Binding protein-dependent transport systems

Bacterial binding protein-dependent transport systems are the best characterized members of a superfamily of transporters which are structurally, functionally, and evolutionary related to each other. These transporters are not only found in bacteria but also in yeasts, plants, and animals including man, and include both import and export systems. Although any single system is relatively specific, different systems handle very different substrates which can be inorganic ions, amino acids, sugars, large polysaccharides, or even proteins. Some are of considerable medical importance, including Mdr, the protein responsible for multidrug resistance in human tumors, and the product of the cystic fibrosis locus. In this article we review the current state of knowledge on the structure and function of the protein components of these transporters, the mechanism by which transport is mediated, and the role of ATP in the transport process.     

用于蛋白质间相互作用研究的新型双杂交系统

近年来,一些不依赖于转录因子活性的新型双杂交系统相继建立,如分离的泛素系统、蛋白质片段互补分析、阻遏物重构分析和SOS恢复系统等。同利用转录因子活性的酵母双杂交系统相似,这些方法也利用了一些活性蛋白的结构与功能特点来研究蛋白质间相互作用,这些活性蛋白不是转录因子,但也可在结构上进行分离可通过重构使其生物活性得以恢复。由于这些新型双杂交系统的各自特点,使得它们成为酵母双杂交系统的有益补充和研究蛋白质间相互作用的有力工具。     

Uncovering network systems within protein structures

Traditionally, proteins have been viewed as a construct based on elements of secondary structure and their arrangement in three-dimensional space. In a departure from this perspective we show that protein structures can be modelled as network systems that exhibit small-world, single-scale, and to some degree, scale-free properties. The phenomenological network concept of degrees of separation is applied to three-dimensional protein structure networks and reveals how amino acid residues can be connected to each other within six degrees of separation. This work also illuminates the unique features of protein networks in comparison to other networks currently studied. Recognising that proteins are networks provides a means of rationalising the robustness in the overall three-dimensional fold of a protein against random mutations and suggests an alternative avenue to investigate the determinants of protein structure, function and folding.     

Synthesis of membrane proteins in eukaryotic cell‐free systems

Cell‐free protein synthesis (CFPS) is a valuable method for the fast expression of difficult‐to‐express proteins as well as posttranslationally modified proteins. Since cell‐free systems circumvent possible cytotoxic effects caused by protein overexpression in living cells, they significantly enlarge the scale and variety of proteins that can be characterized. We demonstrate the high potential of eukaryotic CFPS to express various types of membrane proteins covering a broad range of structurally and functionally diverse proteins. Our eukaryotic cell‐free translation systems are capable to provide high molecular weight membrane proteins, fluorescent‐labeled membrane proteins, as well as posttranslationally modified proteins for further downstream analysis.     

Amino acid balancing for the prediction and evaluation of protein concentrations in cell-free protein synthesis systems

Cell-free protein synthesis (CFPS) systems are an attractive method to complement the usual cell-based synthesis of proteins, especially for screening approaches. The literature describes a wide variety of CFPS systems, but their performance is difficult to compare since the reaction components are often used at different concentrations. Therefore, we have developed a calculation tool based on amino acid balancing to evaluate the performance of CFPS by determining the fractional yield as the ratio between theoretically achievable and experimentally achieved protein molar concentration. This tool was applied to a series of experiments from our lab and to various systems described in the literature to identify systems that synthesize proteins very efficiently and those that still have potential for higher yields. The well-established Escherichia coli system showed a high efficiency in the utilization of amino acids, but interestingly, less considered systems, such as those based on Vibrio natriegens or Leishmania tarentolae, also showed exceptional fractional yields of over 70% and 90%, respectively, implying very efficient conversions of amino acids. The methods and tools described here can quickly identify when a system has reached its maximum or has limitations. We believe that this approach will facilitate the evaluation and optimization of existing CFPS systems and provides the basis for the systematic development of new CFPS systems.     

Quantifying denudation and sediment–accumulation systems (open and closed lakes): basic concepts and first results

The quantitative aspects of denudation in the drainage areas and sediment accumulation in the corresponding closed or open lakes are approached using two methods: (1) from known mean rates of mechanical and chemical denudation and the ratio of the drainage/lake area, average terrigenous and potential chemical (biogenic and evaporites) sedimentation rates in the lake are determined; or (2) from known sedimentation rates the average denudation rate is found. In closed systems the total mass denuded during a certain unit of time is deposited in the lake as terrigenous, biogenic and evaporitic sediments, whereas open lake systems lose a major part of the dissolved river input derived from chemical denudation. Applying elementary equations describing these relationships and using ratios between mechanical and chemical denudation (weathering ratio) established in other regions, e.g. chemical weathering in the lake drainage can be estimated. Likewise, the knowledge of the ‘bio-index' (ratio of biogenic sediment/total dissolved mass) allows the calculation of the loss of dissolved matter through the outflow from the lake, etc. In a first step, existing data from modern lake systems are evaluated. Later, idealized lake systems in various climatic and morphotectonic zones can be simulated and the results utilized to better understand ancient lake systems. Modern Alpine lakes reflect increased mechanical denudation rates (100 to ≥300 mm/ka) in highly elevated drainage areas of the Central Alps (mainly crystalline rocks) and high chemical denudation (up to ca. 100 mm/ka) in carbonate rocks of the Northern Calcareous Alps. Mechanical denudation is significantly enhanced by glaciation, but transport of the detritus into lakes may be delayed until the time immediately after glacial retreat. Average rates of terrigenous lake sedimentation amount to 5 to ≥15 mm/a (crystalline rocks, high relief), or are ≤5 mm/a (carbonate rocks). Delta outbuilding was a main factor in the filling of many Alpine lakes. Lakes draining low-altitude, semi-arid parts along the eastern margin of the Alps (Lake Balaton in Hungary) have low sedimentation rates and reflect the influence of increased temperature and vegetation. In East Africa, the transition from the Late Pleistocene (arid) to the early Holocene (humid) and again back to somewhat drier conditions caused substantial rises in lake levels and changes from closed to open lake systems. In such cases, the calculation of denudation from accumulation rates is biassed as a result of intermittent sediment storage at the lake margins (low sedimentation rates in the lake centre during lake highstands) or redeposition of sediment during lowstands (lowstand shedding). The modern denudation rates of East African rift lake systems vary by a factor of ≥15 (Lake Tanganyika, crystalline rocks and more dense vegetation cover, total denudation rate ca. 4 mm/ka; Lake Turkana, young volcanic rocks and tephra, sparse vegetation, mechanical denudation rate ca. 60 mm/ka).     

Single-vector three-frame expression systems for affinity-tagged proteins

An effort is presented to create expression vectors which would allow expression of an inserted gene fragment in three reading frames in a single vector from a single promoter but with three separate ribosome binding sites (RBS). Each expression frame would generate an in-frame fusion with an affinity tag to allow efficient recovery of the produced fusion proteins. In the first generation vector, three identical polyhistidyl tags (His(6)) were used as affinity tags for the three expression frames. In the second generation vector, three different tags, an albumin binding domain derived from streptococcal protein G, an IgG binding Staphylococcus aureus protein A-derived domain (Z) and a His(6) tag, were employed to allow frame-specific affinity recovery. To evaluate the systems, model genes have been inserted in three different frames in both vectors. The first vector was demonstrated to produce fusion proteins in all three frames, whereas for the second, with a much wider spacing between the RBSs and affinity tags, expression could only be demonstrated from the first two translational start sites. For both systems, the first translation start was found to be significantly favored over the others. Nevertheless, we believe that the presented results represent the first successful attempt to create single-vector three-frame expression systems, a concept that could become valuable in future combined cloning-expression vectors.     

Protein-stimulated exchange of phosphatidylcholine between intact erythrocytes and various membrane systems

Phosphatidylcholine specific exchange protein from beef liver was found to catalyze the exchange of phosphatidylcholine between intact rat and human erythrocytes and various artificial membranes. Both multilamellar liposomes and single bilayer vesicles prepared from egg lecithin, cholesterol and phosphatidic acid (46:50:4, mol/mol) appeared to be effective phospholipid donor systems. Some merits and disadvantages of the various donor systems are discussed.     

Neuronal signaling systems and ethanol dependence

In recent years there have been remarkable developments toward the understanding of the molecular and/or cellular changes in the neuronal second-messenger pathways during ethanol dependence. In general, it is believed that the cyclic adenosine 3′, 5′-monophosphate (cAMP) and the phosphoinositide (PI) signal-transduction pathways may be the intracellular targets that mediate the action of ethanol and ultimately contribute to the molecular events involved in the development of ethanol tolerance and dependence. Several laboratories have demonstrated that acute ethanol exposure increases, whereas protracted ethanol exposure decreases, agonist-stimulated adenylate cyclase activity in a variety of cell systems, including the rodent brain. Recent studies indicate that various postreceptor events of the cAMP signal transduction cascade (i.e., G_s protein, protein kinase A [PKA], and cAMP-responsive element binding protein [CREB]) in the rodent brain are also modulated by chronic ethanol exposure. The PI signal-transduction cascade represents another important second-messenger system that is modulated by both acute and chronic ethanol exposure in a variety of cell systems. It has been shown that protracted ethanol exposure significantly decreases phospholipase C (PLC) activity in the cerebral cortex of mice and rats. The decreased PLC activity during chronic ethanol exposure may be caused by a decrease in the protein levels of the PLC-Β₁ isozyme but not of PLC-δ₁ or PLC-γ₁ isozymes in the rat cerebral cortex. Protein kinase C (PKC), which is a key step in the Pi-signaling cascade, has been shown to be altered in a variety of cell systems by acute or chronic ethanol exposure. It appears from the literature that PKC plays an important role in the modulation of the function of various neurotransmitter receptors (e.g., γ-aminobutyrate type A [GABAa], N-methyl-D-aspartate [NMDA], serotonin2A [5-HT2a], and 5-HT2C, and muscarinic [m1] receptors) resulting from ethanol exposure. The findings described in this review article indicate that neuronal-signaling proteins represent a molecular locus for the action of ethanol and are possibly involved in the neuroadaptational mechanisms to protracted ethanol exposure. These findings support the notion that alterations in the cAMP and the PI-signaling cascades during chronic ethanol exposure could be the critical molecular events associated with the development of ethanol dependence.     

Peptides as antioxidants and carbonyl quenchers in biological model systems

Subjecting selected peptides to in vitro analyses covering their ability to interfere with the lipid oxidation chain reaction as well as to protect proteins from direct and indirect oxidation has provided the basis for a more detailed understanding of peptide-mediated protection in biological systems. The efficiency of peptides as radical scavengers and chain-breaking antioxidants in oxidizing lipid membranes was found to be low. Previous studies on antioxidative activity of peptides tend not to include comparisons with efficiencies of more well-documented antioxidants and/or use irrelevantly high dosages of peptides. The present study demonstrates that the effect of the investigated peptides towards oxidation in biological membrane systems is mainly a protection of vital proteins from being oxidatively modified. This protection is obtained through a prevention of lipid oxidation derived carbonylation (indirect protein oxidation) and through interference with aqueous radical species (direct protein oxidation), and it is only achieved if the peptides are present in high concentrations as sacrificial antioxidants.     

Protein design in systems metabolic engineering for industrial strain development

Accelerating the process of industrial bacterial host strain development, aimed at increasing productivity, generating new bio-products or utilizing alternative feedstocks, requires the integration of complementary approaches to manipulate cellular metabolism and regulatory networks. Systems metabolic engineering extends the concept of classical metabolic engineering to the systems level by incorporating the techniques used in systems biology and synthetic biology, and offers a framework for the development of the next generation of industrial strains. As one of the most useful tools of systems metabolic engineering, protein design allows us to design and optimize cellular metabolism at a molecular level. Here, we review the current strategies of protein design for engineering cellular synthetic pathways, metabolic control systems and signaling pathways, and highlight the challenges of this subfield within the context of systems metabolic engineering.     

Biochemical evidence for the presence of two alpha-glucoside ABC-transport systems in the hyperthermophilic archaeon Pyrococcus furiosus

The hyperthermophilic archaeon Pyrococcus furiosus can utilize different carbohydrates, such as starch, maltose and trehalose. Uptake of alpha-glucosides is mediated by two different, binding protein-dependent, ATP-binding cassette (ABC)-type transport systems. The maltose transporter also transports trehalose, whereas the maltodextrin transport system mediates the uptake of maltotriose and higher malto-oligosaccharides, but not maltose. Both transport systems are induced during growth on their respective substrates.     

The coiled coil motif in polymer drug delivery systems

The coiled coil is a superhelical structural protein motif that has been thoroughly investigated in recent years. Because of the relatively well-understood principles that determine the properties of coiled coil peptides and proteins, macromolecular systems containing the coiled coil motif have been suggested for various applications. This short review focuses on hybrid polymer coiled coil systems designed for drug delivery purposes. After a short introduction, the most important features of the coiled coils (stability, association number, oligomerization selectivity and orientation of helices) are described, and the factors influencing these characteristics are discussed. Several examples of the most interesting biomedical applications of the polymer-coiled coil systems (according to the authors' opinion) are presented.     

Yeast systems biotechnology for the production of heterologous proteins

Systems biotechnology has been established as a highly potent tool for bioprocess development in recent years. The applicability to complex metabolic processes such as protein synthesis and secretion, however, is still in its infancy. While yeasts are frequently applied for heterologous protein production, more progress in this field has been achieved for bacterial and mammalian cell culture systems than for yeasts. A critical comparison between different protein production systems, as provided in this review, can aid in assessing the potentials and pitfalls of applying systems biotechnology concepts to heterologous protein producing yeasts. Apart from modelling, the methodological basis of systems biology strongly relies on postgenomic methods. However, this methodology is rapidly moving so that more global data with much higher sensitivity will be achieved in near future. The development of next generation sequencing technology enables an unexpected revival of genomic approaches, providing new potential for evolutionary engineering and inverse metabolic engineering.     

Radiation inactivation analysis of thylakoid protein kinase systems in light and in darkness

Chloroplast thylakoid contains several membrane-bound protein kinases that phosphorylate thylakoid polypeptides for the regulation of photosynthesis. Thylakoid protein phosphorylation is activated when the plastoquinone pool is reduced either by light-dependent electron flow through photosystem 2 (PS2) or by adding exogenous reductants such as durohydroquinone in the dark. The major phosphorylated proteins on thylakoid are components of light-harvesting complex 2 (LHC2) and a PS2 associated 9 kDa phosphoprotein. Radiation inactivation technique was employed to determine the functional masses of various kinases for protein phosphorylation in thylakoids. Under the photosynthetically active radiation (PAR), the apparent functional masses of thylakoid protein kinase systems (TPKXs) for catalyzing phosphorylation of LHC2 27 and 25 kDa polypeptides were 540±50 and 454±35 kDa as well as it was 448±23 kDa for PS2 9 kDa protein phosphorylation. Furthermore, the functional sizes of dark-regulated TPKXs for 25 and 9 kDa proteins were 318±25 and 160±8 kDa. The 9 kDa protein phosphorylation was independent of LHC2 polypeptides phosphorylation with regard to its TPKX functional mass. Target size analysis of protein phosphorylation mentioned above indicates that thylakoid contains a group of distinct protein kinase systems. A working model is accordingly proposed to interpret the interaction between these protein kinase systems.     

Tolerance for random recombination of domains in prokaryotic and eukaryotic translation systems: Limited interdomain misfolding in a eukaryotic translation system

It has been proposed that eukaryotic translation systems have a greater capacity for cotranslational folding of domains than prokaryotic translation systems, which reduces interdomain misfolding in multidomain proteins and, therefore, leads to tolerance for random recombination of domains. However, there has been a controversy as to whether prokaryotic and eukaryotic translation systems differ in the capacity for cotranslational domain folding. Here, to examine whether these systems differ in the tolerance for the random domain recombination, we systematically combined six proteins, out of which four are soluble and two are insoluble when produced in an Escherichia coli and a wheat germ cell-free protein synthesis systems, to construct a fusion protein library. Forty out of 60 two-domain proteins and 114 out of 120 three-domain proteins were more soluble when produced in the wheat system than in the E. coli system. Statistical analyses of the solubilities and the activities indicated that, in the wheat system but not in the E. coli system, the two soluble domains comprised mainly of beta-sheets tend to avoid interdomain misfolding and to fold properly even at the neighbor of the misfolded domains. These results demonstrate that a eukaryotic system permits the concomitance of a wider variety of domains within a single polypeptide chain than a prokaryotic system, which is probably due to the difference in the capacity for cotranslational folding. This difference is likely to be related to the postulated difference in the tolerance for random recombination of domains.     

Membrane protein synthesis in cell-free systems: From bio-mimetic systems to bio-membranes

When taking up the gauntlet of studying membrane protein functionality, scientists are provided with a plethora of advantages, which can be exploited for the synthesis of these difficult-to-express proteins by utilizing cell-free protein synthesis systems. Due to their hydrophobicity, membrane proteins have exceptional demands regarding their environment to ensure correct functionality. Thus, the challenge is to find the appropriate hydrophobic support that facilitates proper membrane protein folding. So far, various modes of membrane protein synthesis have been presented. Here, we summarize current state-of-the-art methodologies of membrane protein synthesis in biomimetic-supported systems. The correct folding and functionality of membrane proteins depend in many cases on their integration into a lipid bilayer and subsequent posttranslational modification. We highlight cell-free systems utilizing the advantages of biological membranes.     

Non-canonical inhibition strategies and structural basis of anti-CRISPR proteins targeting type I CRISPR-Cas systems

Mobile genetic elements (MGEs) such as bacteriophages and their host prokaryotes are trapped in an eternal battle against each other. To cope with foreign infection, bacteria and archaea have evolved multiple immune strategies, out of which CRISPR-Cas system is up to now the only discovered adaptive system in prokaryotes. Despite the fact that CRISPR-Cas system provides powerful and delicate protection against MGEs, MGEs have also evolved anti-CRISPR proteins (Acrs) to counteract the CRISPR-Cas immune defenses. To date, 46 families of Acrs targeting type I CRISPR-Cas system have been characterized, out of which structure information of 21 families have provided insights on their inhibition strategies. Here, we review the non-canonical inhibition strategies adopted by Acrs targeting type I CRISPR-Cas systems based on their structure information by incorporating the most recent advances in this field, and discuss our current understanding and future perspectives. The delicate interplay between type I CRISPR-Cas systems and their Acrs provides us with important insights into the ongoing fierce arms race between prokaryotic hosts and their predators.     

Matching fusion protein systems for affinity analysis of two interacting families of proteins: the cohesin-dockerin interaction

Cellulosomes are multi-enzyme complexes that orchestrate the efficient degradation of cellulose and related plant cell wall polysaccharides. The complex is maintained by the high-affinity protein-protein interaction between two complementary modules: the cohesin and the dockerin. In order to characterize the interaction between different cohesins and dockerins, we have developed matching fusion-protein systems, which harbor either the cohesin or the dockerin component. For this purpose, corresponding plasmid cassettes were designed, which encoded for the following carrier proteins: (i) a thermostable xylanase with an appended His-tag; and (ii) a highly stable cellulose-binding module (CBM). The resultant xylanase-dockerin and CBM-cohesin fusion products exhibited high expression levels of soluble protein. The expressed, affinity-purified proteins were extremely stable, and the functionality of the cohesin or dockerin component was retained. The fusion protein system was used to establish a sensitive and reliable, semi-quantitative enzyme-linked affinity assay for determining multiple samples of cohesin-dockerin interactions in microtiter plates. A variety of cohesin-dockerin systems, which had been examined previously using other methodologies, were revisited applying the affinity-based enzyme assay, the results of which served to verify the validity of the approach.     

Plant molecular farming: systems and products

Plant molecular farming is a new and promising industry involving plant biotechnology. In this review, we describe several diverse plant systems that have been developed to produce commercially useful proteins for pharmaceutical and industrial uses. The advantages and disadvantages of each system are discussed. The first plant-derived molecular farming products have reached the marketplace and other products are poised to join them during the next few years. We explain the rationale for using plants as biofactories. We also describe the products currently on the market, and those that appear likely to join them in the near future. Lastly, we discuss the issue of public acceptance of molecular farming products.Communicated by P.P. Kumar