Mis-translation of a computationally designed protein yields an exceptionally stable homodimer: implications for protein engineering and evolution

We recently used computational protein design to create an extremely stable, globular protein, Top7, with a sequence and fold not observed previously in nature. Since Top7 was created in the absence of genetic selection, it provides a rare opportunity to investigate aspects of the cellular protein production and surveillance machinery that are subject to natural selection. Here we show that a portion of the Top7 protein corresponding to the final 49 C-terminal residues is efficiently mis-translated and accumulates at high levels in Escherichia coli. We used circular dichroism, size-exclusion chromatography, small-angle X-ray scattering, analytical ultra-centrifugation, and NMR spectroscopy to show that the resulting C-terminal fragment (CFr) protein adopts a compact, extremely stable, homo-dimeric structure. Based on the solution structure, we engineered an even more stable variant of CFr by disulfide-induced covalent circularisation that should be an excellent platform for design of novel functions. The accumulation of high levels of CFr exposes the high error rate of the protein translation machinery. The rarity of correspondingly stable fragments in natural proteins coupled with the observation that high quality ribosome binding sites are found to occur within E. coli protein-coding regions significantly less often than expected by random chance implies a stringent evolutionary pressure against protein sub-fragments that can independently fold into stable structures. The symmetric self-association between two identical mis-translated CFr sub-domains to generate an extremely stable structure parallels a mechanism for natural protein-fold evolution by modular recombination of protein sub-structures.     

Visualizing the Disassembly of S. cerevisiae Rad51 Nucleoprotein Filaments

Rad51 is the core component of the eukaryotic homologous recombination machinery and assembles into elongated nucleoprotein filaments on DNA. We have used total internal reflection fluorescence microscopy and a DNA curtain assay to investigate the dynamics of individual Saccharomyces cerevisiae Rad51 nucleoprotein filaments. For these experiments the DNA molecules were end-labeled with single fluorescent semiconducting nanocrystals. The assembly and disassembly of the Rad51 nucleoprotein filaments were visualized by tracking the location of the labeled DNA end in real time. Using this approach, we have analyzed yeast Rad51 under a variety of different reaction conditions to assess parameters that impact the stability of the nucleoprotein filament. We show that Rad51 readily dissociates from DNA in the presence of ADP or in the absence of nucleotide cofactor, but that free ATP in solution confers a fivefold increase in the stability of the nucleoprotein filaments. We also probe how protein dissociation is coupled to ATP binding and hydrolysis by examining the effects of ATP concentration, and by the use of the nonhydrolyzable ATP analogue adenosine 5'-(beta, gamma-imido) triphosphate and ATPase active-site mutants. Finally, we demonstrate that the Rad51 gain-of-function mutant I345T dissociates from DNA with kinetics nearly identical to that of wild-type Rad51, but assembles 30% more rapidly. Together, these results provide a framework for studying the biochemical behaviors of S. cerevisiae Rad51 nucleoprotein filaments at the single-molecule level.     

Solution Structure of S100A1 Bound to the CapZ Peptide (TRTK12)

As is typical for S100-target protein interactions, a Ca²⁺-dependent conformational change in S100A1 is required to bind to a 12-residue peptide (TRTK12) derived from the actin-capping protein CapZ. In addition, the Ca²⁺-binding affinity of S100A1 is found to be tightened (greater than threefold) when TRTK12 is bound. To examine the biophysical basis for these observations, we determined the solution NMR structure of TRTK12 in a complex with Ca²⁺-loaded S100A1. When bound to S100A1, TRTK12 forms an amphipathic helix (residues N6 to S12) with several favorable hydrophobic interactions observed between W7, I10, and L11 of the peptide and a well-defined hydrophobic binding pocket in S100A1 that is only present in the Ca²⁺-bound state. Next, the structure of S100A1-TRTK12 was compared to that of another S100A1-target complex (i.e., S100A1-RyRP12), which illustrated how the binding pocket in Ca²⁺-S100A1 can accommodate peptide targets with varying amino acid sequences. Similarities and differences were observed when the structures of S100A1-TRTK12 and S100B-TRTK12 were compared, providing insights regarding how more than one S100 protein can interact with the same peptide target. Such comparisons, including those with other S100-target and S100-drug complexes, provide the basis for designing novel small-molecule inhibitors that could be specific for blocking one or more S100-target protein interactions.     

超声波法优选苦豆子中总黄酮的提取工艺研究

目的:超声波法优选苦豆子中总黄酮的提取工艺。方法:以超声波为提取方法,以总黄酮提取率为考察指标,选取乙醇浓度(体积分数)、加溶剂倍量和超声时间为考察因素,采用均匀实验和正交实验,优选苦豆子中总黄酮的提取工艺。结果:影响总黄酮提取率各因素主次为:ACB,确定最佳提取工艺为A2B3C3,即70%乙醇,加溶剂40倍量,超声提取75 min。结论:采用该工艺提取苦豆子中总黄酮,提取率较高且稳定,说明该工艺合理可行。     

四种药用植物花总黄酮类物质提取及抗氧化性研究

目的:采用微波辅助法提取4种药用植物花中总黄酮类化合物,并研究其抗氧化性。方法:利用邻二氮菲-Fe2+-H2O2体系,研究其提取物清除羟基自由基(.OH)的能力,用普鲁士蓝法对其还原能力进行研究。结果:大蓟刺儿菜花总黄酮含量最高,夏至草花总黄酮含量最低,4种药用植物花提取物均有不同抗氧化能力。结论:药用植物花抗氧化能力与黄酮含量、结构有关。     

Potential impacts of climate warming on active soil organic carbon contents along natural altitudinal forest transect of Changbai Mountain

Understanding soil carbon fractions and their responses to the global warming is important for improving soil carbon management of natural altitudinal forest ecosystem. In this study, the contents of soil total organic carbon (SOC), soil labile organic carbon (LOC), and microbial biomass carbon (MBC) in soil upper layers (0–20 cm) were measured along a natural altitudinal transect in the north slope of Changbai Mountain. The results showed that under natural conditions the contents of SOC and LOC were largest in Betula ermanii forest (altitude 1996 m), moderate in spruce-fir forest (altitude 1350 m), and smallest in Korean pine mixed broad-leaf tree forest (altitude 740 m). MBC contents in different forest ecosystems decreased in the order of Betula ermanii forest, Korean pine mixed broad-leaf tree forest, and dark coniferous forest. In addition, the responses of SOC, LOC, and MBC to soil warming were conducted by relocating intact soil cores from high- to low-elevation forests for one year. As expected, the soil core relocation caused significant increase in soil temperature but made no significant effect on soil moisture. After one year incubation, soil relocation significantly decreased SOC contents, whereas the contents of LOC, MBC, and the ratios of LOC to SOC and MBC to SOC increased.     

川西亚高山不同演替阶段天然次生林土壤碳氮含量及酶活性特征

为了解次生林自然更新演替过程中土壤碳氮含量及酶活性的变化规律, 采用空间代替时间的方法, 在川西亚高山米亚罗林区选取环境条件基本一致的20世纪60、70和80年代采伐迹地自然更新演替的次生林(60-NSF、70-NSF和80-NSF)和岷江冷杉(Abies faxoniana)原始林(对照, CK)为对象, 研究了表层(0-20 cm)土壤碳氮含量和土壤酶活性的关系。结果表明: 表层土壤有机碳(SOC)、可溶性有机碳(DOC)、轻组有机碳(LFOC)含量均随森林植被更新演替呈显著降低趋势, 而全氮(TN)和可溶性有机氮(DON)含量则表现为60-NSF < 80-NSF < 70-NSF, 但70-NSF和80-NSF间差异不显著; 次生林表层土壤有机碳氮及其活性组分含量均低于CK, 其中80-NSF的DOC和DON含量与CK差异不显著。次生林的β-葡萄糖苷酶(βG)、β-N-乙酰氨基葡萄糖苷酶(NAG)和多酚氧化酶(PHO)活性均显著低于CK, 纤维素水解酶(CBH)和过氧化物酶(PEO)活性与CK无显著差异; 天然次生林中, 60-NSF的βG和CBH活性显著低于70-NSF和80-NSF; 80-NSF的NAG活性显著高于60-NSF和70-NSF; 4种林型之间的PEO活性无显著性差异。Pearson相关分析和冗余分析显示, 土壤TN、LFOC和DOC含量与土壤酶活性显著相关, 其中TN含量解释了酶活性变化的65.4%, 说明土壤氮含量变化可能会影响到土壤碳的水解酶活性, 同时也表明土壤微生物优先利用易分解碳和氮。因此, 次生林近60年的天然更新演替引起了TN、LFOC及DOC含量的显著下降, 导致表层土壤某些胞外酶(如βG、CBH和NAG)活性降低。从土壤酶活性角度看, 岷江冷杉原始林比早期演替阶段的次生林(<60 a)更有利于川西亚高山高海拔森林生态系统的碳氮循环。     

Dietary stress increases the total opportunity for sexual selection and modifies selection on condition‐dependent traits

Although it is often expected that adverse environmental conditions depress the expression of condition‐dependent sexually selected traits, the full consequences of environmental change for the action of sexual selection, in terms of the opportunity for total sexual selection and patterns of phenotypic selection, are unknown. Here we show that dietary stress in guppies, Poecilia reticulata, reduces the expression of several sexually selected traits and increases the opportunity for total sexual selection (standardized variance in reproductive success) in males. Furthermore, our results show that dietary stress modulates the relative importance of precopulatory (mating success) and postcopulatory (relative fertilization success) sexual selection, and that the form of multivariate sexual selection (linear vs. nonlinear) depends on dietary regime. Overall, our results are consistent with a pattern of heightened directional selection on condition‐dependent sexually selected traits under environmental stress, and underscore the importance of sexual selection in shaping adaptation in a changing world.     

人工红松树干内部节子体积预测模型

基于黑龙江省林口林业局林场和东京城林业局林场29块标准地中49株人工红松1207个节子数据,使用图片处理软件Digimizer对节子纵剖面图片数据进行提取,将节子形状用一个二维散点图表示。根据节子二维形状散点图,把人工红松节子分为3种类型: 活节(整个节子为健全节)、未包藏死节(节子由健全节和疏松节组成)和包藏死节(节子的健全节和疏松节部分被树干包藏)。3个类型节子的健全节体积通过对健全节形状参数方程求积得到;疏松节体积利用圆柱体的体积计算得到;节子总体积等于健全节体积与疏松节体积之和。最后,基于节子变量(节子直径、节子相对高、节子总长度)和树木变量(胸径),采用样地和树木水平的线性混合模型建立了红松人工林健全节体积、疏松节体积和节子总体积的预测模型。与基础模型相比,考虑样地和树木水平的混合效应所建立的健全节体积、疏松节体积和节子总体积预测模型,其参数估计更精准,残差分布更均匀,拟合精度明显提高。检验结果表明,基础模型预估精度均在90%以上,引入样地和树木效应的混合模型的预估精度均在93%以上,说明所建模型可以很好地预测红松人工林节子体积大小。