell‐free systems use an extract composed of the soluble component of lysed Escherichia coli. Although E. coli lysis can be highly efficient (>99.999%), one persistent challenge is that the extract remains contaminated with up to millions of cells per mL. In this work, we examine the potential of multiple decontamination strategies to further reduce or eliminate bacteria in cell‐free systems. Two strategies, sterile filtration and lyophilization, effectively eliminate contaminating cells while maintaining the systems’ protein synthesis capabilities. Lyophilization provides the additional benefit of long‐term stability at storage above freezing. Technologies for personalized, portable medicine and diagnostics can be expanded based on these foundational sterilized and completely “cell‐free” systems. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1716–1719, 2015     

Revisiting “reverse hydrophobic effect”: Applicable only to coil mutations at the surface

In a seminal paper, Pakula and Sauer (Nature, 1990, 344, 363–364) demonstrated that the increase in side‐chain hydrophobicity has a reverse relationship with protein stability. We have addressed this problem with several examples of mutants that span at different locations in protein structure based on secondary structure and solvent accessibility. We confirmed that the stability change upon single coil mutation at exposed region is reversely correlated with hydrophobicity with a single exception. In addition, we found the existence of such relationship in partially buried coil mutants. The stability of exposed helical mutants is governed by conformational properties. In buried and partially buried helical and strand mutants properties reflecting hydrophobicity have direct relationship with stability, whereas an opposite relationship was obtained with entropy and flexibility. The structural analysis of partially buried/exposed mutants showed that the surrounding residues are important for the stability change upon mutation. These results provide insights to understand the general behavior for the stability of proteins upon amino acid substitutions. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 591–599, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Changes in gene expression during germination reveal pea genotypes with either “quiescence” or “escape” mechanisms of waterlogging tolerance

Waterlogging causes germination failure in pea (Pisum sativum L.). Three genotypes (BARI Motorshuti‐3, Natore local‐2 [NL‐2], and Kaspa) contrasting in ability to germinate in waterlogged soil were exposed to different durations of waterlogging. Whole genome RNAseq was employed to capture differentially expressing genes. The ability to germinate in waterlogged soil was associated with testa colour and testa membrane integrity as confirmed by electrical conductivity measurements. Genotypes Kaspa and NL‐2 displayed different mechanisms of tolerance. In Kaspa, an energy conserving strategy was indicated by a strong upregulation of tyrosine protein kinsase and down regulation of linoleate 9S‐lipoxygenase 5, a fat metabolism gene. In contrast, a faster energy utilization strategy was suggested in NL‐2 by the marked upregulation of a subtilase family protein and peroxisomal adenine nucleotide carrier 2, a fat metabolizing gene. Waterlogging susceptibility in germinating seeds of genotype BARI Motorshuti‐3 was linked to upregulation of a kunitz‐type trypsin/protease inhibitor that blocks protein metabolism and may lead to excessive lipid metabolism and the membrane leakage associated with waterlogging damage. Pathway analyses based on gene ontologies showed seed storage protein metabolism as upregulated in tolerant genotypes and downregulated in the sensitive genotype. Understanding the tolerance mechanism provides a platform to breed for adaptation to waterlogging stress at germination in pea.     

A “plug‐n‐play” modular metabolic system for the production of apocarotenoids

Apocarotenoids, such as α‐, β‐ionone, and retinol, have high commercial values in the food and cosmetic industries. The demand for natural ingredients has been increasing dramatically in recent years. However, attempts to overproduce β‐ionone in microorganisms have been limited by the complexity of the biosynthetic pathway. Here, an Escherichia coli‐based modular system was developed to produce various apocarotenoids. Incorporation of enzyme engineering approaches (N‐terminal truncation and protein fusion) into modular metabolic engineering strategy significantly improved α‐ionone production from 0.5 mg/L to 30 mg/L in flasks, producing 480 mg/L of α‐ionone in fed‐batch fermentation. By modifying apocarotenoid genetic module, this platform strain was successfully re‐engineered to produce 32 mg/L and 500 mg/L of β‐ionone in flask and bioreactor, respectively (>80‐fold higher than previously reported). Similarly, 33 mg/L of retinoids was produced in flask by reconstructing apocarotenoid module, demonstrating the versatility of the “plug‐n‐play” modular system. Collectively, this study highlights the importance of the strategy of simultaneous modular pathway optimization and enzyme engineering to overproduce valuable chemicals in microbes.     

Various strategies for obtaining artificial hemoproteins: From “hemoabzymes” to “hemozymes”

The design of artificial hemoproteins that could lead to new biocatalysts for selective oxidation reactions of organic compounds presents a huge interest especially in pharmacology, both for a better understanding of the metabolic profile of drugs and for the synthesis of enantiomerically pure molecules that could be involved in the design of drugs.The present results show that the so-called “host-guest strategy” that involves the non-covalent incorporation of anionic water-soluble iron-porphyrins into xylanase A from Streptomyces lividans, a low cost protein, leads to such an artificial hemoprotein that is able to perform the stereoselective oxidation of sulfides.     

Escherichia coli “TatExpress” strains super‐secrete human growth hormone into the bacterial periplasm by the Tat pathway

Numerous high‐value proteins are secreted into the Escherichia coli periplasm by the General Secretory (Sec) pathway, but Sec‐based production chassis cannot handle many potential target proteins. The Tat pathway offers a promising alternative because it transports fully folded proteins; however, yields have been too low for commercial use. To facilitate Tat export, we have engineered the TatExpress series of super‐secreting strains by introducing the strong inducible bacterial promoter, ptac , upstream of the chromosomal tatABCD operon, to drive its expression in E. coli strains commonly used by industry (e.g., W3110 and BL21). This modification significantly improves the Tat‐dependent secretion of human growth hormone (hGH) into the bacterial periplasm, to the extent that secreted hGH is the dominant periplasmic protein after only 1 hr induction. TatExpress strains accumulate in excess of 30 mg L^?1 periplasmic recombinant hGH, even in shake flask cultures. A second target protein, an scFv, is also shown to be exported at much higher rates in TatExpress strains.

     

An evolutionary interpretation of the “motivation to oviposit”

The ovipositional behavior of parasitoids and other insects is often described by phrases such as “motivation to oviposit” or “ovipositional drive”. This paper shows how an evolutionary (i.e. functional) interpretation can be given to such phrases. A detailed model for the parasitisation of Sycamore aphids by M. pseudoplatani is developed, using experiments by Collins and Dixon (1986). Two models are developed: i) one in which egg complement is the only state variable and ii) one in which egg complement and information concerning host densities are state variables. Comparisons of the behaviour of simulated parasitoids, using the decisions associated with the models, and the experiments suggest that both egg complement and information are important for the parasitoid's decision making. Accepting previously parasitized hosts may be optimal, and not simply an error in parasitoid perception. A number of other detailed predictions are made, such as the relative fitness of first and second eggs in superparasitized hosts and the nature of the memory of the parasitoid.     

Accumulation of “Old Proteins” and the Critical Need for MS‐based Protein Turnover Measurements in Aging and Longevity

Aging and age‐related diseases are accompanied by proteome remodeling and progressive declines in cellular machinery required to maintain protein homeostasis (proteostasis), such as autophagy, ubiquitin‐mediated degradation, and protein synthesis. While many studies have focused on capturing changes in proteostasis, the identification of proteins that evade these cellular processes has recently emerged as an approach to studying the aging proteome. With advances in proteomic technology, it is possible to monitor protein half‐lives and protein turnover at the level of individual proteins in vivo. For large‐scale studies, these technologies typically include the use of stable isotope labeling coupled with MS and comprehensive assessment of protein turnover rates. Protein turnover studies have revealed groups of highly relevant long‐lived proteins (LLPs), such as the nuclear pore complexes, extracellular matrix proteins, and protein aggregates. Here, the role of LLPs during aging and age‐related diseases and the methods used to identify and quantify their changes are reviewed. The methods available to conduct studies of protein turnover, used in combination with traditional proteomic methods, will enable the field to perform studies in a systems biology context, as changes in proteostasis may not be revealed in studies that solely measure differential protein abundances.     

Probing the interaction of distamycin A with S100β: the “unexpected” ability of S100β to bind to DNA‐binding ligands

DNA‐minor‐groove‐binding ligands are potent antineoplastic molecules. The antibiotic distamycin A is the prototype of one class of these DNA‐interfering molecules that have been largely used in vitro. The affinity of distamycin A for DNA is well known, and the structural details of the complexes with some B‐DNA and G‐quadruplex‐forming DNA sequences have been already elucidated. Here, we show that distamycin A binds S100β, a protein involved in the regulation of several cellular processes. The reported affinity of distamycin A for the calcium(II)‐loaded S100β reinforces the idea that some biological activities of the DNA‐minor‐groove‐binding ligands arise from the binding to cellular proteins. Copyright © 2015 John Wiley & Sons, Ltd.