Transmembrane Domain Three Contributes to the Ion Conductance Pathway of Channelrhodopsin-2

Channelrhodopsin-2 (ChR2) is a light-activated nonselective cation channel that is found in the eyespot of the unicellular green alga Chlamydomonas reinhardtii. Despite the wide employment of this protein to control the membrane potential of excitable membranes, the molecular determinants that define the unique ion conductance properties of this protein are not well understood. To elucidate the cation permeability pathway of ion conductance, we performed cysteine scanning mutagenesis of transmembrane domain three followed by labeling with methanethiosulfonate derivatives. An analysis of our experimental results as modeled onto the crystal structure of the C1C2 chimera demonstrate that the ion permeation pathway includes residues on one face of transmembrane domain three at the extracellular side of the channel that face the center of ChR2. Furthermore, we examined the role of a residue at the extracellular side of transmembrane domain three in ion conductance. We show that ion conductance is mediated, in part, by hydrogen bonding at the extracellular side of transmembrane domain three. These results provide a starting point for examining the cation permeability pathway for ChR2.     

华南地区3个棕榈藤种光合途径的判别

C3植物可以通过转入C4植物基因而具备C4植物光合特性,从而提高产量。有鉴于此,本文通过测定我国华南地区分布的黄藤(Daemonoropsmargaritae(Hance)Becc.)、单叶省藤(CalamussimplicifoliusC.F.Wei)和白藤(C.tetradactylusHance)等3个棕榈藤种苗木和成年植株叶片的叶绿素含量、气孔密度、磷酸烯醇式丙酮酸羧化酶(phosphoenolpyruvatecarboxylase,PEPC)、丙酮酸磷酸二激酶(pyruvatephosphatedikinase,PPDK)和稳定碳同位素比值等指标以判别3个藤种的光合途径,为棕榈藤转入C4植物基因工作提供理论依据。结果表明,3种藤种苗木和成年植株的叶绿素含量和气孔密度比常见C3植物和C4植物高,但叶绿素a/b值、叶片上下表面气孔比值、PEPC酶活性、PPDK酶活性和稳定碳同位素比值等指标均较低,与常见C3植物的对应指标相当或略低,而远小于常见C4植物,因此认为黄藤、单叶省藤和白藤等3个藤种是C3植物。     

Enzymes of the Tryptophan Pathway in Three Bacillus Species

The tryptophan synthetic pathway was characterized in three species of Bacillus, B. subtilis, B. pumilus, and B. alvei. They share the common features of a pathway which is subject to tryptophan repression, contains no unexpected complexes among the five enzymes, exhibits dissociable anthranilate synthase enzymes which do not require phosphoribosyl transferase for amidetransfer activity, contains separate indoleglycerol phosphate synthase and phosphoribosylanthranilate isomerase enzymes, and contains similar tryptophan synthetase multimers. In looking at these characteristics in detail however, differences among the three species became apparent, as, for example, in the complementation observed between the alpha and beta(2) components of tryptophan synthetase, and the dissociation patterns of the large and small components of anthranilate synthase. The results demonstrate some pitfalls in attempting to compare multimeric enzymes in crude extracts from different organisms.     

华南地区3个棕榈藤种光合途径的判别

C3植物uT以通过转入C4植物基因而具备C4植物光合特性,从而提高产量.有鉴于此,本文通过测定我国华南地区分布的黄藤(Daemonorops margarttae(Hance)Becc.)、单叶省藤(Calamus simplicifolius C.F.Wei)和白藤(C.tetradactylus Hance)等3个棕榈藤种苗木和成年植株叶片的叶绿素含量、气孔密度、磷酸烯醇式丙酮酸羧化酶(phosphoenolpyruvate carboxylase,PEPC)、丙酮酸磷酸二激酶(pyruvate phosphate dikinase,PPDK)和稳定碳同位素比值等指标以判别3个藤种的光合途径,为棕榈藤转入C4植物基因工作提供理论依据.结果表明,3种藤种苗木和成年植株的叶绿素含量和气孔密度比常见C3植物和C4植物高,但叶绿素a/b值、叶片上下表面气孔比值、PEPC酶活性、PPDK酶活性和稳定碳同位素比值等指标均较低,与常见C3植物的对应指标相当或略低,而远小于常见C4植物,因此认为黄藤、单叶省藤和白藤等3个藤种是C3植物.     

乳腺癌易感蛋白BRCA1的BRCT2结构域染色质伸展活性的定位

40 %～ 5 0 %的遗传性乳腺癌和至少 80 %的既有乳腺癌又有卵巢癌家族史的患者是由BRCA1突变引起的 .BRCA1C末端含有 2个BRCT结构域 (BRCT1和BRCT2 ) ,它们与BRCA1的重要功能密切相关 .许多乳腺癌易感突变发生在BRCA1的BRCT结构域中 .利用染色质结构检测技术表明 ,BRCT结构域具有染色质伸展活性 .利用缺失突变技术构建了 6种BRCT2结构域 (175 6～ 185 2位氨基酸残基 )缺失突变体并将BRCT2结构域中与染色质伸展相关的重要区域定位到 175 6～ 180 8之间的氨基酸残基 ;用丙氨酸扫描技术构建了 6种BRCT2结构域丙氨酸扫描突变体并将重要氨基酸残基序列定位到 1784～ 1788之间的VQLCG .BRCT2结构域的定位有助于预测BRCT2结构域突变后发生乳腺癌的风险 ,也为进一步研究BRCT2结构域的功能机制提供了有用的材料 .     

Unfolding the A2 Domain of Von Willebrand Factor with the Optical Trap

Von Willebrand factor (VWF) is a multimeric plasma glycoprotein involved in both hemostasis and thrombosis. VWF conformational changes, especially unfolding of the A2 domain, may be required for efficient enzymatic cleavage in vivo. It has been shown that a single A2 domain unfolds at most probable unfolding forces of 7-14 pN at force loading rates of 0.35-350 pN/s and A2 unfolding facilitates A2 cleavage in vitro. However, it remains unknown how much force is required to unfold the A2 domain in the context of a VWF multimer where A2 may be stabilized by other domains like A1 and A3. With the optical trap, we stretched VWF multimers and a poly-protein (A1A2A3)₃ that contains three repeats of the triplet A1A2A3 domains at constant speeds of 2000 nm/s and 400 nm/s, respectively, which yielded corresponding average force loading rates of 90 and 22 pN/s. We found that VWF multimers became stiffer when they were stretched and extended by force. After force increased to a certain level, sudden extensional jumps that signify domain unfolding were often observed. Histograms of the unfolding force and the unfolded contour length showed two or three peaks that were integral multiples of ∼21 pN and ∼63 nm, respectively. Stretching of (A1A2A3)₃ yielded comparable distributions of unfolding force and unfolded contour length, showing that unfolding of the A2 domain accounts for the behavior of VWF multimers under tension. These results show that the A2 domain can be indeed unfolded in the presence of A1, A3, and other domains. Compared with the value in the literature, the larger most probable unfolding force measured in this study suggests that the A2 domain is mechanically stabilized by A1 or A3 although variations in experimental setups and conditions may complicate this interpretation.     

Molecular Dynamics Simulations of the Unfolding Mechanism of the Catalytic Domain from Aspergillus awamori var. X100 Glucoamylase

Abstract

In this study, 200 ps molecular dynamics simulations were conducted to investigate the unfolding mechanism of the catalytic domain of glucoamylase from Aspergillus awamori var. X100. The unfolding of this domain was suggested to follow a putative hierarchical manner, in which the heavily O-glycosylated belt region from residues T440 to A471 acted as the initiation site, followed by the a-helix secondary structure destruction, and then the collapse of the catalytic center pocket. The O-glycosylated belt region surrounded the surface of the catalytic domain in its native state at low temperature, whereas it was extended and is more suitable to be classified as part of the subsequent linker domain at high temperatures due to its high flexibility. The inner set helices of the (α/α)₆-barrel seemed to exhibit higher helical content than the outer set ones at all temperatures examined. The distances between the C_α of the three Cys residue pairs fluctuated rapidly at higher temperatures, indicating that these disulfide bonds have little effect on the structural stabilization. The melting temperature, at which the residual total helicity of the catalytic domain is 50%, is much lower than the critical temperature, at which the catalytic center pocket has lost its structural integrity.     

The Ca2+ Influence on Calmodulin Unfolding Pathway: A Steered Molecular Dynamics Simulation Study

The force-induced unfolding of calmodulin (CaM) was investigated at atomistic details with steered molecular dynamics. The two isolated CaM domains as well as the full-length CaM were simulated in N-C-terminal pulling scheme, and the isolated N-lobe of CaM was studied specially in two other pulling schemes to test the effect of pulling direction and compare with relevant experiments. Both Ca²⁺-loaded CaM and Ca²⁺-free CaM were considered in order to define the Ca²⁺ influence to the CaM unfolding. The results reveal that the Ca²⁺ significantly affects the stability and unfolding behaviors of both the isolated CaM domains and the full-length CaM. In Ca²⁺-loaded CaM, N-terminal domain unfolds in priori to the C-terminal domain. But in Ca²⁺-free CaM, the unfolding order changes, and C-terminal domain unfolds first. The force-extension curves of CaM unfolding indicate that the major unfolding barrier comes from conquering the interaction of two EF-hand motifs in both N- and C- terminal domains. Our results provide the atomistic-level insights in the force-induced CaM unfolding and explain the observation in recent AFM experiments.     

不同组成群落3种共有植物光合生理特征研究

为研究物种在不同群落中光合生理特征的变化,以亚高寒草甸围封恢复地为研究对象,对样地内3个不同组成群落进行样方调查,测定了物种高度及各群落垂直方向上光照强度以及群落中3个共有种披碱草(Elymus dahuricus)、刺儿菜(Cirsium setosum)和紫花苜蓿(Medicago sativa)的净光合速率(Aarea)、叶片氮含量(Nmass)、比叶重(LMA)及光合氮利用效率(PNUE)。结果表明:(1)3个样地的群落组成有明显的差异,豆科植物的增多可以一定程度上改善群落氮养分状况,但植物叶片Nmass还受到群落优势种竞争的影响。(2)同一物种在不同群落的高度不同,不同群落垂直方向上光照强度也不相同,导致同一物种在不同群落中能够获得的光照强度有一定差异。(3)在养分、光照强度有差异的情况下,不同植物的Aarea、LMA及PNUE在不同群落中的变化趋势不尽相同,而Narea与Aarea的关系在总体上、群落间及物种间变化不大,基本上显示了较强的正相关关系。由此可见,群落组成、结构引起的光照及氮素差异是导致同一物种光合生理特征在不同群落中变化的重要因素,但不同物种光合生理特征对光照及氮素变化的响应不同。     

Nitrogen and Cation Nutrition of Three Ecologically Different Plant Species

Apple rootstocks M.7 were given a nitrogen application either in the spring or in the preceding autumn. At the time of the spring application some rootstocks were ringed. During the 50-day experimental period from bud-break, shoot growth and the amount of nitrogen incorporated into the new shoots were slightly reduced in the spring-treated trees and strongly reduced in the ringed trees of both treatments. Roots of unringed autumn-fertilized trees showed higher levels of total and amino nitrogen than those of similar trees in the spring treatment; to a lesser degree, the reverse held for xylem sap from the stem. Ringing increased the amino-nitrogen level in the roots, which suggests a reduced translocation rate. The nitrogen treatments led to marked differences in the percentage composition of the amino-nitrogen fraction of roots and xylem sap. The distribution of amino acids and amides in the roots and that in xylem sap of the same trees was divergent, but arginine and asparagine often were the most important constituents. Aspartic acid was rather abundant in xylem sap. Ringing did not affect the composition of the amino-nitrogen fraction in the roots quantitatively but increased the proportion of arginine in the sap. The possible relationship between the composition of xylem sap and soluble nitrogen in the roots is discussed. It is argued that especially in spring-fertilized trees appreciable amounts of nitrogen must be translocated via the phloem in addition to the transport in the xylem.     

Computational Model of Gab1/2-Dependent VEGFR2 Pathway to Akt Activation

Vascular endothelial growth factor (VEGF) signal transduction is central to angiogenesis in development and in pathological conditions such as cancer, retinopathy and ischemic diseases. However, no detailed mass-action models of VEGF receptor signaling have been developed. We constructed and validated the first computational model of VEGFR2 trafficking and signaling, to study the opposing roles of Gab1 and Gab2 in regulation of Akt phosphorylation in VEGF-stimulated endothelial cells. Trafficking parameters were optimized against 5 previously published in vitro experiments, and the model was validated against six independent published datasets. The model showed agreement at several key nodes, involving scaffolding proteins Gab1, Gab2 and their complexes with Shp2. VEGFR2 recruitment of Gab1 is greater in magnitude, slower, and more sustained than that of Gab2. As Gab2 binds VEGFR2 complexes more transiently than Gab1, VEGFR2 complexes can recycle and continue to participate in other signaling pathways. Correspondingly, the simulation results show a log-linear relationship between a decrease in Akt phosphorylation and Gab1 knockdown while a linear relationship was observed between an increase in Akt phosphorylation and Gab2 knockdown. Global sensitivity analysis demonstrated the importance of initial-concentration ratios of antagonistic molecular species (Gab1/Gab2 and PI3K/Shp2) in determining Akt phosphorylation profiles. It also showed that kinetic parameters responsible for transient Gab2 binding affect the system at specific nodes. This model can be expanded to study multiple signaling contexts and receptor crosstalk and can form a basis for investigation of therapeutic approaches, such as tyrosine kinase inhibitors (TKIs), overexpression of key signaling proteins or knockdown experiments.     

Structural Stabilities of Different Regions of the Titin I27 Domain Contribute Differently to Unfolding upon Mitochondrial Protein Import

Protein import into mitochondria requires unfolding of the folded mature domain of precursor proteins. Here we compared the effects of amino-acid replacement between the core region and the N-terminal region of the titin I27 domain (the 27th Ig domain of human titin) on its import into isolated mitochondria when attached to a short presequence (pb₂(35)). We found that several mutations in the core region around Trp34 of the I27 domain enhanced the import rates of the fusion proteins, while the N-terminal K6P mutation, which increases mechanical stability around the N-terminal region, decreases the import rate. When the K6P mutation is combined with core-destabilizing mutations, the import rates of the fusion proteins still decrease, unless a long segment is deleted. These results suggest that mutations in the core region could destabilize the transition state for unfolding from the intermediate with the detached N-terminal segment during import, leading to enhanced unfolding rates, although stabilization of the N-terminal region masks these effects. In other words, the rate-limiting step of the global unfolding upon import into mitochondria switches, depending on the balance between the stability of the N-terminal structure and the stability of the core region of the I27 domain.     

人基质金属蛋白酶2催化区的克隆与表达

为从随机肽库中寻找具有基质金属蛋白酶 2 (MMP 2 )抑制活性的新型小肽抑制剂 ,应用PCR法从含有人MMP 2基因的质粒中扩增了人MMP 2的催化区 .序列分析结果表明无氨基酸突变 .然后构建人MMP 2催化区的表达载体pET MCD ,转化大肠杆菌BL2 1(DE3 ) ,经IPTG诱导表达人MMP 2催化区 .经包涵体分离、变性、金属螯合层析纯化和复性等过程 ,复性后的人MMP 2催化区具有较好的明胶水解活性 .     

Guest Editors' Introduction to the Special Section: Computational Intelligence Approaches in Computational Biology and Bioinformatics

The ten papers in this special section focus on computational intelligence approaches on computational biology and bioinformatics.     

Retention of prolyl hydroxylase PHD2 in the cytoplasm prevents PHD2-induced anchorage-independent carcinoma cell growth

Cellular oxygen tension is sensed by a family of prolyl hydroxylases (PHD1-3) that regulate the degradation of hypoxia-inducible factors (HIF-1α and -2α). The PHD2 isoform is considered as the main downregulator of HIF in normoxia. Our previous results have shown that nuclear translocation of PHD2 associates with poorly differentiated tumor phenotype implying that nuclear PHD2 expression is advantageous for tumor growth. Here we show that a pool of PHD2 is shuttled between the nucleus and the cytoplasm. In line with this, accumulation of wild type PHD2 in the nucleus was detected in human colon adenocarcinomas and in cultured carcinoma cells. The PHD2 isoforms showing high nuclear expression increased anchorage-independent carcinoma cell growth. However, retention of PHD2 in the cytoplasm inhibited the anchorage-independent cell growth. A region that inhibits the nuclear localization of PHD2 was identified and the deletion of the region promoted anchorage-independent growth of carcinoma cells. Finally, the cytoplasmic PHD2, as compared with the nuclear PHD2, less efficiently downregulated HIF expression. Forced HIF-1α or -2α expression decreased and attenuation of HIF expression increased the anchorage-independent cell growth. However, hydroxylase-inactivating mutations in PHD2 had no effect on cell growth. The data imply that nuclear PHD2 localization promotes malignant cancer phenotype.     

不同光照条件下3种冬青属植物的光合特征日变化研究

以冬青、绿冬青、全缘枸骨为材料,运用光生理生态研究方法,对不同遮荫条件下,3种冬青属植物的光生理变化进行了研究。结果表明：3种冬青在全光照下的净光合速率高于透光率45％下的,透光率45％的高于透光率20％的,且全光照处理的净光合速率日变化均为双峰曲线,反映3种冬青均为中性树种。但从午休程度和午休后光合恢复情况来看,全缘枸骨对强光的适应能力好于绿冬青,绿冬青好于冬青。叶片光合色素和比叶重变化进一步证明,全缘枸骨适应能力最好,绿冬青次之,冬青相对较差。该研究对揭示各树种光适应特性和合理的园林配置有指导意义。     

Effect of Available Water on Thermal Resistance of Three Nonsporeforming Species of Bacteria

Cells of Escherichia coli, Pseudomonas fluorescens, and Staphylococcus aureus, previously grown in Trypticase Soy Broth (TSB) at a high level of available moisture (a(w) 0.994) and at low levels produced by addition of NaCl or glucose, were heated in neutral phosphate buffer, and in this buffer adjusted to low levels of available moisture by means of NaCl or glucose. Glucose in the heating medium was more protective than NaCl for E. coli and P. fluorescens, but hastened the thermal destruction of S. aureus. Added protection was given P. fluorescens during heating in glucose-buffer solution at a(w) 0.97 by previous growth in TSB adjusted to that a(w) value with glucose. Added protection was given E. coli during heating in NaCl-buffer solution at a(w) 0.98 by previous growth in TSB adjusted to that value with NaCl. With S. aureus, however, previous growth in TSB plus NaCl or glucose had little effect on heat resistance, but the solute in the heating medium had great influence, in that NaCl was very protective and glucose destructive. Opportunity may have been given during tempering of the cell suspension at 30 C in the heating medium prior to heating for the NaCl and glucose to diffuse into the staphylococcal cells.     

Identification of a Catalytic Active but Non-Aggregating MDM2 RING Domain Variant

As a key regulator of the tumour suppressor protein p53, MDM2 is involved in various types of cancer and has thus been an attractive drug target. So far, small molecule design has primarily focussed on the N-terminal p53-binding domain although on-target toxicity effects have been reported. Targeting the catalytic RING domain of MDM2 resembles an alternative approach to drug MDM2 with the idea to prevent MDM2-mediated ubiquitination of p53 while retaining MDM2′s ability to bind p53. The design of RING inhibitors has been limited by the extensive aggregation tendency of the RING domain, making it challenging to undertake co-crystallization attempts with potential inhibitors. Here we compare the purification profiles of the MDM2 RING domain from several species and show that the MDM2 RING domain of other species than human is much less prone to aggregate although the overall structure of the RING domain is conserved. Through sequence comparison and mutagenesis analyses, we identify a single point mutation, G443T, which greatly enhances the dimeric fraction of human MDM2 RING domain during purification. Neither does the mutation alter the structure of the RING domain, nor does it affect E2(UbcH5B)–Ub binding and activity. Hence, MDM2-G443T facilitates studies involving binding partners that would be hampered by the low solubility of the wild-type RING domain. Furthermore, it will be valuable for the development of MDM2 RING inhibitors.