Bacterial Luciferases from Vibrio harveyi and Photobacterium leiognathi Demonstrate Different Conformational Stability as Detected by Time-Resolved Fluorescence Spectroscopy

Detecting the folding/unfolding pathways of biological macromolecules is one of the urgent problems of molecular biophysics. The unfolding of bacterial luciferase from Vibrio harveyi is well-studied, unlike that of Photobacterium leiognathi, despite the fact that both of them are actively used as a reporter system. The aim of this study was to compare the conformational transitions of these luciferases from two different protein subfamilies during equilibrium unfolding with urea. Intrinsic steady-state and time-resolved fluorescence spectra and circular dichroism spectra were used to determine the stages of the protein unfolding. Molecular dynamics methods were applied to find the differences in the surroundings of tryptophans in both luciferases. We found that the unfolding pathway is the same for the studied luciferases. However, the results obtained indicate more stable tertiary and secondary structures of P. leiognathi luciferase as compared to enzyme from V. harveyi during the last stage of denaturation, including the unfolding of individual subunits. The distinctions in fluorescence of the two proteins are associated with differences in the structure of the C-terminal domain of α-subunits, which causes different quenching of tryptophan emissions. The time-resolved fluorescence technique proved to be a more effective method for studying protein unfolding than steady-state methods.     

复配表面活性剂体系对漆酶降解菲和吲哚的影响研究

为了研究复配表面活性剂体系对漆酶降解有机污染物的影响机理,选用聚氧乙烯月桂醚(Brij-35)和聚山梨酯(Tween-80)复配体系,将漆酶作用于降解多环芳烃菲和氮杂环化合物吲哚,考察了菲和吲哚在不同pH、温度、表面活性剂浓度、漆酶活力浓度4种条件下的降解率,并进行降解动力学分析。结果表明,降解反应最优为pH 3.5,最佳反应温度为40℃,酶活力浓度最佳选择为4 U/mL。表面活性剂浓度分别为1.2、1.0 mmol/L时,菲和吲哚的降解率最高。表面活性剂的加入促进了漆酶对菲和吲哚的降解,菲和吲哚的降解符合一级动力学方程,且降解速率常数K(吲哚)>K(菲)>K(吲哚,空白)>K(菲,空白)。研究结果为漆酶在废水中去除持久性有机污染物的应用提供了参考。     

漆酶与木聚糖酶预处理对稻草蒸煮性能影响的比较

利用木聚糖酶和漆酶对稻草进行预处理,以改善后续的蒸煮性能。研究了不同酶用量对酶预处理效果的影响;同时比较了两种酶预处理对蒸煮性能的影响。结果表明木聚糖酶和漆酶对稻草预处理均能达到改善纸浆性能的目的。较佳的酶用量分别为20IU/g和30IU/g。在提高蒸煮得率、浆料强度方面漆酶优于木聚糖酶,与未处理和木聚糖酶化学浆相比实际得率分别提高13.11%和8.79%;裂断长提高34.2%和5.54%;耐破指数提高23.81%和7.22%;撕裂指数提高78.59%和3.26%。木聚糖酶预处理对改善浆料白度方面则优于漆酶,与未处理浆和漆酶生物化学浆相比分别提高5.3%和0.6%(SBD)。     

Effect of Trehalose and Ceftriaxone on the Stability of Aggregating-Prone Tau Peptide Containing PHF6* Sequence: An SRCD Study

The tau protein, a soluble protein associated with microtubules, which is involved in the assembly and stabilization of cytoskeletal elements, was found to form neurofibrillary tangles in different neurodegenerative diseases. Insoluble tau aggregates were observed to be organized in paired helical filaments (PHFs) and straight filaments (SFs). Recently, two small sequences (306–311 and 275–280) in the microtubule-binding region (MTBR), named PHF6 and PHF6*, respectively, were found to be essential for tau aggregation. Since a possible therapeutic approach consists of impairing amyloid formation either by stabilizing the native proteins or reducing the level of amyloid precursors, here we use synchrotron radiation circular dichroism (SRCD) at Diamond B23 beamline to evaluate the inhibitory effects of two small molecules, trehalose and ceftriaxone, against the aggregation of a small peptide containing the PHF6* sequence. Our results indicate that both these molecules, ceftriaxone and trehalose, increased the stability of the peptide toward aggregation, in particular that induced by heparin. With trehalose being present in many fruits, vegetables, algae and processed foods, these results support the need to investigate whether a diet richer in trehalose might exert a protective effect toward pathologies linked to protein misfolding.     

Conformational Changes of Anoplin,W-MreB1–9, and (KFF)3K Peptides near the Membranes

Many peptides interact with biological membranes, but elucidating these interactions is challenging because cellular membranes are complex and peptides are structurally flexible. To contribute to understanding how the membrane-active peptides behave near the membranes, we investigated peptide structural changes in different lipid surroundings. We focused on two antimicrobial peptides, anoplin and W-MreB_1–9, and one cell-penetrating peptide, (KFF)₃K. Firstly, by using circular dichroism spectroscopy, we determined the secondary structures of these peptides when interacting with micelles, liposomes, E. coli lipopolysaccharides, and live E. coli bacteria. The peptides were disordered in the buffer, but anoplin and W-MreB_1–9 displayed lipid-induced helicity. Yet, structural changes of the peptide depended on the composition and concentration of the membranes. Secondly, we quantified the destructive activity of peptides against liposomes by monitoring the release of a fluorescent dye (calcein) from the liposomes treated with peptides. We observed that only for anoplin and W-MreB_1–9 calcein leakage from liposomes depended on the peptide concentration. Thirdly, bacterial growth inhibition assays showed that peptide conformational changes, evoked by the lipid environments, do not directly correlate with the antimicrobial activity of the peptides. However, understanding the relation between peptide structural properties, mechanisms of membrane disruption, and their biological activities can guide the design of membrane-active peptides.     

Spectroscopic and In Silico Studies on the Interaction of Substituted Pyrazolo[1,2-a]benzo[1,2,3,4]tetrazine-3-one Derivatives with c-Myc G4-DNA

Herein we describe a combined experimental and in silico study of the interaction of a series of pyrazolo[1,2-a]benzo[1,2,3,4]tetrazin-3-one derivatives (PBTs) with parallel G-quadruplex (GQ) DNA aimed at correlating their previously reported anticancer activities and the stabilizing effects observed by us on c-myc oncogene promoter GQ structure. Circular dichroism (CD) melting experiments were performed to characterize the effect of the studied PBTs on the GQ thermal stability. CD measurements indicate that two out of the eight compounds under investigation induced a slight stabilizing effect (2–4 °C) on GQ depending on the nature and position of the substituents. Molecular docking results allowed us to verify the modes of interaction of the ligands with the GQ and estimate the binding affinities. The highest binding affinity was observed for ligands with the experimental melting temperatures (T_ms). However, both stabilizing and destabilizing ligands showed similar scores, whilst Molecular Dynamics (MD) simulations, performed across a wide range of temperatures on the GQ in water solution, either unliganded or complexed with two model PBT ligands with the opposite effect on the T_ms, consistently confirmed their stabilizing or destabilizing ability ascertained by CD. Clues about a relation between the reported anticancer activity of some PBTs and their ability to stabilize the GQ structure of c-myc emerged from our study. Furthermore, Molecular Dynamics simulations at high temperatures are herein proposed for the first time as a means to verify the stabilizing or destabilizing effect of ligands on the GQ, also disclosing predictive potential in GQ-targeting drug discovery.     

光谱法研究羟喜树碱与牛血清白蛋白的相互作用

在模拟人体生理条件下,采用光谱法研究了羟喜树碱（HCPT）与牛血清白蛋白（BSA）的相互作用,计算了不同温度下的结合常数及ΔHθ、ΔGθ、ΔSθ等热力学参数。结果表明,HCPT对BSA的猝灭是由于形成HCPT-BSA复合物而引起的静态猝灭;ΔHθ（-35.91kJ.mol-1）和ΔSθ（-24.30J.mol-1.K-1）的值表明氢键和范德华力在HCPT-BSA的结合中起主要作用;圆二色谱和三维荧光光谱表明,在与HCPT结合后,BSA中的α-螺旋含量减少、微环境和二级结构均发生改变。     

Effects of Tearing Conditions on the Crack Propagation in a Monolayer Graphene Sheet

The path of crack propagation in a graphene sheet is significant for graphene patterning via the tearing approach. In this study, we evaluate the fracture properties of pre-cracked graphene during the tearing process, with consideration of the effects of the aspect ratio, loading speed, loading direction, and ambient temperatures on the crack propagation in the monolayer sheet. Some remarkable conclusions are drawn based on the molecular dynamic simulation results, i.e., a higher loading speed may result in a complicated path of crack propagation, and the propagation of an armchair crack may be accompanied by sp carbon links at high temperatures. The reason for this is that the stronger thermal vibration reduces the load stress difference near the crack tip and, therefore, the crack tip can pass through the sp link. A crack propagates more easily along the zigzag direction than along the armchair direction. The out-of-plane tearing is more suitable than the in-plane tearing for graphene patterning. The path of crack propagation can be adjusted by changing the loading direction, e.g., a rectangular graphene ribbon can be produced by oblique tearing. This new understanding will benefit the application of graphene patterning via the tearing approach.     

介孔分子筛MSU-X合成条件对结构及热稳定性影响的XRD研究

以十八烷基聚氧乙烯醚(C18EO10)为模板剂,以正硅酸乙酯(TEOS)和硅酸钠分别为硅源,研究了不同pH值、反应温度、反应时间、原料比等条件下,合成的全硅MSU-X型介孔分子筛产物的结构变化规律,并通过焙烧和水热处理,考察了MSU-X介孔材料的结构稳定性.实验采用粉末多晶X射线衍射技术,分析了合成条件对MSU-X介孔分子筛结构和稳定性的影响.结果表明:以正硅酸乙酯为硅源,原料摩尔比为TEOS:C18EO10:H2O＝1:0.225:150,凝胶pH值为2,晶化温度为55℃,晶化时间48 h,相应的MSU-X材料具有较好的热稳定性和水热稳定性.相比之下,以硅酸钠为硅源得到的MSU-X材料,具有更高的短程有序度,孔壁厚、孔径小,稳定性优于以正硅酸乙酯为硅源合成的MSU-X产物,经900℃焙烧2 h或760℃水蒸气处理7 h后仍保持其孔道结构.     

Effects of Cardiolipin on the Conformational Dynamics of Membrane-Anchored Bcl-xL

The anti-apoptotic protein Bcl-xL regulates apoptosis by preventing the permeation of the mitochondrial outer membrane by pro-apoptotic pore-forming proteins, which release apoptotic factors into the cytosol that ultimately lead to cell death. Two different membrane-integrated Bcl-xL constructs have been identified: a membrane-anchored and a membrane-inserted conformation. Here, we use molecular dynamics simulations to study the effect of the mitochondrial specific lipid cardiolipin and the protein protonation state on the conformational dynamics of membrane-anchored Bcl-xL. The analysis reveals that the protonation state of the protein and cardiolipin content of the membrane modulate the orientation of the soluble head region (helices α1 through α7) and hence the exposure of its BH3-binding groove, which is required for its interaction with pro-apoptotic proteins.     

Effects of Changes in the Operating Conditions on the Stack Gas Temperature and Stability of Biomass-Fueled Boilers

In this study, the stack gas temperature of a biomass-fueled boiler was estimated from material and energy balance equations written for a boiler system and by using data collected from an operating boiler. A parametric study was subsequently carried out in order to gauge the effects of changes in the moisture content of the biomass, the efficiency of the boiler, and the air flow rate to the boiler furnace on the stack gas temperature and the stability of the boiler operation. The results indicate that there exist values of the moisture content and the air flow rate above which the operation of the boiler becomes unstable. The results further confirm the long-held views that regular maintenance of a boiler and drying of biomass fuel prior to feeding to the boiler furnace have favorable impacts on the operation of biomass-fueled boilers.     

菌种L3深层发酵产漆酶及其对废水脱色的研究

对菌种Trametes versicolor SYBC-L3发酵产漆酶的碳氮源进行了优化,研究了菌株L3漆酶的性质及其在毛纺废水脱色中的作用。结果表明,其最佳发酵碳氮源为:麦芽糖12g/L、豆粕4g/L,优化后的漆酶的酶活在第8天可高达48400U/L;酶最适反应温度为50℃,在70℃以下保温1h,仍然有70%的剩余活力,其最适pH为3.5,在pH4～8下保温24h,仍然有70%以上的活力。用L3漆酶处理毛纺废水,20min脱色率达到70%,有较好的脱色效果。     

Chiral Linked Systems as a Model for Understanding D-Amino Acids Influence on the Structure and Properties of Amyloid Peptides

In this review, we provide an illustration of the idea discussed in the literature of using model compounds to study the effect of substitution of L- for D-amino acid residues in amyloid peptides. The need for modeling is due to the inability to study highly disordered peptides by traditional methods (high-field NMR, X-ray). At the same time, the appearance of such peptides, where L-amino acids are partially replaced by D-analogs is one of the main causes of Alzheimer’s disease. The review presents examples of the use diastereomers with L-/D-tryptophan in model process—photoinduced electron transfer (ET) for studying differences in reactivity and structure of systems with L- and D-optical isomers. The combined application of spin effects, including those calculated using the original theory, fluorescence techniques and molecular modeling has demonstrated a real difference in the structure and efficiency of ET in diastereomers with L-/D-tryptophan residues. In addition, the review compared the factors governing chiral inversion in model metallopeptides and Aβ42 amyloid.     

含Co介孔分子筛的合成与稳定性研究

两步法进行含Co介孔分子筛的合成,采用XRD,FT-IR,TPR,TEM,N2吸附-脱附等方法对样品的物化性能进行表征结果表明:合成出了具有介孔结构的CoMCM-41,550℃焙烧可以将模板剂有效去除并不影响介孔结构,CoMCM-41的比表面积大于700m2/g,孔径分布在3.4～4.5 nm范围内.650℃热处理3 h或100℃水热处理5 d后CoMCM-41的比表面、孔径和孔体积变化较小,表明CoMCM-41的热稳定性和水热稳定性较好.     

槲皮素与牛血清白蛋白相互作用的研究

应用荧光光谱法研究了槲皮素(Qct)与牛血清白蛋白(BSA)的相互作用。结果表明,Qct对BSA的荧光强度产生了静态猝灭。在此基础上计算了二者相互作用的结合常数及结合热力学参数等,结果表明该反应是自发进行的且相互作用力主要为氢键和范德华力。根据位点竞争实验确定了Qct在BSA上的主要结合位置为SiteⅠ。最后利用圆二色谱探讨了Qct对BSA构象的改变。     

Discovering and Targeting Dynamic Drugging Pockets of Oncogenic Proteins: The Role of Magnesium in Conformational Changes of the G12D Mutated Kirsten Rat Sarcoma-Guanosine Diphosphate Complex

KRAS-G12D mutations are the one of most frequent oncogenic drivers in human cancers. Unfortunately, no therapeutic agent directly targeting KRAS-G12D has been clinically approved yet, with such mutated species remaining undrugged. Notably, cofactor Mg2+ is closely related to the function of small GTPases, but no investigation has been conducted yet on Mg2+ when associated with KRAS. Herein, through microsecond scale molecular dynamics simulations, we found that Mg2+ plays a crucial role in the conformational changes of the KRAS-GDP complex. We located two brand new druggable dynamic pockets exclusive to KRAS-G12D. Using the structural characteristics of these two dynamic pockets, we designed in silico the inhibitor DBD15-21-22, which can specifically and tightly target the KRAS-G12D-GDP-Mg2+ ternary complex. Overall, we provide two brand new druggable pockets located on KRAS-G12D and suitable strategies for its inhibition.     

The Effect of Small Cosolutes that Mimic Molecular Crowding Conditions on the Stability of Triplexes Involving Duplex DNA

Triplex stability is studied in crowding conditions using small cosolutes (ethanol, acetonitrile and dimethylsulfoxide) by ultraviolet (UV), circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopies. The results indicate that the triplex is formed preferentially when the triplex forming oligonucleotide (TFO) is RNA. In addition, DNA triplexes (D:D·D) are clearly less stable in cosolute solutions while the stability of the RNA triplexes (R:D·D) is only slightly decreased. The kinetic of triplex formation with RNA-TFO is slower than with DNA-TFO and the thermal stability of the triplex is increased with the salt concentration in EtOH-water solutions. Accordingly, RNA could be considered a potential molecule to form a stable triplex for regulatory purposes in molecular crowding conditions.     

Investigating the structural and functional consequences of circular permutation on lipase B from Candida antarctica

The engineering of lipase B from Candida antarctica (CALB) by circular permutation has yielded over sixty hydrolase variants, and several show significantly improved catalytic performance. Here we report a detailed characterization of ten selected enzyme variants by kinetic and spectroscopic methods to further elucidate the impact of circular permutation on the structure and function of CALB. Our experiments identify lipase variants with up to 175-fold enhanced k(cat)/K(M) values over wild-type. In addition, circular permutation does not change the enzymes' enantiopreference and preserves or even improves their enantioselectivity compared to that of the wild-type enzyme. Finally, our spectroscopic analyses suggest that the structural effects of circular permutation on CALB are mostly local, concentrating on regions near the native and new protein termini. The observed changes in secondary structure and protein thermostability vary among enzyme variants but directly correlate with the locations of the new termini, a first step towards a predictive framework.     

二元醇对分散荧光染料墨水性能的研究

采用纳米粒径分析仪对分散荧光染料色浆的主体颗粒大小进行了研究,分析了二元醇对墨水的黏度、表面张力、保湿性、热储存稳定性、离心稳定性、墨水转移率以及喷墨转移印花后涤纶织物荧光反射率的影响。实验结果表明,采用湿磨法研磨后的染料色浆分散稳定性好,粒径为125.5 nm。二元醇质量分数低于15%时,墨水黏度变化不大;当其质量分数超过15%后,墨水黏度的增幅明显。由于氢键作用力的存在,墨水的保湿性随二元醇质量分数的增加不断提高,其中乙二醇的保湿效果最好。添加15%(质量分数)二元醇的墨水具有较好的热储存稳定性和离心稳定性。二元醇用量的增加有利于墨水转移率和涤纶织物荧光反射率的提高;但其用量过多,也会使转移率和荧光反射率降低。     

操作条件对柴油微乳液制备及稳定性的影响

利用微乳化剂Span 80(失水山梨醇单油酸酯)、D 08/1021(双烷基氯化铵),助乳化剂正戊醇,自来水制备出了柴油微乳液;进一步探讨了温度、加料方式、搅拌方式等对制备微乳液的影响.结论为温度在30-35℃时制备的微乳液澄清较快,而加料方式以及加料过程中的搅拌对微乳液的制备无影响.对所制备的微乳液样品进行稳定性验证:先混配制得微乳化剂,再将柴油和微乳化剂混合均匀,搅拌一段时间后加入水,再搅拌一段时间后加助乳化剂正戊醇,操作温度控制在30℃,由此制得的柴油微乳液澄清较快,且稳定性高.