利用核磁共振法探讨类泛素媒介的信息传导分子机制（英文）

转译后类泛素修饰调控多项细胞活动,此信息传导的途径为:第一步将类泛素(SUMO)接于E1活化蛋白,第二步将SUMO转移到E2共轭蛋白(Ubc9),然后帮助受体蛋白完成类泛素化,最后借由蛋白酶去除类泛素完成整个传导过程.受体蛋白的类泛素化调控基本上靠Ubc9来辨识受体蛋白上的特殊类泛素序列(SM),在某些情况下亦可借由E3辨识完成.而类泛素辨识序列功效的发挥则依赖于招慕含有类泛素辨识序列(SIM)的感应蛋白来实现.此外原核细胞的类泛素化皆有形成多聚类泛素(poly-SUMO)化的能力.多聚类泛素化修饰可被含多聚类泛素辨识序列(poly-SIM)的蛋白质如RNF4识别,促进受体蛋白的多聚类泛素化,并导致目标蛋白的分解.该文综述了作者所在研究组近年来利用核磁共振法研究类泛素介导的信息传导分子机制方面的成果.     

细胞色素c甲硫氨酸氧化机制的NMR研究

细胞在呼吸作用中产生活性氧，活性氧含量处于低水平时有利于信号传导，累积过量时会引发蛋白质氧化修饰．细胞色素c是位于线粒体内的多功能金属蛋白，其发生氧化修饰特别是甲硫氨酸Met80的亚砜化修饰可能影响蛋白构象变化，但其机制仍不清楚．本研究通过13C选择性标记细胞色素c上甲硫氨酸的末端甲基，利用液体核磁共振技术，追踪了细胞色素c在氧化环境中的氧化修饰变化．发现在氧化环境中，该蛋白质先由还原态转化为氧化态，当活性氧达到一定量时，再发生甲硫氨酸Met80的亚砜化修饰，但在活性氧作用初期未导致明显的蛋白结构变化．这表明细胞色素c具有一定抵御活性氧的作用．     

49≤Z≤53类钴离子3d9-3p53d10,3d9-3d84p跃迁谱线和振子强度的计算

用等电子序列高离化态离子能级△E随Zc的一般拟合公式,系统计算了中Z段49≤Z≤53类钴离子的能级,并给出了3d9-3p53d10,3d9-3d84p跃迁的波长和振子强度.     

Nutlin-3a对p53-MDM2复合物稳定性影响

p53蛋白是一种与细胞周期停滞和细胞凋亡有关的蛋白质.在受到细胞压力或环境扰动后, p53促进下游多个靶基因的转录,介导肿瘤抑制. MDM2是主要的E3泛素连接酶,也是p53的负调控因子. MDM2可促进p53的泛素化和核输出,抑制p53的抑癌活性.因此MDM2对p53的负调控始终是肿瘤治疗中急切需要解决的问题. Nutlin-3a是被证明可以有效抑制p53-MDM2相互作用的小分子抑制剂.本文使用全原子分子动力学模拟,研究Nutlin-3a对p53-MDM2复合物的稳定性的影响.结果表明,通过引起p53和MDM2间Phe19-Gln72的氢键和Glu17-Lys94的盐桥发生的断裂, Nutlin-3a可以削弱p53和MDM2间的相互作用.我们的工作对Nutlin-3a小分子抑制剂的作用机制进行了说明,揭示了抗癌药物Nutlin-3a介导的p53-MDM2复合物亲和力降低的分子机制,并为针对p53蛋白的有效抗癌治疗提供了理论基础.     

Cs~(134)的衰变

本工作用双磁镜β谱仪,带铁中间象式β谱仪、有铁双聚焦β谱仪和闪烁技术来研究Cs~(134)(半衰期为2.3年)的衰变,定出4个β分支的能量和它们的相对强度的百分比是79(16％),410(5％),652(64％)和683(15％)千电子伏;它们的相应的log ft是6.48,9.34,8.91,9.58.还定出Ba~(134)的9个γ跃迁的能量和它们的多极性是473(E1),564(M1+E2),571(M1),604.8(E2),796(E2),803(E2),1036(E1+M2),1169(E2)和1367(E2)千电子伏。在测量结果的基础上提出了Cs~(134)的衰变纲图。     

纳米晶Fe73.5Cu1Nb3Si13.5B9晶间非晶相结构弛豫与模量的振荡变化

采用X射线衍射(XRD),杨氏模量测量方法研究了非晶态Fe73.5Cu1Nb3Si13.5B9合金500℃等温晶化时,纳米α-Fe(Si)晶相的平均晶粒尺寸(d),晶化体积分数(Vc),杨氏模量(E)随保温时间(t)的变化.d-t关系具有阶梯式长大特征,而E-t关系则呈现相应的周期性变化.分别计算了纳米α-Fe(Si)晶生长前沿非晶区的化学成分以及晶间非晶区平衡化学成分.由Fick扩散方程计算晶间非晶区成分均匀化的时间,与纳米α-Fe(Si)阶梯式长大周期相当.E-t关系的振荡性变化可能是由扩散控制的纳米α-Fe(Si)晶相的阶梯式长大与晶间非晶区周期性弛豫引起的.     

高压诱导有机CT共晶荧光发射原位可调控性的光谱研究

有机电荷转移(CT)共晶是通过给体(D)与受体(A)分子之间的CT作用形成具有特定结构的分子有序组合体,其带隙较窄便于调控,表现出新颖各异的光学性质。最近该课题小组利用高压对顶砧技术,对有机CT共晶材料9ACA-TFP的荧光发射进行原位持续调控~([1])。与传统基于合成或掺杂法来调控有机共晶体荧光发射相比,通过压力刺激有机CT共晶的荧光发射,其方法简单有效且实现了对荧光发射的原位持续调控。基于此研究,为了揭示有机CT共晶在高压下荧光调控机制的共性和规律,以TFP(2,3,5,6-四氟对苯二甲腈)为接受体,设计合成具有蓝色发光CT共晶系列,通过压力刺激对有机CT共晶材料体系的荧光发射进行了原位持续调控,并利用光谱学技术手段(紫外及拉曼)监控调控过程中给受体间CT作用的变化,讨论了变压力对有机共晶给受体间分子间相互作用的敏感性及可控性。     

Cs134的衰变

本工作用双磁镜β谱仪,带铁中间象式β谱仪、有铁双聚焦β谱仪和闪烁技术来研究Cs¹³⁴(半衰期为2.3年)的衰变,定出4个β分支的能量和它们的相对强度的百分比是79(16％),410(5％),652(64％)和683(15％)千电子伏;它们的相应的logft是6.48,9.34,8.91,9.58.还定出Ba¹³⁴的9个γ跃迁的能量和它们的多极性是473(E1),564(M1+E2),571(M1),604.8(E2),796(E2),803(E2),1036(E     

基于SERS技术的靶向修饰金纳米探针 用于Hela细胞的检测

表皮生长因子受体(EGFR)是一种肿瘤表面标记性蛋白。本文报道了基于anti-EGFR功能化金纳米棒探针AuNRs probes的表面增强拉曼散射(SERS),用于EGFR阳性肿瘤细胞的检测。通过AuNRs probes上anti-EGFR特异性结合到EGFR阳性癌细胞上,可使修饰于金纳米棒表面的拉曼活性染料4-巯基苯甲酸(4-MBA)位于1 100 cm~(-1)和1 600 cm~(-1)的特征峰强度得到信号增强。该SERS探针由于具有生物兼容性好、细胞拉曼信号稳定、特异性高等优点而具有巨大的临床应用前景。     

基于DNA损伤的蛋白调控网络研究

细胞生长的每个阶段都离不开蛋白质相互作用.研究细胞周期的功能、调控机理及参与调控的蛋白质之间的关系对生物工程等领域有重大的应用价值.本文通过研究电离辐射下生物体细胞的DNA损伤后,细胞内以p53为核心的扩展蛋白调控网络的功能、原理及其自修复机理,在现有蛋白网络基础上引入更多蛋白网络调控因子来建立蛋白调控网络,仿真模拟更为全面的细胞周期进程;并且从复杂网络图论和细胞周期调控两个方面分析扩展PMP调控网络的抗扰能力及自修复机理,结果表明:1)蛋白网络在对抗环境中出现的小扰动时具有较强的稳定性.但在面对蓄意攻击时网络的稳定性较差.2)受损的DNA能否被修复取决于p53蛋白的动力学行为,即低损伤与中损伤情况下,p53可诱导细胞周期进程阻滞来完成细胞的自修复;而当高损伤或过损伤时,p53蛋白浓度表现为周期振荡行为并诱导细胞凋亡.     

Study of DNA accessibility in the condensed chromatin structures by resonance energy transfer

The linker DNA accessibility of chicken erythrocyte chromatin was studied by diffusion-enhanced resonance energy transfer (DERET). The 4″-{9?-[((4-carboxy-3-hydroxyphenyl)-acetatamido)-3?,6?,9?-(triacetyl)-3″,6?,9?-triazanonamido]-2″,6″-diazanonyl}-4,5′,8-trimethyl psoralen-terbium complex was photocovalently bound to linker DNA and transferred its energy to fluorescein free in solution or bound on proteins of different sizes. We observed a diminution of linker DNA accessibility in chromatin as the protein size increased. Free fluorescein and proteins (up to a molecular weight of 24,000) labeled with fluorescein isothiocyanate (FITC) showed no variation in linker accessibility as chromatin condensation from 10- to 30-nm fibers was induced by an increase in ionic strength. We can conclude from these observations that linker DNA is located on the outside of the condensed chromatin fiber or, alternatively, that small proteins are free to diffuse toward an inside-located linker DNA, even in the condensed state of chromatin, possibly through the central cavity of the solenoïd model.     

Protein adsorption resistant surface on polymer composite based on 2D- and 3D-controlled grafting of phospholipid moieties

To prepare the biocompatible surface, a phosphorylcholine (PC) group was introduced on this hydroxyl group generated by surface hydrolysis on the polymer composite composed of polyethylene (PE) and poly (vinyl acetate) (PVAc) prepared by supercritical carbon dioxide. Two different procedures such as two-dimensional (2D) modification and three-dimensional (3D) modification were applied to obtain the steady biocompatible surface. 2D modification was that PC groups were directly anchored on the surface of the polymer composite. 3D modification was that phospholipid polymer was grafted from the surface of the polymer composite by surface-initiated atom transfer radical polymerization (SI-ATRP) of 2-methacryloyloxyethyl phosphorylcholine (MPC). The surfaces were characterized by X-ray photoelectron spectroscopy, dynamic water contact angle measurements, and atomic force microscope. The effects of the poly(MPC) chain length on the protein adsorption resistivity were investigated. The protein adsorption on the polymer composite surface with PC groups modified by 2D or 3D modification was significantly reduced as compared with that on the unmodified PE. Further, the amount of protein adsorbed on the 3D modified surface that is poly(MPC)-grafted surface decreased with an increase in the chain length of the poly(MPC). The surface with an arbitrary structure and the characteristic can be constructed by using 2D and 3D modification. We conclude that the polymer composites of PE/PVAc with PC groups on the surface are useful for fabricating biomedical devices due to their good mechanical and surface properties.     

Glass-forming liquids,anomalous liquids,and polyamorphism in liquids and biopolymers

Summary Glass formation in nature and materials science is reviewed and the recent recognition of polymorphism within the glassy state, polyamorphism, is discussed. The process by which the glassy state originates during the continuous cooling or viscous slowdown process, is examined and the three canonical characteristics of relaxing liquids are correlated through the fragility. The conversion of strong liquids to fragile liquids by pressure-induced coordination number increases is discussed, and then it is shown that for the same type of system it is possible to have the same conversion accomplished via a first-order transition within the liquid state. The systems in which this can happen are of the same type which exhibit polyamorphism, and the whole phenomenology can be accounted for by a recent simple modification of the van der Waals model for tetrahedrally bonded liquids. The concept of complex amorphous systems which can lose a significant number of degrees of freedom through weak first-order transitions is then used to discuss the relation between native and denatured hydrated proteins, since the latter have much in common with plasticized chain polymer systems. Finally, we close the circle by taking a short-time-scale phenomenon given much attention by protein physicists,viz., the onset of an anomaly in the Debye-Waller factor with increasing temperature, and showing that for a wide variety of liquids, including computer-simulated strong and fragile ionic liquids, this phenomenon is closely correlated with the experimental glass transition temperature. This implies that the latter owes its origin to the onset of strong anharmonicity in certain components of the vibrational density of states (evidently related to the boson peak) which then permits the system to gain access to its configurational degrees of freedom. The more anharmonic these vibrational components, the closer to the Kauzmann temperature will commence the exploration of configuration space and, for a given configurational microstate degeneracy, the more fragile the liquid will be. Paper presented at the I International Conference on Scaling Concepts and Complex Fluids, Copanello, Italy, July 4–8, 1994.     

Ultrasonic pre-treatment modifies the pH-dependent molecular interactions between β-lactoglobulin and dietary phenolics: Conformational structures and interfacial properties

There is a need to understand the ultrasound-induced changes in the interactions between proteins and phenolic compounds at different pH. This study systematically explored the role of high-intensity ultrasound pre-treatment on the binding mechanisms of β-lactoglobulin (β-LG) to two common phenolic compounds, i.e., (−)-epigallocatechin-3-gallate (EGCG) and chlorogenic acid (CA) at neutral and acidic pH (pH 7.2 and 2.4). Tryptophan fluorescence revealed that compared to proteins sonicated at 20% and 50% amplitudes, 35%-amplitude ultrasound pre-treatment (ULG-35) strengthened the binding affinities of EGCG/CA to β-LG without altering the main interaction force. After phenolic addition, ULG-35 displayed a similar but a greater extent of protein secondary and tertiary structural changes than the native protein, ascribed to the ultrasound-driven hydrophobic stacking among interacted molecules. The dominant form of β-LG (dimer/monomer) played a crucial role in the conformational and interfacial properties of complexes, which can be explained by the distinct binding sites at different pH as unveiled by molecular docking. Combining pre-ultrasound with EGCG interaction notably increased the foaming and emulsifying properties of β-LG, providing a feasible way for the modification of bovine whey proteins. These results shed light on the understanding of protein–phenolic non-covalent binding under ultrasound and help to develop complex systems with desired functionality and delivery.     

Modified titanium surface with gelatin nano gold composite increases osteoblast cell biocompatibility

This study examined the gelatin nano gold (GnG) composite for surface modification of titanium in addition to insure biocompatibility on dental implants or biomaterials. The GnG composite was constructed by gelatin and hydrogen tetrachloroaurate in presence of reducing agent, sodium borohydrate (NabH₄). The GnG composite was confirmed by UV-VIS spectroscopy and transmission electron microscopy (TEM). A dipping method was used to modify the titanium surface by GnG composite. Surface was characterized by scanning electron microscopy (SEM) and energy dispersive X-ray (EDX). The MC-3T3 E1 cell viability was assessed by trypan blue and the expression of proteins to biocompatibility were analyzed by Western blotting. The GnG composite showed well dispersed character, the strong absorption at 530 nm, roughness, regular crystal and clear C, Na, Cl, P, and Au signals onto titanium. Further, this composite allowed MC-3T3 E1 growth and viability compared to gelatin and pure titanium. It induced ERK activation and the expression of cell adherent molecules, FAK and SPARC, and growth factor, VEGF. However, GnG decreased the level of SAPK/JNK. This shows that GnG composite coated titanium surfaces have a good biocompatibility for osteoblast growth and attachment than in intact by simple and versatile dipping method. Furthermore, it offers good communication between cell and implant surfaces by regulating cell signaling and adherent molecules, which are useful to enhance the biocompatibility of titanium surfaces.     

Peptide arrays with designed α-helical structures for characterization of proteins from FRET fingerprint patterns

A practical high-throughput protein detection system is described, based on synthetic peptide arrays consisting of designed alpha-helical peptides, detected by fluorescence resonance energy transfer (FRET). Initially a model alpha-helical peptide known to interact with a structured protein, calmodulin, was selected to establish the strategy for high-throughput detection. In comparison to peptides with a single probe, a much higher FRET response has been observed with two fluorescent probes (7-diethylaminocoumarin-3-carboxylic acid and 5(6)-carboxy-fluorescein) at both termini of the synthetic peptides. To establish a reproducible high-throughput detection system, peptides were also immobilized onto a solid surface for detection of the target proteins. A small library of 112 different peptides was constructed, based on a model of the alpha-helical peptide with systematic replacement of residues carrying specific charges and/or hydrophobicities. The library was used to effectively characterize various proteins, giving their own 'protein fingerprint' patterns. The resulting 'protein fingerprints' correlate with the recognition properties of the proteins. The present microarray with designed synthetic peptides as the capturing agents is promising for the development of protein detection chips.     

Biomolecular dynamics and binding studies in the living cell

Isolation and preparation of proteins of higher organisms often is a tedious task. In the case of success, the properties of these proteins and their interactions with other proteins can be studied in vitro. If however, these proteins are modified in the cell in order to gain or change function, this is non-trivial to correctly realise in vitro. When, furthermore, the cellular function requires the interplay of more than one or two proteins, in vitro experiments for the analysis of this situation soon become complex. Instead, we thus try to obtain information on the molecular properties of proteins in the living cell. Then, the cell takes care of correct protein folding and modification. A series of molecular techniques are, and new ones become, available which allow for measuring molecular protein properties in the living cell, offering information on concentration (FCS), dynamics (FCS, RICS, FRAP), location (PALM, STED), interactions (F3H, FCCS) and protein proximities (FRET, BRET, FLIM, BiFC). Here, these techniques are presented with their advantages and drawbacks, with examples from our current kinetochore research. The review is supposed to give orientation to researchers planning to enter the field, and inform which techniques help us to gain molecular information on a multi-protein complex. We show that the field of cellular imaging is in a phase of transition: in the future, an increasing amount of physico-chemical data can be determined in the living cell.