技术与方法理性设计改造牛肠激酶的热稳定性

以牛肠激酶作为研究对象,利用理性设计的方法提高其热稳定性。首先通过分子动力学模拟软件Gromacs v 4.5.5,FlexService以及B-FITTER软件预测出了肠激酶的柔性区Fragment 64～69,Fragment 85～90;然后结合β-转角序列统计学信息以及引入位置原有的残基不参与形成氢键的原则,确立了3个突变位点S67P,R87P以及Y136P;通过Quik Change^TM 定点突变的方法引入突变位点,并进行了酶热稳定性分析。结果表明,R87P突变体酶的失活半衰（t_1/2）和T₅₀¹⁰ 较野生型分别提高了3.1 min和11.8℃,同时,动力学常数（K_m/k_cat）测定结果显示酶活未受到显著影响。该策略有潜力应用于其他工业酶分子的热稳定性改造,为推动生物酶的工业化应用奠定基础。     

Engineering Synthetic Adaptors and Substrates for Controlled ClpXP Degradation

Facile control of targeted intracellular protein degradation has many potential uses in basic science and biotechnology. One promising approach to this goal is to redesign adaptor proteins, which can regulate proteolytic specificity by tethering substrates to energy-dependent AAA+ proteases. Using the ClpXP protease, we have probed the minimal biochemical functions required for adaptor function by designing and characterizing variant substrates, adaptors, and ClpX enzymes. We find that substrate tethering mediated by heterologous interaction domains and a small bridging molecule mimics substrate delivery by the wild-type system. These results show that simple tethering is sufficient for synthetic adaptor function. In our engineered system, tethering and proteolysis depend on the presence of the macrolide rapamycin, providing a foundation for engineering highly specific degradation of target proteins in cells. Importantly, this degradation is regulated by a small molecule without the need for new adaptor or enzyme biosynthesis.Targeted proteolytic degradation plays important roles in protein quality control and in regulating cellular circuitry in organisms ranging from bacteria to humans (1–4). In some instances, substrates are recognized directly by a protease enzyme via a degradation tag (Fig. 1A) (see Refs. 5 and 6). In other cases, adaptor proteins or multiple types of substrate sequences are also required to ensure efficient degradation (Fig. 1, B and C) (see Refs. 7 and 8).Open in a separate windowFIGURE 1.A, the ClpX component of the ClpXP protease recognizes some substrates via a degradation tag, denatures the substrate, and then translocates the unfolded protein into ClpP for degradation. B, adaptor-assisted binding of a substrate to ClpXP. C, self-tethering of a substrate to ClpXP.Experimentally induced degradation can be used as a tool to probe the role of specific proteins in cellular processes. For example, a protein that is normally stable can be modified to make its degradation conditionally dependent on the presence of an adaptor, allowing studies of the consequences of depletion after induction of adaptor synthesis (7, 9). Such systems complement methods, such as RNA interference, that rely upon repressing biosynthesis of the target protein but offer significant advantages when rapid depletion of otherwise long-lived proteins is the goal (10–12). We are interested in engineering synthetic adaptor systems to control targeted intracellular degradation.ClpXP is a AAA+ protease present in bacteria and mitochondria that consists of two components, ClpX and ClpP. Hexamers of ClpX recognize degradation tags in specific substrate proteins, unfold them in a reaction that requires ATP hydrolysis, and then use additional cycles of ATP hydrolysis to translocate the unfolded polypeptide into an interior chamber of ClpP, where proteolysis takes place (see Fig. 1A). The simplest way in which an adaptor could stimulate degradation is by tethering a specific substrate to a protease, thereby increasing its effective concentration and facilitating proteolysis (see Fig. 1B; for review, see Ref. 3). The SspB adaptor, for example, appears to function by this mechanism. SspB enhances ClpXP degradation of certain substrates, including N-RseA and proteins bearing the ssrA-degradation tag (2, 8, 13). ClpXP degrades these substrates in the absence of SspB, but K_m for degradation is substantially lower when this adaptor is present. Two features of SspB are consistent with a tethering mechanism. It has a substrate-binding domain with a groove that binds a portion of the ssrA tag or a sequence in N-RseA, and it contains a flexible C-terminal extension terminating with a peptide motif (XB) that binds to the N-terminal domain of ClpX (14–19). Mutations that prevent SspB binding to ClpX or block substrate binding to SspB eliminate stimulation of degradation (13, 16, 20).It has not been rigorously established, however, that tethering per se is sufficient for the activity of any adaptor. Based on biochemical experiments, for instance, Thibault et al. (21) proposed that the adaptor activity of SspB is mediated, in part, by its ability to direct the movement of the N-terminal domains of ClpX, and thereby to regulate the delivery of tagged substrates to ClpXP. For some adaptors, tethering of the substrate to the protease is not sufficient for degradation. For example, the ClpS adaptor tethers N-end rule substrates to the AAA+ ClpAP protease (22–24), but some ClpS mutants mediate efficient substrate tethering to ClpAP without facilitating degradation (25). In such cases, more complicated transactions between the adaptor and the protease appear to be needed to ensure that the substrate is properly delivered to the protease. Moreover, in some instances, adaptors play roles in substrate delivery but are also required for assembly of the active protease (26).The studies reported here were motivated by two major goals. First, we wished to test if a completely synthetic adaptor system could be used to regulate substrate degradation. Second, we sought to design a proteolysis system that could be controlled by the presence or absence of a small molecule. To define the minimal biochemical properties required for adaptor-protein function, we engineered and characterized synthetic variants of adaptors, substrates, and the ClpXP protease. We reasoned that if specialized interactions between SspB and the N-terminal domain of ClpX were a requisite part of substrate delivery, then replacing either component would preclude efficient degradation. By contrast, we found that rapid degradation of an otherwise poor substrate was possible in the absence of SspB and the N-domain as long as substrate·enzyme tethering was maintained by other interaction domains. These results show that tethering alone is sufficient for synthetic- adaptor function. We were also able to control degradation in vitro and in vivo using systems in which a small molecule, rapamycin, drives assembly of tethered proteolytic complexes. Thus, targeted degradation can be engineered to depend, in a conditional fashion, on the presence of a small molecule. In principle, degradation under small molecule control has many of the advantages of chemical genetics (27), but should be even simpler and more widely applicable as a method of functional inhibition. In addition, controlling degradation in this fashion is possible even when biosynthesis of new macromolecules is precluded.     

Corrosion and Alloy Engineering in Rational Design of High Current Density Electrodes for Efficient Water Splitting

Alongside rare‐earth metals, Ni, Fe, Co, Cu are some of the critical materials that will be in huge demand thanks to growth in clean‐energy sector. Herein scrap stainless steel wires (SSW) from worn‐out tires are employed as a support material for catalyst integration in the hydrogen evolution reaction (HER). In addition, SSW by corrosion engineering is exercised as an in situ formed freestanding robust electrode for the oxygen evolution reaction (OER). By superficial corrosion of SSW, inherent active species are unmasked in the form of Ni/FeOOH nanocrystallites displaying efficient water oxidation by reaching 500 mA cm^?2 at low overpotential (η₅₀₀) of 287 mV in 1 m KOH. Similarly, cathode scrap SSW with active (alloy) coatings of MoNi₄ catalyzes the HER at η_‐200 = 77 mV, with a low activation energy (E_a = 16.338 kJ mol^?1) and high durability of 150 h. Promisingly, when used in industrial conditions, 5 m KOH, 343 K, these electrodes demonstrate abnormal activity by yielding high anodic and cathodic current density of 1000 mA cm^?2 at η = 233 mV and η = 161 mV, respectively. This work may inspire researchers to explore and reutilize high‐demand metals from scrap for addressing critical material shortfalls in clean‐energy technologies.     

Antimicrobial Peptide Engineering: Rational Design,Synthesis, and Synergistic Effect

Russian Journal of Bioorganic Chemistry - The swift killing kinetics is an attractive characteristics of wide-spectrum antimicrobial activity of peptides, which made them generate great attention...     

尊重自然力启示下的城市自生群落改良设计实验

城市人工植物群落的设计与管理中抑制场地自然力和群落演替过程,增加了维护成本的同时削弱了群落的生态功能,尊重场地自然力的城市自生植物群落具有良好的生态价值与潜力,但其外貌不被公众所接受。是否存在一种可行的群落设计方法,能够协调抑制自然力所付出的代价与尊重自然力带来的问题之间的矛盾？研究首先梳理尊重自然力对城市植物群落生态设计的启示：尊重自愈力,利用场地自生群落;尊重过程,将演替过程引入群落设计;尊重自然力,管理中减少除草。其次,提出解决矛盾的出路在于尊重自然力基础上,引入栽培植物对自生群落进行改良设计,重构新的种间关系平衡。最后,以“自愈力演替—人工改良—自然力演替—人工管理维护”的理论途径设计自愈力演替实验和自生群落改良设计实验,并在城市场地展开实验性研究。2年的观测结果表明,基于自然演替的改良设计途径可以有效协调“尊重自然力”的矛盾,显著提高群落的美学价值和物种丰富度,还能降低建植和维护成本。     

A Biophysically Based Mathematical Model for the Kinetics of Mitochondrial Calcium Uniporter

Ca²⁺ transport through mitochondrial Ca²⁺ uniporter is the primary Ca²⁺ uptake mechanism in respiring mitochondria. Thus, the uniporter plays a key role in regulating mitochondrial Ca²⁺. Despite the importance of mitochondrial Ca²⁺ to metabolic regulation and mitochondrial function, and to cell physiology and pathophysiology, the structure and composition of the uniporter functional unit and kinetic mechanisms associated with Ca²⁺ transport into mitochondria are still not well understood. In this study, based on available experimental data on the kinetics of Ca²⁺ transport via the uniporter, a mechanistic kinetic model of the uniporter is introduced. The model is thermodynamically balanced and satisfactorily describes a large number of independent data sets in the literature on initial or pseudo-steady-state influx rates of Ca²⁺ via the uniporter measured under a wide range of experimental conditions. The model is derived assuming a multi-state catalytic binding and Eyring's free-energy barrier theory-based transformation mechanisms associated with the carrier-mediated facilitated transport and electrodiffusion. The model is a great improvement over the previous theoretical models of mitochondrial Ca²⁺ uniporter in the literature in that it is thermodynamically balanced and matches a large number of independently published data sets on mitochondrial Ca²⁺ uptake. This theoretical model will be critical in developing mechanistic, integrated models of mitochondrial Ca²⁺ handling and bioenergetics which can be helpful in understanding the mechanisms by which Ca²⁺ plays a role in mediating signaling pathways and modulating mitochondrial energy metabolism.     

A Biophysically Based Mathematical Model for the Kinetics of Mitochondrial Na-Ca Antiporter

Sodium-calcium antiporter is the primary efflux pathway for Ca²⁺ in respiring mitochondria, and hence plays an important role in mitochondrial Ca²⁺ homeostasis. Although experimental data on the kinetics of Na⁺-Ca²⁺ antiporter are available, the structure and composition of its functional unit and kinetic mechanisms associated with the Na⁺-Ca²⁺ exchange (including the stoichiometry) remains unclear. To gain a quantitative understanding of mitochondrial Ca²⁺ homeostasis, a biophysical model of Na⁺-Ca²⁺ antiporter is introduced that is thermodynamically balanced and satisfactorily describes a number of independent data sets under a variety of experimental conditions. The model is based on a multistate catalytic binding mechanism for carrier-mediated facilitated transport and Eyring's free energy barrier theory for interconversion and electrodiffusion. The model predicts the activating effect of membrane potential on the antiporter function for a 3Na⁺:1Ca²⁺ electrogenic exchange as well as the inhibitory effects of both high and low pH seen experimentally. The model is useful for further development of mechanistic integrated models of mitochondrial Ca²⁺ handling and bioenergetics to understand the mechanisms by which Ca²⁺ plays a role in mitochondrial signaling pathways and energy metabolism.     

Novel Fluorescence-Assisted Whole-Cell Assay for Engineering and Characterization of Proteases and Their Substrates

We have developed a sensitive and highly efficient whole-cell methodology for quantitative analysis and screening of protease activity in vivo. The method is based on the ability of a genetically encoded protease to rescue a coexpressed short-lived fluorescent substrate reporter from cytoplasmic degradation and thereby confer increased whole-cell fluorescence in proportion to the protease''s apparent activity in the Escherichia coli cytoplasm. We demonstrated that this system can reveal differences in the efficiency with which tobacco etch virus (TEV) protease processes different substrate peptides. In addition, when analyzing E. coli cells expressing TEV protease variants that differed in terms of their in vivo solubility, cells containing the most-soluble protease variant exhibited the highest fluorescence intensity. Furthermore, flow cytometry screening allowed for enrichment and subsequent identification of an optimal substrate peptide and protease variant from a large excess of cells expressing suboptimal variants (1:100,000). Two rounds of cell sorting resulted in a 69,000-fold enrichment and a 22,000-fold enrichment of the superior substrate peptide and protease variant, respectively. Our approach presents a new promising path forward for high-throughput substrate profiling of proteases, engineering of novel protease variants with desired properties (e.g., altered substrate specificity and improved solubility and activity), and identification of protease inhibitors.Proteases constitute a group of enzymes that irreversibly catalyze the cleavage of peptide bonds and represent approximately 2% of all protein-encoding genes in living organisms (39). Besides acting as virulence factors for many pathogens (16), proteases are crucial for the regulation of numerous biological processes that influence the life and death of a cell (4). These enzymes also underlie several pathological conditions, such as cancer (13) and neurodegenerative (20) and cardiovascular (8) diseases. A key issue for increasing our knowledge about such complex biological processes, and thereby hopefully also providing possibilities for new therapeutic strategies, is to deduce the proteases’ substrate repertoires. Consequently, a lot of efforts around the world are dedicated to the characterization of proteases and their substrates (2, 31). In addition to their biological importance, proteases have attracted much interest in several biotechnological and industrial applications, such as removal of “fusion tags” from recombinant target proteins (38), as supplements in dishwashing and laundry detergents, or for bating of hides and skin in the leather industry (41, 44). Sometimes, however, their use is hindered due to limitations inherent to a specific protease: for example, low solubility, poor enzyme stability and specificity, or limited activity. It would therefore be of great aid to have powerful and straightforward methods available that facilitate the engineering of novel protease variants not suffering from such limitations.Traditionally, protease substrate specificity has been studied by comparison and alignment of naturally occurring substrate peptide sequences (7) or through biochemical analysis of cleavage products with synthetic peptides (47). More recent and powerful methods instead rely on the use of combinatorial substrate libraries, which can either be chemically or biologically generated (6, 15). Although all of these methods have proven useful in determining protease function, many suffer from being laborious and of limited throughput capacity, having an insufficient dynamic range, and resulting in limited information on the substrate profile. Moreover, only a small fraction of all proteases have been studied to date, and there is a need for novel approaches that allow for determination of protease specificity in a rapid, accurate, and quantitative manner.Concerning the engineering of enzymes toward novel desired properties, like altered substrate specificity and improved activity, solubility, and stability; researchers have relied on the use of rational design and/or directed-evolution methods in combination with appropriate screening and selection procedures (1, 10, 11, 22). For instance, various mutagenesis procedures and subsequent screening via assays that report on the successful folding of a protein of interest (9, 32, 45) have been used to engineer protein variants exhibiting improved solubility (35, 37, 46). Despite the obvious success of using such folding reporters in solubility/folding engineering projects, there is a risk that the engineered protein may lose its inherent activity since these screening procedures in general do not select for retained activity but only improved solubility/folding. Therefore, as in the case of a protease, it would be advantageous to establish a screening or selection system that has the ability to simultaneously address traits such as improved folding/solubility without loss of proteolytic activity. However, directed evolution of desired catalytic properties has proven quite a challenge. A popular strategy has been to use phage display technologies, often in combination with transition state analogues (18) or mechanism-based suicide inhibitors, for selection (30). Although successful, the enrichment conferred by these methods is generally based on binding rather than catalysis. Georgiou and coworkers circumvented this potential problem by developing an interesting system that actually enables function-based isolation of novel protease variants from large libraries (34, 42, 43). However, their methodology is dependent on the use of cell surface-displayed proteases, which is not applicable to all proteases and therefore may limit its usefulness.Herein, we present a novel, function-based, and highly efficient fluorescence-assisted whole-cell assay for characterization and engineering of proteases and their cognate substrate peptides. The method takes advantage of genetically encoded short-lived fluorescent substrates that upon coexpression of a substrate-specific protease result in a fluorescence signal, which can easily be monitored on a flow cytometer. Cells having a desired fluorescence profile can then be collected through sorting and sequenced to identify the protease-sensitive substrate peptide or protease capable of processing a particular peptide. Using this approach, we show that it is possible to analyze the efficiency with which the highly sequence-specific tobacco etch virus protease (TEVp) processes different substrate peptides and in model experiments also identify and enrich cells expressing the most favorable substrate peptide or protease from a large background of cells harboring less-efficient variants.     

Rational Design of Hybrid Nanostructures for Advanced Photocatalysis

Nanocatalysis has been a growing field over the past few decades with significant developments in understanding the surface properties of nanocatalysts. With recent advances in synthetic methods, size, shape and composition of the nanoparticles can be controlled in a well defined manner which facilitates achieving selective reaction products in multipath reactions. Nanoparticles with specific exposed crystal facets can have different reactivity than other facets for reaction intermediates, which favours selective pathways during the course of reaction. Heterogeneous catalysts have been studied extensively; nano‐sized metal particles are absorbed on mesoporus supports, facilitating access to the large surface area of the nanoparticles and hence exposure of more catalytic sites. Photocatalysis is attractive area of catalysis, in which photoinduced charge carriers are used for a variety of catalytic applications. More interestingly, clean and renewable liquid fuels energy sources such as hydrogen and methyl alcohol can be generated using photocatalysts through water splitting and CO₂ reduction, respectively. Herein, we highlight the progress of nanocatalysis through metal, bimetallic nanoparticle, metal‐semiconductor hybrid nanostructures and oxide nanoparticles for various reactions.     

Improved method for chemiluminescent determination of peroxidase-mimicking DNAzyme activity

The optimization of experimental conditions for the chemiluminescent determination of peroxidase-mimicking DNAzyme (PMDNAzyme) formed at the interaction of hemin and its aptamer EAD2 was performed. The effect of concentrations of hydrogen peroxide and luminol, acidity of the substrate solution, and composition and concentration of the assay buffer was estimated. Under optimized conditions, a value of detection limit for the PMDNAzyme was 350 pM. A comparison of the conditions determined in this work with those reported previously showed that the optimization of the composition of the substrate solution improved the sensitivity of the chemiluminescent determination of the PMDNAzyme. The obtained results open up promising perspectives for using the proposed method to improve the sensitivity of PMDNAzyme-based assays.     

Multi-scale Predictions of Drug Resistance Epidemiology Identify Design Principles for Rational Drug Design

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Rational Design of Advanced Thermoelectric Materials

Advanced thermoelectric technologies can drastically improve energy efficiencies of industrial infrastructures, solar cells, automobiles, aircrafts, etc. When a thermoelectric device is used as a solid‐state heat pump and/or as a power generator, its efficiency depends pivotally on three fundamental transport properties of materials, namely, the thermal conductivity, electrical conductivity, and thermopower. The development of advanced thermoelectric materials is very challenging because these transport properties are interrelated. This paper reviews the physical mechanisms that have led to recent material advances. Progresses in both inorganic and organic materials are summarized. While the majority of the contemporary effort has been focused on lowering the lattice thermal conductivity, the latest development in nanocomposites suggests that properly engineered interfaces are crucial for realizing the energy filtering effect and improving the power factor. We expect that the nanocomposite approach could be the focus of future materials breakthroughs.     

Structure-Based Rational Design of a Phosphotriesterase

In silico substrate docking of both stereoisomers of the pesticide chlorfenvinphos (CVP) in the phosphotriesterase from Agrobacterium radiobacter identified two residues (F131 and W132) that prevent productive substrate binding and cause stereospecificity. A variant (W131H/F132A) was designed that exhibited ca. 480-fold and 8-fold increases in the rate of Z-CVP and E-CVP hydrolysis, respectively, eliminating stereospecificity.Synthetic organophosphate pesticides (OPs) can cause acute neurotoxicity in insects and humans as a result of their inhibition of acetylcholinesterase at the nerve synapse (15). The >90% identical bacterial phosphotriesterases (PTEs) from Pseudomonas diminuta (oph; PTE_Pd) (5) and Agrobacterium radiobacter (opdA; PTE_Ar) (9) efficiently catalyze the hydrolysis of a broad range of OPs, effectively detoxifying them. This has led to the commercialization of PTE_Ar as a free-enzyme bioremediant (14) and its use in treating OP poisoning in animal studies (1). However, not all OPs are efficiently turned over by the PTEs. For instance, despite having a reasonably reactive leaving group (Fig. ​(Fig.1),1), the turnover of chlorfenvinphos (CVP) by PTE_Ar was not detected in a previous study (9).Open in a separate windowFIG. 1.Structures of the leaving groups of all the substrates discussed in this work. (a) 3,5,6-Trichloro-2-pyridinol for methyl chlorpyrifos oxon; (b) 4-nitrophenol for methyl paraoxon and methyl parathion; (c) 2,2-dichloroethenol for dichlorvos; (d) Z/E-2-chloro-1-(2,4-dichlorophenyl)ethanol for E/Z-CVP; (e) 4-methoxyphenol for EPO.