基于19F化学标记磷酸化泛素的温度传感器开发

泛素是一种真核细胞信号分子,主要参与蛋白质降解和DNA修复等生命活动.泛素Ser65位被磷酸化之后,在溶液中呈现两个稳定的溶液构象,这两种构象的比例能够被pH调控,本研究利用NMR进一步发现它还受到温度影响.基于该发现,对磷酸化泛素进行了¹⁹F化学标记,利用¹⁹F NMR方法表征了不同温度下磷酸化泛素两种构象的比例,发现两者比例变化与温度之间的关系可以通过线性方程来描述,利用该方程可以通过构象比例计算样品内部温度,因此可以作为一种基于NMR检测的温度传感器.本文所开发的基于¹⁹F化学标记磷酸化泛素的温度传感器不仅能够作为体外样品温度检测的有力工具,还有望用于检测细胞内部的温度.从而有助于揭示生物学特性和功能.     

利用核磁共振法探讨类泛素媒介的信息传导分子机制

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．     

NMR技术在蛋白质-蛋白质相互作用研究中的应用——泛素-蛋白水解酶体通路研究实例介绍

蛋白质-蛋白质相互作用在多种细胞内生理活动中发挥关键性作用,而蛋白质复合物结构信息的获得主要依赖于X-射线衍射技术和核磁共振技术2种主要技术手段的使用. 需要指出的是,虽然大部分蛋白质复合物的结构解析使用了X-射线衍射技术,然而在包括无法获得蛋白质复合物晶体、 蛋白质与蛋白质结合强度较弱以及蛋白质复合物系统具有复杂的动力学行为等几种情况下,核磁共振技术是可用于蛋白质复合物结构测定的唯一手段. 用于蛋白质-蛋白质相互作用研究的NMR技术主要有化学位移扰动分析、分子间NOE的检测、顺磁弛豫增强技术、残余偶极耦合检测技术等几种. 该文将结合这几种技术在泛素-蛋白水解酶体通路领域的应用实例对它们的工作原理以及可提供的信息做出总结介绍.     

铽离子-镧系金属结合标签的荧光探针的改进

镧系金属因其具有较窄的发射光谱带,较大的斯托克斯位移以及毫秒级的荧光寿命,而被广泛应用于荧光检测中,其中Tb~(3+)、Eu~(3+)最为常用.镧系金属结合标签(Lanthanide Binding Tag,LBT)可以与蛋白质融合表达,并且一般不会影响蛋白的结构和功能,这些特点使LBT被广泛应用于蛋白质结构与功能研究中.LBT能够特异性地结合镧系金属离子,并利用LBT上色氨酸的吲哚环作为"天线"吸收外部能量,再将能量传递给镧系金属离子,进而激发镧系金属离子产生荧光.该文以经典模式蛋白泛素(Ubiquitin,Ub)作为媒介,将LBT引入到Ub的碳端,采用定点突变的方法增加LBT上吲哚环的数量,观察Ub-LBT[结合铽离子(Tb~(3+))]荧光量子产率的变化.结果表明在LBT结构中增加吲哚环的数量能够提高LBT(结合Tb~(3+))的荧光量子产率.     

蛋白质INSM1中的锌指结构域ZF (4-5)的表达、纯化与表征

胰岛素瘤相关蛋白1（INSM1）是一类转录调节蛋白,通过其C-端的锌指结构域（氨基酸250-510）来识别序列特异性的DNA分子.INSM1的C-端包含有5个串联的锌指结构域,然而这些结构域的结构及其如何识别DNA的分子机制目前仍不清楚.通过重组构建的质粒pET-32m-INSM1（424-497）表达的蛋白质（氨基酸424-497）包含了最后两个锌指结构域4和5,简称为ZF（4-5）.该文详细研究了蛋白质ZF（4-5）的诱导表达条件,得到了较高产率的纯化蛋白.核磁共振（NMR）谱和圆二色谱（CD）揭示了Zn²⁺对稳定锌指蛋白结构的必要性,以及C2H2-Zn²⁺结合的组氨酸呈现为δ-异构方式.     

以液体核磁共振波谱分析与帕金森氏病相关的I93M 突变对人类泛素碳端水解酶结构的影响

人类泛素碳端水解酶（UCH-L1）是涉及帕金森氏病并且在神经元高度表达的蛋白．UCH-L1 的家族性突变与转译后修饰会引起聚集倾向增加与去泛素活性损失,这二者都可能成为致病因素．作者所在实验室之前的研究指出与帕金森氏病相关的突变I93M 显著降低UCH-L1 的折叠稳定性并且加速其构型展开动力学．该研究使用液体核磁共振分析方法,包括侧链甲基化学位移,松弛骨干动力学和残余偶极耦合,以进一步阐明I93M 突变如何影响UCH-L1 的结构和动态．结果显示I93M 显著影响突变位点周围的疏水核心侧链构型．然而,这样的结构扰动并不会影响在纳秒时间尺度的快速骨干动力学．透过残余偶极耦合分析显示UCH-L1 在水溶液中的结构与之前报道的晶体结构有相当显著的偏离,另外I93M 突变也导致超出突变位点的远距离结构扰动．这一系列水溶液结构的分析结果可补充之前已知的晶体学数据,并对UCH-L1 在帕金森氏病相关的基因突变影响并提供详细的见解．     

二价金属离子与YycFN相互作用的NMR研究

YycGF最早发现于枯草芽孢杆菌,是与细胞存活密切相关的双组分信号转导系统存在于少量低鸟嘌呤和胞嘧啶(G+C)含量的革兰氏阳性菌中,包括金黄色葡萄球菌和肺炎链球菌等人类病原菌,在外界环境刺激下,胞膜上的组氨酸激酶YycG通过自身组氨酸磷酸化活化,将磷酸根转移至反应调节蛋白YycF的N端调节区(YycF_N)使之磷酸化,调控下游基因表达,实现特定细胞应答反应.二价金属离子在双组分信号转导系统反应调节蛋白的磷酸化过程中起着非常关键的作用,但它们与YycF_N相互作用的机制尚不清楚.该文利用液体核磁共振(NMR)方法研究了Ca~(2+)、Mg~(2+)两种离子与YycF_N的相互作用,对详细的相互作用界面进行了分析,并计算了Ca~(2+)、Mg~(2+)与YycF_N的解离常数(K_d).发现金属离子的关键作用位点是Asp9、Asp16和Asp53等残基,蛋白的整体构象也发生了一定变化,为阐明二价金属离子在反应调节蛋白信号转导过程中的作用机制提供了重要线索.     

HBx 诱发肝脏糖原代谢的昼夜节律性异常

在本文中,基于Hill 动力学与 Michaelis-Menten 方程,建立理论模型研究乙肝病毒x蛋白(HBx)诱发肝脏糖原代谢的昼夜节律性改变。理论模型考虑：HBx、组蛋白脱乙酰基酶1 (HDAC1) 与乙酰化的p53(p53AC) 结合形成复合体,并抑制 GYS2 表达;CLOCK 基因通过调控昼夜节律mRNA(Circadian mRNA)和频率蛋白(FRQ)的表达合成,调节 GYS2 磷酸化/去磷酸化的昼夜节律性。研究发现,在较低 HBx 浓度条件下,磷酸化的 GYS2 (pGYS2) 和去磷酸化的 GYS2 (dGYS2) 随时间演化,呈现了周期性的振荡特性。GYS2 通过磷酸化作用抑制其活性,通过去磷酸化,GYS2 被激活,这种磷酸化/去磷酸化转变保持了肝脏糖原代谢的昼夜节律性。在较高 HBx 浓度条件下,dGYS2 随时间演变的周期振荡节律性被改变,并且振荡幅度降低。由此表明,较高浓度的 HBx 则会在很大程度上改变 GYS2 去磷酸化的活性,GYS2 磷酸化/去磷酸化转变的昼夜节律性会被 HBx 破坏。另外,HBx 与 HDAC1、p53AC 形成复合体协同抑制 GYS2,也会在很大程度上改变 GYS2 磷酸化/去磷酸化转变的昼夜节律性。糖原代谢昼夜节律性的改变,导致肝脏内糖原代谢紊乱,进而促使肝癌(HCC)的发生发展。理论结果符合实验,并进一步揭示了 HBx 诱发肝脏糖原代谢紊乱,进而导致 HCC 的发生发展的一种致癌机理,可为设计阻断 HBV 向 HCC 转变通路的治疗方案提供理论依据。     

探测应答调控蛋白PhoBNF20D自由态中存在的Pre-Active构象

PhoB是PhoR/PhoB双组分信号转导系统（TCS）中的应答调控蛋白，来自大肠杆菌，可以参与环境中自由Pi的调控应答.PhoB拥有保守的磷酸化位点D53，当Mg²⁺存在时，会变成活性构象，但是其中的机理尚不清楚.目前已经有报道指出，自由态的应答调控蛋白存在少量的活性构象，而且它们和蛋白的激活过程相关，但是这种活性构象一般不稳定，很难观测到.本文以PhoB_N^F20D作为模型，¹H-¹⁵N HSQC和¹⁹F NMR谱图显示，自由态PhoB_N^F20D在溶液中存在pre-active构象，BeF₃^-可以稳定这种活性构象，而且Mg²⁺可以促进非活性构象到活性构象之间的转化，在BeF₃^-存在的情况下可以使PhoB_N^F20D形成完全的活性构象.同时我们利用Carr-Purcell-Meiboom-Gill（CPMG）弛豫弥散实验证明了PhoB_N^F20D存在两态交换.     

细菌反应调节蛋白RR468磷酸化和去磷酸化关键位点的NMR研究

反应调节蛋白是细菌双组分信号转导系统的重要组分,用于传递来自组氨酸激酶的信号并产生适应性反应.在整个信号转导过程中,反应调节蛋白的磷酸化和去磷酸化最终决定了该系统的信号输出和信号转导终止,因此其磷酸化和去磷酸化作用位点是控制其功能的关键要素．我们以来源于Thermotoga maritima中的反应调节蛋白RR468作为研究对象,将其分别位于loop b3-a3和loop b4-a4上的两个关键位点M55和K85进行突变,通过功能实验验证了这两个残基突变会对蛋白磷酸化和去磷酸化产生影响,并且利用液体核磁共振（NMR）手段对两个突变体的结构和动力学性质进行了研究．     

Raman spectroscopic study of G--A mismatches

G-A mismatches are non-canonical base pairs that widely occur in native nucleic acids. They have been found to be functionally important in adopting unusual structures. In this paper, G-A mispairing was studied by Raman spectral characterization of Polyadenylic acid （PolyA）, Polyguanylic acid （PolyG） and their equimolar mixture in solution of 0.08 mol/L Na^＋, pH7.0. The experiment showed the following three results. （1） At the experimental conditions used in the present work, PolyA and G A complexes existed as single-stranded and double-stranded helix of A form, respectively. And PolyG mainly occurred to be anti-parallel quadruplex conformers. （2） The formation of G A complexes, on one hand strengthened parts of base stacking interactions especially for PolyG, leading to Raman hypochromism effect with some corresponding bands shifting, and on the other hand weakened other base stacking interactions especially for PolyA to a certain degree. During this process, the backbone of PolyG underwent a significant change, but PolyA still conserved its main chain conformation. （3） The formation of G A complexes was stabilized by two interbase hydrogen- bond interactions （i.e. N6H2（A）-N3 （G） and N7 （A）--N2H2（G）） and a third hydrogen bond between O2^＋ （G） and N6 （A）. The third hydrogen bond was responsible for the remarkable changes of PolyG backbone conformation.[第一段]     

Computational investigations into new fluorescence quenching process induced by complexation of alkali metal ion

A novel fluorescent switchable chemosensor 1 , which is composed of an anthracene‐modified calix[4]crown in the 1,3‐alternate conformation, was calculated by density functional theory and time‐dependent density functional theory method. Geometries, molecular orbitals and binding thermal energies were evaluated at the restricted hybrid Becke's three‐parameter exchange functional using 6‐31 G(d) basis set and relativistic effective core potentials. The metal–ligand and cation–π interactions were investigated acting as two main types of driving force. Our calculations clearly show that solvent effects strongly influence cation selectivity, and K⁺ selectivity is recovered when even a few waters of hydration are considered. The calculations indicate that because of the photoinduced electron transfer effect, the addition of alkali metal ions have hardly any effect on the fluorescence of ligand 1 under neutral or basic conditions. Also, the high selectivity of ligand 1 for K⁺ and Rb⁺, under acidic conditions, the complexed metal ion can result in ammonium ion deprotonation, which leads to quenching of fluorescence of 1 ?H⁺. Copyright © 2012 John Wiley & Sons, Ltd.     

无规聚甲基丙烯酸甲酯立构复合的红外光谱研究

用红外光谱研究了无规聚甲基丙烯酸甲酯的丙酮、苯、氯仿溶液成膜样品的立构复合状况。结果表明无规聚甲基丙烯酸甲酯在丙酮和苯中确能形成立构复合结构,结构的形成主要是依靠分子链中间规和等规链段的相互作用。对丙酮样品的退火实验表明,在退火过程中能够发生间规链段的自聚集现象。     

Enthalpy difference between conformations of normal alkanes: effects of basis set and chain length on intramolecular basis set superposition error

The quantum chemistry of conformation equilibrium is a field where great accuracy (better than 100?cal?mol^?1) is needed because the energy difference between molecular conformers rarely exceeds 1000–3000?cal?mol^?1. The conformation equilibrium of straight-chain (normal) alkanes is of particular interest and importance for modern chemistry. In this paper, an extra error source for high-quality ab initio (first principles) and DFT calculations of the conformation equilibrium of normal alkanes, namely the intramolecular basis set superposition error (BSSE), is discussed. In contrast to out-of-plane vibrations in benzene molecules, diffuse functions on carbon and hydrogen atoms were found to greatly reduce the relative BSSE of n-alkanes. The corrections due to the intramolecular BSSE were found to be almost identical for the MP2, MP4, and CCSD(T) levels of theory. Their cancelation is expected when CCSD(T)/CBS (CBS, complete basis set) energies are evaluated by addition schemes. For larger normal alkanes (N?>?12), the magnitude of the BSSE correction was found to be up to three times larger than the relative stability of the conformer; in this case, the basis set superposition error led to a two orders of magnitude difference in conformer abundance. No error cancelation due to the basis set superposition was found. A comparison with amino acid, peptide, and protein data was provided.     

Many-electron effect in the angle resolved L3-M23M23 Auger-photoelectron coincidence spectroscopy (APECS) spectrum of metallic Zn

The many-body effect in the L₃-M₂₃M₂₃ Auger-electron spectroscopy (AES) spectrum of metallic Zn is discussed. The lifetime width and residual relaxation energy shift of the two M₂₃-hole state are governed by the (super) Coster-Kronig (sCK) transitions of two M₂₃-hole state. The residual relaxation energy shift and decay width of the two M₂₃-hole state are calculated in an average configuration by an ab initio atomic many-body theory. The agreement with experiment is good. To elucidate the many-body effect in the two-hole states, it is necessary to be able to discriminate individual components of the multiplet-split AES spectrum. We discuss how to discriminate individual components of the multiplet-split L₃-M₂₃M₂₃ AES spectrum of metallic Zn by angle-resolved Auger-photoelectron coincidence spectroscopy (AR-APECS) in order to determine accurately their line shapes, multiplet splitting energies, and spin states (singlet etc.).     

On the discrimination of bound and free sodium ions in solutions of biomolecules by means of the T1 relaxation.

The interactions of charged macromolecules with counterions have been extensively examined previously by ²³Na NMR, leading to the model that the counterions can be classified as beeing both “free” and “bound” in solution. We will demonstrate that the two dimensional time evolution approach for resolving a composite “free-induction-decay” opens the possibilities to measure both the T₁ of the free sodium ion and the T₁ of the sodium ion bound to a macromolecule simultaneously. A lot of various compounds as DNA and Heparin serve as models to demonstrate the usefulness of this new method.     

Mechanical properties and the evolution of matrix molecules in PTFE upon irradiation with MeV alpha particles

The morphology, chemical composition, and mechanical properties in the surface region of α-irradiated polytetrafluoroethylene (PTFE) have been examined and compared to unirradiated specimens. Samples were irradiated with 5.5 MeV ⁴He²⁺ ions from a tandem accelerator to doses between 1 × 10⁶ and 5 × 10¹⁰ Rad. Static time-of-flight secondary ion mass spectrometry (ToF-SIMS), using a 20 keV C₆₀⁺ source, was employed to probe chemical changes as a function of α dose. Chemical images and high resolution spectra were collected and analyzed to reveal the effects of α particle radiation on the chemical structure. Residual gas analysis (RGA) was utilized to monitor the evolution of volatile species during vacuum irradiation of the samples. Scanning electron microscopy (SEM) was used to observe the morphological variation of samples with increasing α particle dose, and nanoindentation was engaged to determine the hardness and elastic modulus as a function of α dose.The data show that PTFE nominally retains its innate chemical structure and morphology at α doses <10⁹ Rad. At α doses ≥10⁹ Rad the polymer matrix experiences increased chemical degradation and morphological roughening which are accompanied by increased hardness and declining elasticity. At α doses >10¹⁰ Rad the polymer matrix suffers severe chemical degradation and material loss. Chemical degradation is observed in ToF-SIMS by detection of ions that are indicative of fragmentation, unsaturation, and functionalization of molecules in the PTFE matrix. The mass spectra also expose the subtle trends of crosslinking within the α-irradiated polymer matrix. ToF-SIMS images support the assertion that chemical degradation is the result of α particle irradiation and show morphological roughening of the sample with increased α dose. High resolution SEM images more clearly illustrate the morphological roughening and the mass loss that accompanies high doses of α particles. RGA confirms the supposition that the outcome of chemical degradation in the PTFE matrix with continuing irradiation is evolution of volatile species resulting in morphological roughening and mass loss. Finally, we reveal and discuss relationships between chemical structure and mechanical properties such as hardness and elastic modulus.     

锌基-有机金属大环荧光探针对谷胱甘肽生物分子的识别

利用1H NMR,ESI-MS,UV-Vis,荧光光谱等测试手段研究了基于锌基-有机金属三元大环探针M-1对生物分子谷胱甘肽(GSH)的识别与传感。并且,通过研究识别过程中M-1与组成谷胱甘肽的氨基酸(半胱氨酸、谷氨酸、甘氨酸)的作用关系,确立了M-1对GSH的识别机理。结果表明,化合物M-1在H₂O/DMF(1∶9, φ)溶液中形成了稳定的[3+3]大环结构;紫外滴定光谱表明,向M-1中加入GSH后303 nm处吸收峰强度增加,380 nm处吸收峰强度减弱,在330 nm处出现了一个等吸收点,紫外滴定和ESI-MS质谱证实了M-1能够1∶1包合GSH,平衡常数(log K_GSH)为4.62±0.15。1H NMR表明谷胱甘肽在M-1中的构型为组成谷胱甘肽的谷氨酸通过羧基与金属中心之间的静电作用深深地进入M-1空穴内部。荧光光谱表明,向M-1中加入GSH时,以330 nm的光激发,发射波长从510 nm红移至540nm,荧光强度增加1倍;加入半胱氨酸、谷氨酸时,荧光强度分别增加0.4倍和0.2倍;而加入甘氨酸时,荧光没有变化。综合上述结果证明了M-1空穴的限域作用及其底部三元环上的氨基和GSH上的巯基(半胱氨酸)间的氢键作用使M-1的电子构型发生转变,进而引起紫外光谱和荧光光谱发生变化,实现了大环化合物M-1对生物分子谷胱甘肽的可视化、高灵敏度检测,检测限达到3.0×10-6 mol·L-1。     

Isotactic poly(p-fluorostyrene): Conformation and molecular packing analysis

The chain conformation and molecular packing of isotactic poly(p-fluorostyrene) have been examined using calculations made with semiempirical potential energy functions. Isolated chain conformational energies indicate no difference in conformation for the fluoropolymer from the conformation for isotactic polystyrene. The energy for packing poly(p-fluorostyrene) chains into a crystalline array as 3₁ or 4₁ helices was also compared with the energies for packing polystyrene in both of these helical forms. While not being the lowest energy mode of packing for poly(p-fluorostyrene), the packing of 4₁-helices does yield a local energy minimum. Such packing of 4₁ helical polystrene chains is considerably less energetically feasible. The results indicate the causes for the experimentally observed difference in the crystalline conformations of the two isotactic polymers as being due to intermolecular influences.     

The phonon-mediated intervalley electron-electron interaction in silicon

A tight-binding formulation is used to estimate both the electron-phonon matrix element and the intervalley phonon-mediated exchange interaction in bulk silicon. The resulting interactions are about an order of magnitude too small to be able to account, of their own, for the broken symmetry states observed in inversion layers at the Si/SiO₂ interface, where the selection rules are relaxed and other modes are available to enhance the interactions.