盐酸胍诱导的变性卵清溶菌酶分子重折叠过程及其各稳定构象态的分布和过渡

采用变性和非变性电泳、 高效凝胶排阻色谱、 内源荧光发射光谱和荧光相图以及生物活性测定等方法, 研究了盐酸胍诱导的变性卵清溶菌酶分子的重折叠过程及此过程中卵清溶菌酶分子各稳定构象态的分布和过渡. 结果表明, 当复性液中盐酸胍浓度分别约为5.0和2.4 mol/L时, 变性卵清溶菌酶分子的重折叠过程各存在1个稳定折叠中间态, 重折叠过程符合"四态模型". 在卵清溶菌酶分子四态重折叠过程基础上, 结合盐酸胍与卵清溶菌酶分子之间的缔合-解离平衡, 给出了一个定量描述变性剂诱导的蛋白质分子复性过程中蛋白质分子复性率随溶液中变性剂浓度变化的方程. 该方程包含2个特征折叠参数, 一个是蛋白质分子从一个稳定构象态过渡到另一个稳定构象态的热力学过渡平衡常数k; 另一个是在此过程中平均每个蛋白质分子所结合的变性剂分子数目m. 通过这2个特征折叠参数能够定量描述盐酸胍诱导的变性卵清溶菌酶完全去折叠态、 折叠中间态和天然态分子随复性液中盐酸胍浓度变化的分布和过渡情况.     

不同介质中大肠杆菌碱性磷酸酶构象变化的研究

利用色氨酸残基作为内源荧光探针的膜模拟剂（各种表面活性剂）和不同变性剂中对大肠杆菌碱性磷酸酶（ＡＰ）的构象变化进行了系统的研究。通过测定在不同变性时间下盐酸胍浓度对荧光强度的影响以及荧光强度随pＨ有规律的变化,进一步证实了该蛋白质变性过程中形成较稳定中间态的结论。     

多光谱法研究溶菌酶不对称变复性机理

利用紫外-可见光谱法、荧光相图法、荧光偏振法以及共振瑞利散射法多光谱技术对溶菌酶(Lysozyme)的变复性特征进行表征,分析溶菌酶变复性机理,构建其变性动力学模型.结果表明,变性过程溶菌酶分子体积变大,结构松弛,变性速度快,不存在中间态,符合"二态模型";复性过程变性溶菌酶分子体积变小,结构紧缩,复性过程慢,且存在中间体,符合"多态模型",说明了溶菌酶的复性过程比其变性过程复杂.实验定量研究了不同变复性条件下,溶菌酶不对称变复性机理,为溶菌酶分子结构分析的研究提供了参考.     

光谱法研究尿素对水溶液中血红蛋白构象的影响

应用荧光猝灭法和动态光散射法测定尿素-水混合溶剂中血红蛋白(Hb)与联苯胺的结合距离和Hb的流体动力学半径. 结合Hb的荧光光谱和吸收光谱, 探讨尿素与蛋白质分子在水溶液中相互作用的机理及其对蛋白质构象的影响. 结果显示, 尿素分子取代水分子在蛋白质周围形成溶剂化层, 并与骨架肽链和亲水侧链形成氢键, 从而积聚在蛋白质分子表面. 尿素分子与蛋白质分子之间的直接相互作用对蛋白质的构象具有复杂的影响, 高浓度的尿素-水混合溶剂破坏蛋白质的构象, 而低浓度的混合溶剂则有利于蛋白质形成更紧密的构象. 在高浓度的尿素-水混合溶剂中, Hb血红素疏水空穴失去原有的三级结构后形成一个与熔球态相类似的结构.     

盐酸胍诱导的淀粉液化芽孢杆菌α-淀粉酶去折叠过程的研究

分别用内源荧光光谱法、荧光相图法、荧光探针法、荧光猝灭法、蛋白质电泳法以及体积排阻色谱法研究了盐酸胍诱导的淀粉液化芽孢杆菌a-淀粉酶的去折叠过程. 内源荧光光谱和荧光相图结果表明, 当变性液中盐酸胍浓度约为1.0 mol/L时, 芽孢杆菌a-淀粉酶的去折叠过程中出现一个部分折叠中间体, 其去折叠过程符合“三态模型”; 荧光探针结果表明, 在溶液中盐酸胍浓度约为1.0 mol/L时, 中间态芽孢杆菌a-淀粉酶分子中存在着能够与探针分子1-苯胺 基-8-萘磺酸(ANS)结合的稳定的疏水区域; 荧光猝灭研究给出了不同程度变性的淀粉液化芽孢杆菌a-淀粉酶中的Trp的分布情况, 结果表明中间态芽孢杆菌a-淀粉酶分子中能够被碘化钾猝灭的位于分子表面的色氨酸残基数目达到最大的8个; 蛋白电泳和体积排阻色谱结果表明, 在盐酸胍诱导的芽孢杆菌a-淀粉酶分子的整个去折叠过程中, 不会以共价键或非共价键形式形成芽孢杆菌a-淀粉酶分子之间的集聚体或集聚体沉淀. 在此基础上, 对盐酸胍诱导的淀粉液化芽孢杆菌a-淀粉酶的去折叠过程进行了描述.     

用尺寸排阻色谱法研究蛋白质的热稳定性

用尺寸排阻色谱ＺｏｒｂａｘＧＦ－２５０凝胶过滤柱研究了核糖核酸酶Ａ,马心肌红蛋白和溶菌酶的热稳定性。测定了蛋白质在尺寸排阻系统中的分配系数Ｋｄ并用荧光检测器分析了加热引起的蛋白质内源性荧光的改变,从而计算出了蛋白质的热变性转化点温度,测定了蛋白质天然态、热变性和盐酸胍诱导变性状态下的斯托期斯半径,实验结果进一步证帝了热变性后蛋白质不是呈真正的随机线状,而是保留了部分紧密的结构区域。用本方法研究了Ｐ     

由激光诱导荧光确定对称陀螺分子定向和取向的理论研究

采用密度矩阵方法推导了从激光诱导荧光强度中抽出对称陀螺分子定向和取向参数的一般表达式. 分子定向和取向由分子态多极矩描述. 激发激光和探测荧光分别为圆偏振和线偏振光. 在一般情况下, 激光诱导荧光强度是初始分子态多极矩、动力学因子和激发-探测几何因子的复杂函数. 它包含一个布居(population)、10个定向(orientation)和14个取向(alignment)态多极矩. 讨论了如何从分辨荧光强度中抽出所有初始分子态多极矩问题.     

由激光诱导荧光方法探测光碎片分子取向的理论研究

采用密度矩阵方法,推导了从激光诱导荧光(LIF)强度中抽出光碎片取向参数的表达式.光碎片的取向由分子态多极矩描述.用于解离母分子和激发碎片分子的激光均为线偏振光,而探测荧光为非偏振光.激光诱导荧光强度是光碎片分子初始态多极矩、线强度因子和解离-激发几何因子的函数.光碎片的取向参数可以由测量荧光偏振比和计算动力学因子而获得.     

单分子荧光共振能量转移用于生物大分子构象动态变化过程研究进展

生物大分子参与生命活动的各个过程,在单分子水平上实时观测和分析生物大分子自身的结构动态以及生物大分子相互作用的动态过程,对于深入理解生物大分子的作用机制具有重要意义。自提出以来,单分子荧光共振能量转移技术逐渐展现出其在研究生物大分子构象变化和相互作用过程等方面的巨大潜力,一系列新的作用机理陆续被提出。本文对单分子荧光共振能量转移技术在蛋白质与核酸分子构象动态变化、蛋白质-蛋白质相互作用以及蛋白质-核酸相互作用等方面取得的研究进展进行了综述。     

脲或盐酸胍溶液中变性溶菌酶复性过程中集聚体的研究

建立在蛋白质变性-复性三态模型的基础上, 给出了一个描述在变性液中变性蛋白质复性时蛋白质浓度和其复性率的关系式. 通过这个关系式, 可以获得两个重要的描述蛋白质变性-复性体系特征的参数, 一个是包含在一个集聚体分子中的变性蛋白质的分子数目n, 另一个是蛋白质从原始态到形成集聚体过程中的表观集聚平衡常数K. 以三种溶菌酶在脲和盐酸胍溶液中的变性-复性过程对此方程进行了验证, 结果表明所给出的方程能够很好地描述三种溶菌酶在这两种变性液中的复性结果, 三种溶菌酶在两种变性液中有形成二分子集聚体的趋势. 变性溶菌酶在复性过程中的电泳和高效凝胶排阻色谱也同时能够监测到复性过程中集聚体的形成, 并且监测结果与上述方程所得的结果一致.     

Quantitative determination of the single-molecule detection regime in fluorescence fluctuation microscopy by means of photon counting histogram analysis

Fluorescence fluctuation experiments are performed in single-molecule detection regime if the fluorescence of at most one molecule is registered at a time. Although the significance of such experiments for investigations of complex nonergodic systems like those met in the biosciences has been stressed out by many scientists, the quantitative and accurate determination of the single-molecule detection regime received rather little attention. In this work we present a method based on the photon counting histogram (PCH) analysis, which enables the determination of the average number N of molecules within the observation volume, for which only the fluorescence of individual molecules is detected at a time. Thus, the accurate design of fluorescence fluctuation experiments performed in single-molecule detection regime is possible. Demonstrative fluorescence fluctuation experiments based on two-photon excitation are performed on diluted solutions of coumarin 153, in order to verify the potential of the PCH analysis in experiments on the single-molecule detection level. If the mean number N of molecules within the excitation volume is larger than 0.048, the probability to simultaneously detect the fluorescence of two or more molecules is no longer negligible, i.e., no single-molecule detection regime. If the mean number N of molecules is lower than 0.0057, the detection limit of the method is reached, i.e., the fluorescence signal cannot be distinguished from the background. Consequently, the concentration of coumarin 153 characteristic for the single-molecule detection regime lies in the range 13-110 pmol/l for the given experimental conditions. We also investigate the influence of the molecular brightness, i.e., detected photons per fluorophore molecule and sampling time, on the single-molecule detection regime.     

Single-molecule fluorescence spectroelectrochemistry of cresyl violet

Here we report a new path to study single molecule electron transfer dynamics by coupling scanning fluorescence microscopy with a potentiostat via a conventional electrochemical cell to enable single-molecule fluorescence spectroelectrochemistry of cresyl violet in aqueous solution, demonstrating that the single-molecule fluorescence intensity of cresyl violet is modulated synchronously with the cyclic voltammetric potential scanning.     

应用全内反射单分子荧光成像研究蛋白复合物亚基组成

细胞的生化过程大都是由蛋白复合物完成的,研究蛋白复合物亚基的组成对于了解蛋白质的结构和生物学功能具有重要的意义,然而如何准确确定蛋白复合物中蛋白质亚基的数量（stoichiometry）仍然是一个挑战.近年来,活细胞体系单分子荧光成像技术的不断发展为原位实时动态地研究蛋白质的结构和性质提供了新的手段.本文主要介绍了应用活细胞全内反射单分子荧光成像技术表征细胞膜区蛋白复合物组成的3种方法,包括单分子漂白步数分析、荧光强度统计分布以及蛋白运动分析,并结合其基本原理介绍了这几种方法在活细胞体系膜蛋白研究中的应用.     

Metal-enhanced fluorescence from CdTe nanocrystals: a single-molecule fluorescence study

We described, for the first time, the metal-enhanced fluorescence from the CdTe nanocrystals spin coated on silver island films (SIFs). CdTe nanocrystals show approximately 5-fold increase in fluorescence intensity, 3-fold decrease in lifetimes, and reduction in blinking on SIF surfaces that can be observed by ensemble and single-molecule fluorescence studies. The single-molecule study also provides further insight on the heterogeneity in the fluorescence enhancement and lifetimes of the CdTe nanocrystals on both glass and SIF surfaces, which is otherwise not possible to observe using ensemble measurements.     

Using single-molecule fluorescence spectroscopy to study electron transfer.

Techniques in single-molecule fluorescence spectroscopy now allow sophisticated studies of photophysical processes in single molecules. As interest grows in the possibilities of molecular electronics, researchers have begun to turn these techniques to the study of electron transfer. Electron-transfer reactions have now been detected and measured at the single-molecule level in a variety of systems and on a variety of timescales by adapting techniques from previous single-molecule fluorescence studies.     

Single-molecule interfacial electron transfer dynamics manipulated by an external electric current

Interfacial electron transfer (IET) dynamics in a 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine (DiD) dye molecule/indium tin oxide (ITO) film system have been probed at the ensemble and single-molecule levels. By comparing the difference in the external electric current (EEC) dependence of the fluorescence intensities and lifetimes of the ensembles and single molecules, it is shown that the single-molecule probe can effectively demonstrate IET dynamics. The backward electron transfer and electron transfer from the ground state induce single-molecule fluorescence quenching when an EEC is applied to the DiD/ITO film system.     

The oxidation state of a protein observed molecule-by-molecule.

We report the observation of the redox state of the blue copper protein azurin on the single-molecule level. The fluorescence of a small fluorophore attached to the protein is modulated by the change in absorption of the copper center via fluorescence resonance energy transfer (FRET). In our model system, the fluorescence label Cy5 was coupled to azurin from Pseudomonas aeruginosa via cysteine K27C. The Cy5 fluorescence was partially quenched by the absorption of the copper center of azurin in its oxidized state. In the reduced state, absorption is negligible, and thus no quenching occurs. We report on single-molecule measurements, both in solution by using fluorescence correlation spectroscopy (FCS) combined with fluorescence intensity distribution analysis (FIDA), and on surfaces by using wide-field fluorescence microscopy.     

细胞膜上信号转导蛋白的单分子成像与分析

结合本课题组的研究工作, 介绍了单分子荧光成像原理、 荧光标记方法及数据分析方法, 并进一步综述了单分子荧光成像在几种重要的膜蛋白信号转导分子机制和相关药物研究中的进展.     

Single-molecule fluorescence imaging of nanocatalytic processes

This tutorial review covers recent developments in using single-molecule fluorescence microscopy to study nanoscale catalysis. The single-molecule approach enables following catalytic and electrocatalytic reactions on nanocatalysts, including metal nanoparticles and carbon nanotubes, at single-reaction temporal resolution and nanometer spatial precision. Real-time, in situ, multiplexed measurements are readily achievable under ambient solution conditions. These studies provide unprecedented insights into catalytic mechanism, reactivity, selectivity, and dynamics in spite of the inhomogeneity and temporal variations of catalyst structures. Prospects, generality, and limitations of the single-molecule fluorescence approach for studying nanocatalysis are also discussed.