The spontaneous gating activity of OmpC porin is affected by mutations of a putative hydrogen bond network or of a salt bridge between the L3 loop and the barrel

Porins are trimeric channel-forming proteins of the outer membrane of Escherichia coli. Each subunit contains 16 beta-strands forming a transmembrane beta-barrel whose pore is constricted by the third extracellular loop (L3). We investigated the effects of site-directed mutations at two critical regions of the OmpC porin: (i) the D315A mutation targets a key component of a putative hydrogen bond network linking the L3 loop to the adjacent barrel wall and (ii) the D118Q, R174Q and R92Q mutations target putative salt bridges at the root of the L3 loop. We purified the outer membrane fractions obtained from each mutant and reconstituted them in liposomes suitable for electrophysiology. Patch clamp experiments showed that the frequency of spontaneous transitions between open and closed states is increased in the D315A, D118Q and R92Q mutants but unchanged in the R174Q mutant. These transitions are not driven by transmembrane voltage changes and represent the thermal oscillations between functionally distinct conformations. The asymmetric voltage-dependent inactivation of the channels is not affected by the mutations, however, suggesting different molecular mechanisms for the spontaneous and voltage- dependent gating processes. We propose that the positioning or flexibility of the L3 loop across the pore, as governed by the putative hydrogen-bond network and a salt bridge, play a role in determining the frequency of spontaneous channel gating.      

Modulating functional loop movements: the role of highly conserved residues in the correlated loop motions

Loop flexibility in enzymes plays a vital role in correctly positioning catalytically important residues. This strong relationship between enzyme flexibility and function provides an opportunity to engineer new substrates and inhibitors. It further allows the design of site-directed mutagenesis experiments to explore enzymatic activity through the control of flexibility of a functional loop. Earlier, we described a novel mechanism in which a small loop triggers the motions of a functional loop in three enzymes (beta-1,4-galactosyltransferase, lipase, and enolase) unrelated in sequence, structure, or function. Here, we further address the question of how the interactions between various flexible loops modulate the movements of the functional loop. We examine beta-1,4-galactosyltransferase as a model system in which a Long loop undergoes a large conformational change (moves in space up to 20 A) upon substrate binding in addition to a small loop (Trp loop) that shows a considerably smaller conformational change. Our molecular-dynamics simulations carried out in implicit and explicit solvent show that, in addition to these two loops, two other neighboring loops are also highly flexible. These loops are in contact with either the Long loop or the Trp loop. Analysis of the covariance of the spatial displacement of the residues reveals that coupled motions occur only in one of these two loops. Sequence analysis indicates that loops correlated in their motions also have highly conserved residues involved in the loop-loop interactions. Further, analysis of crystal structures and simulations in explicit water open the possibility that the Trp loop that triggers the movement of the Long loop in the unbound conformation may also play the same role in the substrate-bound conformation through its contact with the conserved and correlated third loop. Our proposition is supported by the observation that four of the five conserved positions in the third loop are at the interface with the Trp loop. Evolution appears to select residues that drive the functional Long loop to a large conformational change. These observations suggest that altering selected loop-loop interactions might modulate the movements of the functional loop.     

Determination of the conformation of the human VDAC1 N-terminal peptide, a protein moiety essential for the functional properties of the pore

Mitochondrial porin or VDAC (voltage-dependent anion-selective channel) is the most abundant protein in the mitochondrial outer membrane. The structure of VDAC has been predicted to be a transmembrane beta-barrel with an alpha-helix at the N terminus. It is a matter of debate as to whether this putative alpha-helix plays a structural role as a component of the pore walls or a function in the pore activity. We have synthesised the human VDAC1 (HVDAC1) N-terminal peptide Ac-AVPPTYADLGKSARDVFTK-NH2 (Prn2-20) and determined its structure by CD and NMR spectroscopy. CD studies show that the Prn2-20 peptide exists in aqueous solvent as an unstructured peptide with no stable secondary structure. In membrane-mimetic SDS micelles or water/trifluoroethanol, however, it assumes an amphipathic alpha-helix conformation between Tyr5 and Val16, as deduced from NMR. No ordered structure was observed in dodecyl beta-maltoside. Differential scanning calorimetric measurements were carried out in order to examine the membrane affinity of the peptide. Upon interaction with the negatively charged 1,2 dipalmitoyl-sn-glycero-3-phosphoserine membrane, Prn2-20 exhibited distinctive behaviour, suggesting that electrostatics play an important role. Interaction between the peptide and artificial bilayers indicates that the peptide lies on the membrane surface. Recombinant HVDAC1 deletion mutants, devoid of seven or 19 N-terminal amino acids, were used for transfection of eukaryotic cells. Over-expression of HVDAC1 increases the number of Cos cells with depolarised mitochondria, and this effect is progressively reduced in cells transfected with HVDAC1 lacking those seven or 19 amino acids. The mitochondrial targeting of the deletion mutants is unaffected. The overall picture emerging from our experiments is that the VDAC N-terminal peptide plays a role in the proper function of this protein during apoptotic events.     

Deletions of single extracellular loops affect pH sensitivity, but not voltage dependence, of the Escherichia coli porin OmpF

The molecular basis for the voltage and pH dependence of the Escherichia coli OmpF porin activity remains unknown. The L3 loop was previously shown not be involved in voltage dependence. Here we used seven OmpF mutants where single extracellular loops, except L3, were deleted one at a time. The proteins are expressed at levels comparable to wild-type and purified as trimers. Wild-type and mutant proteins were inserted into planar lipid bilayers for electrophysiological measurement of their activity. Current-voltage relationships show the typical porin channel closure at voltages greater than the critical voltage. Measurements of critical voltages for the seven deletion mutants showed no significant differences relative to wild-type, hence eliminating the role of single loops in voltage sensitivity. However, deletions of loops L1, L7 or L8 affected the tendency of channels to close at acidic pH. Wild-type channels close more readily at acidic pH and their open probability is decreased by approximately 60% at pH 4.0 relative to pH 7.0. For mutants lacking loop L1, L7 or L8, the channel open probability was found not to be significantly different at pH 4.0 than at pH 7.0. The other deletion mutants retained a pH sensitivity similar to the wild-type channel. Possible mechanistic scenarios for the voltage- and pH dependence of E.coli OmpF porin are discussed based on these results.     

Homology modelling of transferrin-binding protein A from Neisseria meningitidis

Neisseria meningitidis, a causative agent of bacterial meningitis, obtains transferrin-bound iron by expressing two outer membrane located transferrin-binding proteins, TbpA and TbpB. TbpA is thought to be an integral outer membrane pore that facilitates iron uptake. Evidence suggests that TbpA is a useful antigen for inclusion in a vaccine effective against meningococcal disease, hence the identification of regions involved in ligand binding is of paramount importance to design strategies to block uptake of iron. The protein shares sequence and functional similarities to the Escherichia coli siderophore receptors FepA and FhuA, whose structures have been determined. These receptors are composed of two domains, a 22-stranded beta-barrel and an N-terminal plug region that sits within the barrel and occludes the transmembrane pore. A three-dimensional TbpA model was constructed using FepA and FhuA structural templates, hydrophobicity analysis and homology modelling. TbpA was found to possess a similar architecture to the siderophore receptors. In addition to providing insights into the highly immunogenic nature of TbpA and allowing the prediction of potentially important ligand-binding epitopes, the model also reveals a narrow channel through its entire length. The relevance of this channel and the spatial arrangement of external loops, to the mechanism of iron translocation employed by TbpA is discussed.     

Influence of catalyst pore network structure on the hysteresis of multiphase reactions

The effects of the catalyst pore network structure on multiphase reactions in catalyst pellets are investigated by using the experimentally validated pore network model proposed in our recent work (AIChE J, 62 , 451, 2016). The simulations display hysteresis loops of the effectiveness factor. The hysteresis loop area becomes significantly larger, when having small volume‐averaged pore radius, wide pore‐size distribution, and low pore connectivity; however, the loop area is insensitive to pellet size, even though it affects the value of the effectiveness factor. The hysteresis loop area is also strongly affected by the spatial distribution of the pore size, in particular for a bimodal pore‐size distribution. The pore network structure directly influences mass transfer, capillary condensation, and pore blocking, and subsequently passes these influences on to the hysteresis loop of the effectiveness factor. Recognizing these effects is essential when designing porous catalysts for multiphase reaction processes. © 2016 American Institute of Chemical Engineers AIChE J, 63: 78–86, 2017     

The importance of loop length in the folding of an immunoglobulin domain

Immunoglobulin (Ig)-like proteins have been shown to fold following formation of a nucleus comprising interactions between residues that are distant in the primary sequence. What role do the loops connecting these nucleus residues play? Here, the importance of loops connecting beta-strands in different sheets of the Ig fold is investigated, by insertion of five glycine residues into the B-C loop of an Ig domain from human titin, TI I27. The folding pathway of this elongated 'pseudo wild-type' TI I27 is probed using protein engineering and Phi-value analysis. The Phi-values calculated for mutants within the pseudo wild-type protein indicate that the folding nucleus in wild-type TI I27 is conserved, supporting the hypothesis that the inter-sheet loop is not critical to the formation of a long-range folding nucleus.     

Dislocation loop evolution in Kr-irradiated ThO2

The early stage of microstructural evolution of ThO₂, under krypton irradiation at 600, 800, and 1000°C, was investigated using in situ transmission electron microscopy (TEM). Dislocation loops grew faster, whereas their number density decreased with increasing irradiation temperature. Loop density was found to decrease with ion dose. Interstitial dislocation loops, including Frank loops with Burgers vector of a/3〈111〉 and perfect loops with Burgers vector of a/2〈110〉, were determined by traditional TEM and atomic resolution–scanning TEM techniques. Atomistic and mesoscale level modeling are performed to interpret experimental observations. The migration energy barriers of defects in ThO₂ were calculated using density-functional theory. The energetics of different dislocation loop types were studied using molecular dynamics simulations. Loop density and diameter were analyzed using a kinetic rate theory model that considers stoichiometric loop evolution. This analysis reveals that loop growth is governed by the mobility of cation interstitials, whereas loop nucleation is determined by the mobility of anion defects. Lastly, a rate theory model was used to extract the diffusion coefficients of thorium interstitials, oxygen interstitials, and vacancies.     

Beta-hairpins in proteins revisited: lessons for de novo design

Beta-Hairpins with short connecting loops (1-5 residues) have been identified from a data set of 250 non-homologous, high resolution (< or =2.0 A) protein crystal structures. The conformational preferences of the loop segments have been analyzed with the specific aim of identifying frequently occurring motifs. Type I' and II' beta-turns were found to have a high propensity for occurrence in two residue loops. For three and four residue loops, the major conformational motif in the linking segments is alphaR-alphaR-alphaL (type I beta-turn followed by a residue in a left-handed helical conformation) and alphaR- alphaR-alphaR-alphaL (a pi-turn motif), respectively. The present larger data set confirms the high occurrences of these motifs which have been identified in earlier analyses. In addition to type I' and type II' beta-turns, several examples of type I beta-turn nucleated two residue loop hairpins, in spite of having an opposing sense of twist to that of type I' beta-turn, have also been observed. Examination of these frequently occurring motifs (flanked by extended conformation [beta]) in the data set reveals that the motifs beta-alphaR-alphaR- alphaL-beta and beta-type I'-beta have equal propensity and type II' indeed having highest propensity to nucleate beta-hairpins. The larger number of examples in this study allows the estimation of the specific amino acid preferences for loop positions in two, three and four residue loops. Small polar residues Asn, Asp, Ser, Thr, Gly and Pro in general have a high propensity for the loop positions but they reveal specific positional preferences in these frequently occurring motifs. There are no strong compositional preferences in the strand segments. Amino acid pair correlations across strands also do not show any significant pattern, with the exception of Cys-Cys pairs. Several Cys- Cys pairs have been identified at the non-hydrogen bonded positions of beta-hairpins; as many as six are disulfide bonded pairs. An examination of longer loop length hairpins reveals that the distortions of hairpins nucleated by tight turns (two residues) are much less frequently observed. The results presented in this study provide inputs for the de novo design of consensus loop segments in synthetic hairpins.      

Artificial beta-barrels

In biology, beta-barrels, cylindrically rolled-up forms of beta-sheets, are ubiquitous structural motifs within various binding proteins, pores, and enzymes. This biological multifunctionality suggested that synthetic artificial beta-barrels would provide access to many different functions beyond the limitations of peptide chemistry. Unlike the relative ease of formation of synthetic (de novo) alpha-helix bundles, the synthesis of artificial beta-barrels remains a challenge. To bypass the folding problems involved, we have employed "unfoldable" rigid-rod scaffolds as privileged staves (staves are the wood strips that form the sides of macroscopic barrels); the resulting barrel-stave supramolecules exhibit their expected multifunctionality. Several "rigid rod" beta-barrels that act as receptors, ion channels, pores, catalysts, and sensors have been prepared and studied. The most recent topic of interest concerns the use of artificial beta-barrels as multicomponent sensors ("artificial tongues") in complex analyte matrices. For multicomponent sensing, we have designed artificial beta-barrels to form pores that can open and close in response to chemical stimulation within lipid bilayers. With use of fluorogenic vesicles, changes in pore activity are readily detectable with either the naked eye or multiwell screening formats. The varying responsiveness to substrates and products makes synthetic pores versatile detectors of chemical reactions, of the activity of the enzymes that catalyze these reactions, and of their inhibitors. In sensing applications, the "perfect" selectivity of enzymes is exploited to generate analyte-specific signals. Reactive signal amplifiers are then covalently linked to the products of enzymatic signal generation to enhance their pore blockage potency. With the help of signal generators and amplifiers, we have employed artificial beta-barrel pores to sense sweet (sucrose, lactose), sour (acetate, lactate, citrate), and umami ("deliciousness", glutamate) components in various food samples. This breakthrough naturally led us to design and synthesize refined pores for advanced sensing applications. We have developed methods to build guest-binding sites not only at internal and external barrel surfaces but also near the core or near the periphery of the pore. Further refinements include the introduction of asymmetric staves for voltage gating and anchoring of the pore at the membrane-water interface.     

On the stability and plastic properties of the interior L3 loop in R.capsulatus porin. A molecular dynamics study

Structural properties of Rhodobacter capsulatus porin are studiedby molecular dynamics simulation using the GROMOS force field.Unconstrained simulations of the trimer and monome r show thetrimer to be more stable than the isolated monomer. Simulationsof the L3 loop insi de the pore are used to assess its stabilityand plastic properties. Simulated annealing shows that the conformationalspace available to the L3 loop inside the pore is very large.Simulations at different temperatures show that the energy hypersurfacearound the open state is complex and flat. These studies alsoindicate four zones that are more flexible than the rest ofthe loop. Two of these are stabilized by the addition of thedetergent molecule present in the X-ray structure. It is possiblethat the two remaining flexible zones, situated in the halfof the loop facing the extracellular end of the porin molecule,residues Asp93–Gly98 and Arg110–Leu111, are involvedin a mechanism for opening and closing of the pore.     

高温干燥对褐煤孔隙结构及水分复吸的影响

利用卧式固定床实验炉制得不同温度下干燥处理的煤样,采用低温氮吸附法测试煤样的比表面积、孔体积和孔径分布等孔隙特征参数,使用复吸实验装置测定不同干燥程度褐煤煤样的平衡含水量,探索了高温干燥处理后褐煤孔隙结构的演变与其复吸特性之间的关联规律。结果表明：褐煤原煤及不同干燥温度(600~800℃)下半焦的等温吸附曲线均属于第Ⅱ类吸附等温线,褐煤原煤、600℃和700℃干燥半焦的吸附回线均属于L1型,800℃干燥半焦的吸附回线有从L1型转变为L2型的趋势;随着干燥温度的增加,干燥半焦的比表面积先增大后减小,介孔峰值的孔径微分同样先增大后减小,而大孔孔径微分基本保持不变;分形维数D₁和D₂呈相反的变化趋势,且D₂>D₁;不同干燥程度半焦的复吸曲线变化趋势相同,且平衡含水量随着干燥温度的升高而减小;半焦复吸特性与孔隙结构有关,平衡含水率与其孔容积之间呈较好的线性关系。     

Skeletal isomerization of 1-butene over ferrierite and ZSM-5 zeolites: influence of zeolite acidity

Three ferrierite (FER) and five ZSM-5 (MFI) zeolites with SiO₂Al₂O₃ ratio ranging from 27 to 2000 are tested as catalysts for the skeletal isomerization of 1-butene at 350–450°C and atmospheric total pressure in order to study the influence of acidity and pore structure of zeolite on conversion and selectivity. The catalytic and NH₃ temperature-programmed desorption results from FER and MFI catalysts with the same SiO₂/Al₂O₃ ratio reveal that the pore structure of FER zeolite rather than its acidity may play an important role in achieving high selectivity for the skeletal isomerization of 1-butene to isobutene.     

离子色谱法测定降水中阴离子影响因素的研究

监测降水各种离子特别是F-、Cl-、NO3-、SO24-等阴离子的含量,是研究酸雨危害的前提条件[1]。离子色谱分析具有高灵活性、高选择性、高灵敏度的特点,已经广泛应用于环境等众多领域,是测定很多阴离子和阳离子的有效方法[2];但离子色谱仪在调节和分析过程中还存在许多影响因素,从而对实验结果产生影响。为此,本文对ICS-90型离子色谱仪在降水中F-、Cl-、NO3-、SO42-阴离子分析中出现的三个主要影响因素进行了初步研究和探讨。     

Mechanistic insights into phosphoprotein-binding FHA domains

[Structure: see text]. FHA domains are protein modules that switch signals in diverse biological pathways by monitoring the phosphorylation of threonine residues of target proteins. As part of the effort to gain insight into cellular avoidance of cancer, FHA domains involved in the cellular response to DNA damage have been especially well-characterized. The complete protein where the FHA domain resides and the interaction partners determine the nature of the signaling. Thus, a key biochemical question is how do FHA domains pick out their partners from among thousands of alternatives in the cell? This Account discusses the structure, affinity, and specificity of FHA domains and the formation of their functional structure. Although FHA domains share sequence identity at only five loop residues, they all fold into a beta-sandwich of two beta-sheets. The conserved arginine and serine of the recognition loops recognize the phosphorylation of the threonine targeted. Side chains emanating from loops that join beta-strand 4 with 5, 6 with 7, or 10 with 11 make specific contacts with amino acids of the ligand that tailor sequence preferences. Many FHA domains choose a partner in extended conformation, somewhat according to the residue three after the phosphothreonine in sequence (pT + 3 position). One group of FHA domains chooses a short carboxylate-containing side chain at pT + 3. Another group chooses a long, branched aliphatic side chain. A third group prefers other hydrophobic or uncharged polar side chains at pT + 3. However, another FHA domain instead chooses on the basis of pT - 2, pT - 3, and pT + 1 positions. An FHA domain from a marker of human cancer instead chooses a much longer protein fragment that adds a beta-strand to its beta-sheet and that presents hydrophobic residues from a novel helix to the usual recognition surface. This novel recognition site and more remote sites for the binding of other types of protein partners were predicted for the entire family of FHA domains by a bioinformatics approach. The phosphopeptide-dependent dynamics of an FHA domain, SH2 domain, and PTB domain suggest a common theme: rigid, preformed binding surfaces support van der Waals contacts that provide favorable binding enthalpy. Despite the lack of pronounced conformational changes in FHA domains linked to binding events, more subtle adjustments may be possible. In the one FHA domain tested, phosphothreonine peptide binding is accompanied by increased flexibility just outside the binding site and increased rigidity across the beta-sandwich. The folding of the same FHA domain progresses through near-native intermediates that stabilize the recognition loops in the center of the phosphoprotein-binding surface; this may promote rigidity in the interface and affinity for targets phosphorylated on threonine.     

Design and synthesis of nanostructured catalysts

Advanced catalysts have been designed in the forms of nanocrystalline and nanoporous materials. These nanostructured catalysts are derived with superior control in crystallite size, pore structure, pore size, surface area, compositional flexibility, and component dispersion. They are tailored with excellent activity, selectivity and stability for a wide variety of catalytic reactions.     

Investigation of D₁ receptor-agonist interactions and D₁/D₂ agonist selectivity using a combination of pharmacophore and receptor homology modeling

The aim of this study was to use a combined structure and pharmacophore modeling approach to extract information regarding dopamine D? receptor agonism and D?/D? agonist selectivity. A 3D structure model of the D? receptor in its agonist-bound state was constructed with a full D? agonist present in the binding site. Two different binding modes were identified using (+)-doxanthrine or SKF89626 in the modeling procedure. The 3D model was further compared with a selective D? agonist pharmacophore model. The pharmacophore feature arrangement was found to be in good agreement with the binding site composition of the receptor model, but the excluded volumes did not fully reflect the shape of the agonist binding pocket. A new receptor-based pharmacophore model was developed with forbidden volumes centered on atom positions of amino acids in the binding site. The new pharmacophore model showed a similar ability to discriminate as the previous model. A comparison of the 3D structures and pharmacophore models of D? and D? receptors revealed differences in shape and ligand-interacting features that determine selectivity of D? and D? receptor agonists. A hydrogen bond pharmacophoric feature (Ser-TM5) was shown to contribute most to the selectivity. Non-conserved residues in the binding pocket that strongly contribute to D?/D? receptor agonist selectivity were also identified; those were Ser/Cys3·3?, Tyr/Phe?·3?, Ser/Tyr?·?1, and Asn/His?·?? in the transmembrane (TM) helix region, together with Ser/Ile and Leu/Asn in the second extracellular loop (EC2). This work provides useful information for the design of new selective D? and D? agonists. The combined receptor structure and pharmacophore modeling approach is considered to be general, and could therefore be applied to other ligand-protein interactions for which experimental information is limited.     

Prediction of hypervariable CDR-H3 loop structures in antibodies

The structure of the most variable antibody hypervariable loop,CDR-H3, has been predicted from amino acid sequence alone. Incontrast to other approaches predictions are made for loop lengthsup to 17 residues. The predictions have been achieved usingartificial neural networks which are trained on a large setof loops from the Brookhaven Protein Databank which have structuressimilar to CDRH3. The loop structures are described by the twobackbone dihedral angles and for each residue. For 21 CDR-H3loops unique to the neural network, the prediction of dihedralangles leads to an average root mean square deviation in theCartesian coordinates of 2.65 Å. The present method, whencombined with existing modelling protocols, provides an importantaddition to the structural prediction of the complementaritydetermining regions of antibodies.     

L3 loop-mediated mechanisms of pore closing in porin: a molecular dynamics perturbation approach

L3 loop-mediated mechanisms for pore closing in porin are investigatedwith molecular dynamics simulation, using an approach that canbe related to the phenomenon of voltage gating. Voltage gatingis seen as a perturbation of the electrostatic screening insidethe porin pore where, by the influence of the potential gradient,water and counter-ion distribution can be slightly displacedfrom their equilibrium distribution. This is simulated by perturbingthe screening electrostatics of ionizable groups inside thepore. Under these conditions, a localized conformational changetakes place, involving 12 (Ile102–Alall3) out of the 44residues of the loop. The pore is reduced to a sixth of itsopen state size. The conformational change can be achieved witha small perturbation and it is reversible once the perturbationis switched off (relaxation process). Other types of behaviourpredominating at higher simulation temperatures are found forthe loop, involving an extra conformational change in the Thr92–Asp96loop segment. This conformational change completely closes thepore, but is not reversible under the simulation conditions.Both zones involved in the conformational changes contain oroverlap the zones which were described previously, using othertechniques, to be the most flexible zones of the loop.     

水合物流动环路实验研究进展

作为水合物研究的重要手段之一,水合物环路实验研究因其能够模拟管输体系中的水合物生成、流动及堵塞规律,得到国内外众多研究者的青睐。本文介绍了法国Archimede环路、法国IFP-Lyre环路、美国ExxonMobil环路、美国塔尔萨大学FAL环路、澳大利亚Hytra环路、挪威NTNU环路、挪威SINTEF环路、挪威Petreco A/S高压轮形环路、中国科学院GIEC水合物实验环路、中国CUPB化工实验环路和中国CUPB储运实验环路11条国内外水合物实验环路的主要设计参数和实验工艺流程等基本概况。从流动体系水合物生成研究、水合物浆液流动特性研究、水合物堵管机理及风险控制研究、水合物颗粒聚并及粒径分布研究等4个方面,总结了基于水合物环路实验所获得的研究进展及主要成果。为未来更具功能特色的水合物实验环路的搭建提供了设计思路和参考意见。并指出后续深入开展水合物环路实验研究,应将宏观实验数据分析与微观测试手段相结合,优化实验方案,明确实验目标,以揭示水合物相关基础研究的本征机理。