A conserved threonine spring‐loads precursor for intein splicing

Protein splicing is an autocatalytic process where an “intein” self‐cleaves from a precursor and ligates the flanking N‐ and C‐“extein” polypeptides. Inteins occur in all domains of life and have myriad uses in biotechnology. Although the reaction steps of protein splicing are known, mechanistic details remain incomplete, particularly the initial peptide rearrangement at the N‐terminal extein/intein junction. Recently, we proposed that this transformation, an N‐S acyl shift, is accelerated by a localized conformational strain, between the intein's catalytic cysteine (Cys1) and the neighboring glycine (Gly‐1) in the N‐extein. That proposal was based on the crystal structure of a catalytically competent trapped precursor. Here, we define the structural origins and mechanistic relevance of the conformational strain using a combination of quantum mechanical simulations, mutational analysis, and X‐ray crystallography. Our results implicate a conserved, but largely unstudied, threonine residue of the Ssp DnaE intein (Thr69) as the mediator of conformational strain through hydrogen bonding. Further, the strain imposed by this residue is shown to position the splice junction in a manner that enhances the rate of the N‐S acyl shift substantially. Taken together, our results not only provide fundamental understanding of the control of the first step of protein splicing but also have important implications in various biotechnological applications that require precursor manipulation.     

Conformational stability of CopC and roles of residues Tyr79 and Trp83

CopC is a periplasmic copper Chaperone protein that has a β‐barrel fold and two metal‐binding sites distinct for Cu(II) and Cu(I). In the article, four mutants (Y79F, Y79W, Y79WW83L, Y79WW83F) were obtained by site‐directed mutagenesis. The far‐UV CD spectra of the proteins were similar, suggesting that mutations did not bring any significant changes in secondary structures. Meanwhile the effects of mutations on the protein's function were manifested by Cu(II) binding. Fluorescence lifetime measurement and quenching of tryptophan fluorescence by acrylamide and KI showed that the microenvironment around Trp83 was more hydrophobic than that around Tyr79 in apoCopC. Unfolding experiments induced by guanidinium chloride (GdnHCl), urea provided the conformational stability of each protein. The Δ<ΔG⁰_element> obtained using the model of structural elements was used to show the role of Tyr79 and Trp83. On the one hand, the <ΔG⁰_element> induced by urea for Y79F, Y79W have a loss of 6.51, 2.03 kJ/mol, respectively, compared with apoCopC, proving that replacement of Tyr79 by Phe or Trp all decreased the protein stability, meaning that the hydrogen bonds interactions between Tyr79 and Thr75 played an important role in stabilizing apoCopC. On the other hand, the <ΔG⁰_element> induced by urea for Y79WW83L have a loss of 11.44 kJ/mol, but for Y79WW83F did a raise of 1.82 kJ/mol compared with Y79W. The replacement of Trp83 by Phe and Leu yields opposite effects on protein stability, which suggested that the aromatic ring of Trp83 was important in maintaining the hydrophobic core of apoCopC.     

His-234是毛白杨对香豆酸 ∶ CoA连接酶的重要酶催化活性残基

对香豆酸∶CoA连接酶(4-coumarate: coenzyme A ligase,4CL)是植物苯丙烷类代谢途径中的一个重要的酶．4CL以肉桂酸衍生物(香豆酸、咖啡酸、阿魏酸等)、ATP和CoA为底物合成相应的酰基-CoA酯,这些酰基-CoA酯是一系列重要化合物(如木质素)的前体．4CL的酶催化反应分两步进行：第一步以肉桂酸衍生物和Mg² -ATP为底物合成酰基-AMP,第二步用CoA取代AMP,产生酰基-CoA酯,催化过程中酶的构象产生明显的变化．因为4CL在木质素的合成中所起的作用,这个酶是通过蛋白质工程方法改进林产品质量的重要靶标．我们通过X射线衍射技术,解析了毛白杨对香豆酸∶CoA连接酶1(Pt4CL1)与其中间产物对香豆酰-AMP的复合物晶体结构,与同家族成员结构比对,确定所获得的蛋白质结构为Pt4CL1催化第二步反应,即酰基-CoA酯合成的构象．结构分析表明：His-234残基在Pt4CL1的酶催化机理中起着多重作用,即通过侧链与AMP磷酸基团形成氢键,降低磷酸基团的负电荷,催化CoA的亲核取代反应;侧链可以采取两种不同的构象以调节CoA进入Pt4CL1的催化中心;His-234的侧链还可能夺取CoA巯基的质子,从而增强CoA的亲核反应活性．突变体酶活数据结果也显示His-234对Pt4CL1的活性非常重要,是Pt4CL1催化中心的活性残基．     

Purification and Chemical Analysis of Microbial Cell Flocculant Produced by Aspergillus sojae AJ7002

A bio-flocculant was isolated from the culture broth of Asp. sojae AJ 7002. It was partially purified by acetone or ethanol precipitation, by ion-exchange and gel chromatography, and by dialysis. The isolated polymer possessed chemical characteristics of a poly-hexosamine and a protein. Glucosamine and galactosamine were not acetylated. The flocculant contained 2-ketogluconic acid, but sulfur or phosphorus was not detected. This flocculant was thermo-stable and its activity varied with pH. It was suggested that the hexosamine moiety in the polymer played a major role in bio-flocculation, assisted by protein portion in enlargement of the molecular weight of the flocculant, and by 2-ketogluconic acid in endowing it with amphoteric character.     

The species-rich assemblages of tintinnids (marine planktonic protists) are structured by mouth size

Many microbial taxa in the marine plankton appear super-saturated in species richness. Here, we provide a partial explanation by analyzing how species are organized, species packing, in terms of both taxonomy and morphology. We focused on a well-studied group, tintinnid ciliates of the microzooplankton, in which feeding ecology is closely linked to morphology. Populations in three distinct systems were examined: an Eastern Mediterranean Gyre, a Western Mediterranean Gyre and the California Current. We found that species abundance distributions exhibited the long-tailed, log distributions typical of most natural assemblages of microbial and other organisms. In contrast, grouping in oral size-classes, which corresponds with prey-size exploited, revealed a geometric distribution consistent with a dominant role of a single resource in structuring an assemblage. The number of species found in a particular oral size-class increases with the numerical importance of the size-class in the overall population. We suggest that high species diversity reflects the fact that accompanying each dominant species are many ecologically similar species, presumably able to replace the dominant species, at least with regard to the size of prey exploited. Such redundancy suggests that species diversity greatly exceeds ecological diversity in the plankton.     

Acoustic detection of cell adhesion to a coated quartz crystal microbalance – implications for studying the biocompatibility of polymers

Biocompatibility of polymers is an important parameter for the successful application of polymers in tissue engineering. In this work, quartz crystal microbalance (QCM) devices were used to follow the adhesion of NIH 3T3 fibroblasts to QCM surfaces modified with fibronectin (FN) and poly-D -lysine (PDL). The variations in sensor resonant frequency (Δf) and motional resistance (ΔR), monitored as the sensor signal, revealed that cell adhesion was favored in the PDL-coated QCMs. Fluorescence microscopy images of seeded cells showed more highly spread cells on the PDL substrate, which is consistent with the results of the QCM signals. The sensor signal was shown to be sensitive to extracellular matrix (ECM)-binding motifs. Ethylenediaminetetraacetic acid (EDTA) and soluble Gly-Arg-Gly-Asp-Ser (GRGDS) peptides were used to interfere with cell-ECM binding motifs onto FN-coated QCMs. The acquired acoustic signals successfully showed that in the presence of 30 mM EDTA or 1 mM GRGDS, cell adhesion is almost completely abolished due to the inhibition/blocking of integrin function by these compounds. The results presented here demonstrate the potential of the QCM sensor to study cell adhesion, to monitor the biocompatibility of polymers and materials, and to assess the effect of adhesion modulators. QCM sensors have great potential in tissue engineering applications, as QCM sensors are able to analyze the biocompatibility of surfaces and it has the added advantage of being able to evaluate, in situ and in real time, the effect of specific drugs/treatments on cells.     

RNA approaches the B‐form in stacked single strand dinucleotide contexts

Duplex RNA adopts an A‐form structure, while duplex DNA interconverts between the A‐ and B‐forms depending on the environment. The C2′‐endo sugar pucker seen in B‐form DNA can occur infrequently in ribose sugars as well, but RNA is not understood to assume B‐form conformations. Through analysis of over 45,000 stacked single strand dinucleotide (SSD) crystal structure conformations, this study demonstrates that RNA is capable of adopting a wide conformational range between the canonical A‐ and B‐forms at the localized SSD level, including many B‐form‐like conformations. It does so through C2′‐endo ribose conformations in one or both nucleotides, and B‐form‐like neighboring base stacking patterns. As chemical reactions on nucleic acids involve localized changes in chemical bonds, the understanding of how enzymes distinguish between DNA and RNA nucleotides is altered by the energetic accessibility of these rare B‐form‐like RNA SSD conformations. The existence of these conformations also has direct implications in parametrization of molecular mechanics energy functions used extensively to model nucleic acid behavior., 2016. © 2015 Wiley Periodicals, Inc. Biopolymers 105: 65–82, 2016     

Molecular basis of ion permeability in a voltage‐gated sodium channel

Voltage‐gated sodium channels are essential for electrical signalling across cell membranes. They exhibit strong selectivities for sodium ions over other cations, enabling the finely tuned cascade of events associated with action potentials. This paper describes the ion permeability characteristics and the crystal structure of a prokaryotic sodium channel, showing for the first time the detailed locations of sodium ions in the selectivity filter of a sodium channel. Electrostatic calculations based on the structure are consistent with the relative cation permeability ratios (Na⁺ ≈ Li⁺ ≫ K⁺, Ca²⁺, Mg²⁺) measured for these channels. In an E178D selectivity filter mutant constructed to have altered ion selectivities, the sodium ion binding site nearest the extracellular side is missing. Unlike potassium ions in potassium channels, the sodium ions in these channels appear to be hydrated and are associated with side chains of the selectivity filter residues, rather than polypeptide backbones.     

Chlorophyll a is the crucial redox sensor and transmembrane signal transmitter in the cytochrome b6f complex. Components and mechanisms of state transitions from the hydrophobic mismatch viewpoint

The cytochrome (cyt) b₆f complex is involved in the transmembrane redox signaling that triggers state transitions in cyanobacteria and chloroplasts. However, the components and molecular mechanisms are still unclear. In an attempt to solve this long-standing problem, we first focused on the unknown role of a single chlorophyll a (Chla) in cyt b₆f with a new approach based on Chla structural properties. Various b₆f X-ray crystal structures were analyzed to identify their differences, which correlate with differences in Chla molecular volume. We found that the distance of the Rieske [2Fe-2S] cluster to Chla correlates with the distance between a pair of residues at the Q_o-site and the distance between a pair of residues at the opposite membrane side. These correlations were accompanied by the rotation of a key peripheral residue and by changes in the hydrophobic thickness of cyt b₆f. Parallel analysis of cyt bc₁ crystal structures allowed us to conclude that Chla acts as the crucial redox sensor and transmembrane signal transmitter in b₆f for changes in the plastoquinone pool redox state. The hydrophobic mismatch induced by the changed hydrophobic thickness of cyt b₆f is the driving force for the structural reorganizations of the photosynthetic apparatus during induction and the progression of state transitions in cyanobacteria and chloroplasts. A mechanism for LHCII kinase activation in chloroplasts is also proposed. Our understanding of the dynamic structural changes in bc-complexes during turnover at the Q_o-site and state transitions is augmented by the time-sequence ordering of 56 bc crystal structures.