单个蛋白分子的测定及配体与受体在细胞表面结合和运动的研究
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摘要
本文第一章对单分子检测(SMD)的意义及常用的单分子检测技术作了简单介绍。SMD技术在生命科学上的应用可以在单分子水平上揭示分子的动态行为、动力学过程和机制,并发现大量分子集合体中分子个体的性质和行为。这一章中我们对在生物大分子SMD中应用较为广泛的技术:全内反射荧光显微术(TIRFM)的原理等进行了介绍。对TIRFM用于分子马达的运动、分子间相互作用、生物大分子的构象变化等离体生物单分子探测作了详细的综述。对TIRFM用于细胞信号转导、细胞趋化性、离子通道等在体生物单分子探测作了详细的综述。
第二章中我们建立了一种新的、灵敏的全内反射荧光显微术(TIRFM)的基于蛋白与玻片之间达到吸附平衡的单分子检测(SMD)定量方法。在我们的工作之前文献中用TIRFM检测了罗丹明6G分子和罗丹明6G标记的DNA分子,检测限仅2.5×10~(-9)mol/L。我们用Alexa Fluor 488标记的羊抗鼠IgG(H+L)(Alexa Fluor 488 goat anti-rat IgG(H+L),IgG(H+L)-488)作为检测蛋白。首先将IgG(H+L)-488在盖玻片上孵育一定时间。IgG(H+L)-488在溶液和玻片之间达到吸附平衡后,使用安装有电子多级放大电感偶合器(electronmultiplying charge coupled device,EMCCD)的TIRFM拍摄IgG(H+L)-488的荧光图像。图像中荧光点的数目为隐失场中IgG(H+L)-488分子吸附在盖玻片上的数目和溶液中的数目的总和。当溶液的浓度在5.4×10~(11)～8.1×10~(-10)mol/L之间时,图像中荧光点的数目与溶液中的IgG(H+L)-488浓度有良好的线性关系。线性范围的下限比文献报道的低了46倍。此部分内容已发表在Anal.Chim.Acta杂志上。
第三章中我们在单分子水平对转铁蛋白与细胞膜上的转铁蛋白受体介导的转运过程早期阶段进行了研究。转铁蛋白-转铁蛋白受体介导的转运过程是生物体细胞最具特点的转运过程之一。转铁蛋白-转铁蛋白受体介导的转运过程的早期阶段包括质膜上转铁蛋白与转铁蛋白受体复合物(TfR-Tf)的运动、聚集以及内涵体的形成等,这些现象曾用形态学、生物化学等方法进行研究,但大多将大量细胞溶解后通过直接测量放射性标记物或荧光标记物的强度进行判断。由于受大量细胞平均化检测方法的限制,无法动态观察这些过程。在本章中我们以单分子检测方法中灵敏度高、对样品损伤小的细胞内TIRFM为主要检测方式,对下列过程进行了实时动态的观察。
1.实时动态观察了溶液中Tf-QD被质膜上TfR捕获的过程。
2.在实时追踪质膜上TfR-Tf的聚集过程时,观察到了质膜上TfR-Tf通过扩散运动与质膜上另一个已形成的TfR-Tf复合体聚集的过程。
3.在4℃下和37℃下通过观测荧光点的运动来分析细胞膜上的TfR-Tf复合物及其侧向运动,观测到人的胃癌细胞膜上的TfR在4℃和37℃时扩散的方式有自由扩散和受限扩散形式。并且通过荧光点的连续移动对应于照片中的像素坐标分别计算出了两个温度下TfR-Tf复合体的瞬时扩散系数。
上述在单分子水平上的研究结果,目前均未见文献报道。
在第四章中我们在α_1-肾上腺素受体的拮抗剂—药物小分子哌唑嗪(prazosin)和α_1-肾上腺素受体的激动剂—去氧肾上腺素(phenylephrine)键合上生物素(biotin)。利用生物素与链酶亲和素(streptavidin)之间极强的亲和力将链酶亲和素包被的量子点(QD-streptavidin)标记到哌唑嗪和去氧肾上腺素上,并用于观察此两药物小分子与活细胞表面α_(1A)-肾上腺素受体结合的情况。由于量子点独特的优良光学性质,近年来用量子点标记药物小分子及用于细胞成像已有报道。然而,他们利用多步化学键合反应将药物小分子连接到量子点上的方法存在反应产率低及分离提纯等问题。而且用这种方法很难控制连接到量子点上药物小分子的数目。另一种思路是首先将生物素连接到药物小分子(生物素化药物小分子),这些生物素化药物小分子然后与QD-streptavidin反应将生物素化药物小分子结合到量子点上。文献也报道了用这种思路将多巴胺转运蛋白的拮抗剂结合到量子点上,但未用于细胞的荧光成像。根据这种背景我们进行了下列的研究工作:
1.用与文献完全不同的合成路线将生物素与药物小分子哌唑嗪和去氧肾上腺素键合在一起,再与QD-streptavidin反应将量子点标记到哌唑嗪和去氧肾上腺素。这种标记量子点的方法克服了文献中化学键合反应标记法存在的反应产率低及分离提纯等问题,而且还可以控制每个量子点上连接到量子点上药物小分子的数目,因为每个量子点上包被的链酶亲和素的量是一定的。由于QD-streptavidin已有商品,所以我们这种标记量子点的方法十分方便,有利于在生命科学的研究中应用。研究结果证明用这种方法标记量子点的哌唑嗪和去氧肾上腺素仍具有药理活性,可用于细胞的荧光成像。
2.研究了去氧肾上腺素与生物素之间连接不同链长的化合物时去氧肾上腺素与细胞表面α_(1A)-肾上腺素受体结合的情况。发现在生物素与去氧肾上腺素之间的链越长,越易于与细胞表面α_(1A)-肾上腺素受体结合,反映了细胞表面受体-配体结合时可能存在空间位阻的影响。
3.用标记量子点的哌唑嗪和去氧肾上腺素观察了哌唑嗪和去氧肾上腺素在表达α_(1A)-肾上腺素受体的HEK293A细胞表面与受体结合与定位的情况,并且用几种方法证明了哌唑嗪和去氧肾上腺素与α_(1A)-肾上腺素受体结合的特异性。还观测到了激动剂去氧肾上腺素和拮抗剂哌唑嗪与α_(1A)-肾上腺素受体结合作用的差异。
In chapter one,significance of single molecule detection(SMD)and techniques were reviewed briefly.These techniques were powerful tools for investigation of dynamics and kinetics of molecules.New information can be obtained from the single molecule research,which is otherwise hidden or averaged out.Single molecule detection is a technology of studying biomolecules with high spatial and temporal resolution.Fluorescence microscopy was rapidly expanding from single molecule detection techniques into all fields of cell and molecular biology.Total internal reflection fluorescence microscopy(TIRFM)was introduced.An intimate summary of their applications was also provided with single-molecule analysis in vitro and single-molecule analysis in vivo.
In chapter two,we developed a sensitive single-molecule imaging method for quantification of protein by total internal reflection fluorescence microscopy(TIRFM) with adsorption equilibrium.Rhodamine 6G(RtG)and DNA molecules labeled by R6G had been detected by TIRFM in literature.The limit of detection(LOD)was only 2.5×10~(-9)mol/L.We choosed Alexa Fluor 488-labeled goat anti-rat IgG(H+L) (IgG(H+L)-488)as the model protein.First,the solution of IgG(H+L)-488 was incubated with a glass microscope coverslip for a certain time.In this case,the adsorption equilibrium of protein was achieved between the solution and the coverslip.Then,fluorescence images of protein molecules in an evanescent wave field were taken by a highly sensitive electron multiplying charge coupled device. Finally,the number of fluorescent spots corresponding to the protein molecules in the images was counted.The spot number showed an excellent linear relationship with protein concentration.The concentration linear range was 5.4×10~(-11)to 8.1×10~(-10) mol/L.The low end of the linear relationship was 46-fold lower than reported in literature.
In chapter three,the total internal reflection fluorescence microscopy combined with Epi-FM was used to study the early events of Transferrin and Transferrin Receptor-mediated transcytosis in living cells at single molecule level.The early stages of Transferrin and Transferrin Receptor processes included moving, congregation and internalization of Transferrin-Transferrin Receptor complexes, which have already been studied by morphological methods and biochemical methods. But most researches made judgement by the direct measurement of radioactive marker or the stability of fluorescent labelling marker after a large number of cytolysis.Due to the limitation of equation measuring method,these processes could not be observed dynamically.We has carried out dynamic study on the early events of Transferrin and Transferrin Receptor-mediated transcytosis at single molecule level, and the moving and congregation of Transferrin and Transferrin Receptor complexes in the real time.And there were no reports about real time tracking until now.These early processes using single molecule fluorescence detection were as follow.
1.The process that Transferrin-QD(Tf-QD)in the solution bound to its receptor (TfR)on the cell membrane to produce a complex(TfR-Tf)was visualized in real time.
2.The lateral moving and the congregation of TfR-Tf complexes on the plasma membrane were traced at single molecule level.Then,the congregation of two TfR-Tf complexes happened.
3.At the temperature of 4℃and 37℃,the analysis of TfR-Tf complexes and the lateral moving on the plasma membrane were observed through surveying the movement of fluorescence spots.On the previous condition,the suffused modes of TfR on living MGC832 cell were free diffusion mode and restricted diffusion mode. Consequently,the transient diffusion coefficients of TfR-Tf complexes were calculated respectively by pixel coordinates correspond to sequential movements of fluorescence spots in pictures.
In chapter four,we report the incorporation of biotin onto small drug molecules, the antagonist prazosin,and the agonist phenylephrine,through a series of reactions. Prazosin and phenylephrine were respectively labeled with quantum dot (QD)-streptavidins through the strong affinity between biotin and streptavidin to observe the binding between small drug molecules andα_(1A)-AR.Because of the unique optical properties of QD,more and more studies have been reported now concerning such fluorescent QD-marked drug molecules and application in fluorescence imaging of cells.However,most of such processes included complicated modifications on drug molecules and therefore gave rise to the problems of yield and separation. Moreover,the attached amount of drug molecules to QD is difficult to control. Another method is to biotinylate small drug molecules and then to bind them to QD-streptavidin.Though there is now report that the antagonist of dopamine transporter can bind to QD in such way,application in fluorescence imaging on living cell has not been seen.Motivated by these considerations,we have performed investigations as follows:
1.Using a novel synthetic clue,we have attached biotin to prazosin and phenylephrine molecules.Then,QD-streptavidins were introduced to bind prazosin and phenylephrine specifically and effectively.In such way,the problems of yield and separation usually met in covalent binding were avoided.Also,the amount of small molecules attached to QD became much controllable because the number of streptavidin coated on QD was invariable.Because QD-streptavidin is available commercially,it is convenient to apply to the life science studies.Obtained results indicated that the QD-labeled prazosin and phenylephrine molecules still had pharmacological activity.That is,QD-streptavidin could work effectively as fluorescent probe in imaging of living cell.
2.Comparisons are made on the binding abilities respectively betweenα_(1A)-AR on the cell surface and four biotinylated phenylephrines with different chain lengths. It is found that the phenylephrine with longer chain between biotin and phenylephrine can bind toα_(1A)-AR on the cell surface more easily.Correspondingly,more fluorescent spots would appear under the same condition.This reflected the possible steric hindrance on the binding ability between acceptor on the cell surface and ligands.
3.The phenylephrine-QD and prazosin-QD were used to observe their binding and anchoring status with the acceptor on the cell surface ofα_(1A)-AR HEK293A. Several methods have adopted to support the binding specificity of prazosin-QD and phenylephrine-QD toα_(1A)-AR respectively.Furthermore,we also observed the function difference when agonist-phenylephrine and antagonist-prazosin were respectively bound toα_(1A)-AR.
第二章中我们建立了一种新的、灵敏的全内反射荧光显微术(TIRFM)的基于蛋白与玻片之间达到吸附平衡的单分子检测(SMD)定量方法。在我们的工作之前文献中用TIRFM检测了罗丹明6G分子和罗丹明6G标记的DNA分子,检测限仅2.5×10~(-9)mol/L。我们用Alexa Fluor 488标记的羊抗鼠IgG(H+L)(Alexa Fluor 488 goat anti-rat IgG(H+L),IgG(H+L)-488)作为检测蛋白。首先将IgG(H+L)-488在盖玻片上孵育一定时间。IgG(H+L)-488在溶液和玻片之间达到吸附平衡后,使用安装有电子多级放大电感偶合器(electronmultiplying charge coupled device,EMCCD)的TIRFM拍摄IgG(H+L)-488的荧光图像。图像中荧光点的数目为隐失场中IgG(H+L)-488分子吸附在盖玻片上的数目和溶液中的数目的总和。当溶液的浓度在5.4×10~(11)～8.1×10~(-10)mol/L之间时,图像中荧光点的数目与溶液中的IgG(H+L)-488浓度有良好的线性关系。线性范围的下限比文献报道的低了46倍。此部分内容已发表在Anal.Chim.Acta杂志上。
第三章中我们在单分子水平对转铁蛋白与细胞膜上的转铁蛋白受体介导的转运过程早期阶段进行了研究。转铁蛋白-转铁蛋白受体介导的转运过程是生物体细胞最具特点的转运过程之一。转铁蛋白-转铁蛋白受体介导的转运过程的早期阶段包括质膜上转铁蛋白与转铁蛋白受体复合物(TfR-Tf)的运动、聚集以及内涵体的形成等,这些现象曾用形态学、生物化学等方法进行研究,但大多将大量细胞溶解后通过直接测量放射性标记物或荧光标记物的强度进行判断。由于受大量细胞平均化检测方法的限制,无法动态观察这些过程。在本章中我们以单分子检测方法中灵敏度高、对样品损伤小的细胞内TIRFM为主要检测方式,对下列过程进行了实时动态的观察。
1.实时动态观察了溶液中Tf-QD被质膜上TfR捕获的过程。
2.在实时追踪质膜上TfR-Tf的聚集过程时,观察到了质膜上TfR-Tf通过扩散运动与质膜上另一个已形成的TfR-Tf复合体聚集的过程。
3.在4℃下和37℃下通过观测荧光点的运动来分析细胞膜上的TfR-Tf复合物及其侧向运动,观测到人的胃癌细胞膜上的TfR在4℃和37℃时扩散的方式有自由扩散和受限扩散形式。并且通过荧光点的连续移动对应于照片中的像素坐标分别计算出了两个温度下TfR-Tf复合体的瞬时扩散系数。
上述在单分子水平上的研究结果,目前均未见文献报道。
在第四章中我们在α_1-肾上腺素受体的拮抗剂—药物小分子哌唑嗪(prazosin)和α_1-肾上腺素受体的激动剂—去氧肾上腺素(phenylephrine)键合上生物素(biotin)。利用生物素与链酶亲和素(streptavidin)之间极强的亲和力将链酶亲和素包被的量子点(QD-streptavidin)标记到哌唑嗪和去氧肾上腺素上,并用于观察此两药物小分子与活细胞表面α_(1A)-肾上腺素受体结合的情况。由于量子点独特的优良光学性质,近年来用量子点标记药物小分子及用于细胞成像已有报道。然而,他们利用多步化学键合反应将药物小分子连接到量子点上的方法存在反应产率低及分离提纯等问题。而且用这种方法很难控制连接到量子点上药物小分子的数目。另一种思路是首先将生物素连接到药物小分子(生物素化药物小分子),这些生物素化药物小分子然后与QD-streptavidin反应将生物素化药物小分子结合到量子点上。文献也报道了用这种思路将多巴胺转运蛋白的拮抗剂结合到量子点上,但未用于细胞的荧光成像。根据这种背景我们进行了下列的研究工作:
1.用与文献完全不同的合成路线将生物素与药物小分子哌唑嗪和去氧肾上腺素键合在一起,再与QD-streptavidin反应将量子点标记到哌唑嗪和去氧肾上腺素。这种标记量子点的方法克服了文献中化学键合反应标记法存在的反应产率低及分离提纯等问题,而且还可以控制每个量子点上连接到量子点上药物小分子的数目,因为每个量子点上包被的链酶亲和素的量是一定的。由于QD-streptavidin已有商品,所以我们这种标记量子点的方法十分方便,有利于在生命科学的研究中应用。研究结果证明用这种方法标记量子点的哌唑嗪和去氧肾上腺素仍具有药理活性,可用于细胞的荧光成像。
2.研究了去氧肾上腺素与生物素之间连接不同链长的化合物时去氧肾上腺素与细胞表面α_(1A)-肾上腺素受体结合的情况。发现在生物素与去氧肾上腺素之间的链越长,越易于与细胞表面α_(1A)-肾上腺素受体结合,反映了细胞表面受体-配体结合时可能存在空间位阻的影响。
3.用标记量子点的哌唑嗪和去氧肾上腺素观察了哌唑嗪和去氧肾上腺素在表达α_(1A)-肾上腺素受体的HEK293A细胞表面与受体结合与定位的情况,并且用几种方法证明了哌唑嗪和去氧肾上腺素与α_(1A)-肾上腺素受体结合的特异性。还观测到了激动剂去氧肾上腺素和拮抗剂哌唑嗪与α_(1A)-肾上腺素受体结合作用的差异。
In chapter one,significance of single molecule detection(SMD)and techniques were reviewed briefly.These techniques were powerful tools for investigation of dynamics and kinetics of molecules.New information can be obtained from the single molecule research,which is otherwise hidden or averaged out.Single molecule detection is a technology of studying biomolecules with high spatial and temporal resolution.Fluorescence microscopy was rapidly expanding from single molecule detection techniques into all fields of cell and molecular biology.Total internal reflection fluorescence microscopy(TIRFM)was introduced.An intimate summary of their applications was also provided with single-molecule analysis in vitro and single-molecule analysis in vivo.
In chapter two,we developed a sensitive single-molecule imaging method for quantification of protein by total internal reflection fluorescence microscopy(TIRFM) with adsorption equilibrium.Rhodamine 6G(RtG)and DNA molecules labeled by R6G had been detected by TIRFM in literature.The limit of detection(LOD)was only 2.5×10~(-9)mol/L.We choosed Alexa Fluor 488-labeled goat anti-rat IgG(H+L) (IgG(H+L)-488)as the model protein.First,the solution of IgG(H+L)-488 was incubated with a glass microscope coverslip for a certain time.In this case,the adsorption equilibrium of protein was achieved between the solution and the coverslip.Then,fluorescence images of protein molecules in an evanescent wave field were taken by a highly sensitive electron multiplying charge coupled device. Finally,the number of fluorescent spots corresponding to the protein molecules in the images was counted.The spot number showed an excellent linear relationship with protein concentration.The concentration linear range was 5.4×10~(-11)to 8.1×10~(-10) mol/L.The low end of the linear relationship was 46-fold lower than reported in literature.
In chapter three,the total internal reflection fluorescence microscopy combined with Epi-FM was used to study the early events of Transferrin and Transferrin Receptor-mediated transcytosis in living cells at single molecule level.The early stages of Transferrin and Transferrin Receptor processes included moving, congregation and internalization of Transferrin-Transferrin Receptor complexes, which have already been studied by morphological methods and biochemical methods. But most researches made judgement by the direct measurement of radioactive marker or the stability of fluorescent labelling marker after a large number of cytolysis.Due to the limitation of equation measuring method,these processes could not be observed dynamically.We has carried out dynamic study on the early events of Transferrin and Transferrin Receptor-mediated transcytosis at single molecule level, and the moving and congregation of Transferrin and Transferrin Receptor complexes in the real time.And there were no reports about real time tracking until now.These early processes using single molecule fluorescence detection were as follow.
1.The process that Transferrin-QD(Tf-QD)in the solution bound to its receptor (TfR)on the cell membrane to produce a complex(TfR-Tf)was visualized in real time.
2.The lateral moving and the congregation of TfR-Tf complexes on the plasma membrane were traced at single molecule level.Then,the congregation of two TfR-Tf complexes happened.
3.At the temperature of 4℃and 37℃,the analysis of TfR-Tf complexes and the lateral moving on the plasma membrane were observed through surveying the movement of fluorescence spots.On the previous condition,the suffused modes of TfR on living MGC832 cell were free diffusion mode and restricted diffusion mode. Consequently,the transient diffusion coefficients of TfR-Tf complexes were calculated respectively by pixel coordinates correspond to sequential movements of fluorescence spots in pictures.
In chapter four,we report the incorporation of biotin onto small drug molecules, the antagonist prazosin,and the agonist phenylephrine,through a series of reactions. Prazosin and phenylephrine were respectively labeled with quantum dot (QD)-streptavidins through the strong affinity between biotin and streptavidin to observe the binding between small drug molecules andα_(1A)-AR.Because of the unique optical properties of QD,more and more studies have been reported now concerning such fluorescent QD-marked drug molecules and application in fluorescence imaging of cells.However,most of such processes included complicated modifications on drug molecules and therefore gave rise to the problems of yield and separation. Moreover,the attached amount of drug molecules to QD is difficult to control. Another method is to biotinylate small drug molecules and then to bind them to QD-streptavidin.Though there is now report that the antagonist of dopamine transporter can bind to QD in such way,application in fluorescence imaging on living cell has not been seen.Motivated by these considerations,we have performed investigations as follows:
1.Using a novel synthetic clue,we have attached biotin to prazosin and phenylephrine molecules.Then,QD-streptavidins were introduced to bind prazosin and phenylephrine specifically and effectively.In such way,the problems of yield and separation usually met in covalent binding were avoided.Also,the amount of small molecules attached to QD became much controllable because the number of streptavidin coated on QD was invariable.Because QD-streptavidin is available commercially,it is convenient to apply to the life science studies.Obtained results indicated that the QD-labeled prazosin and phenylephrine molecules still had pharmacological activity.That is,QD-streptavidin could work effectively as fluorescent probe in imaging of living cell.
2.Comparisons are made on the binding abilities respectively betweenα_(1A)-AR on the cell surface and four biotinylated phenylephrines with different chain lengths. It is found that the phenylephrine with longer chain between biotin and phenylephrine can bind toα_(1A)-AR on the cell surface more easily.Correspondingly,more fluorescent spots would appear under the same condition.This reflected the possible steric hindrance on the binding ability between acceptor on the cell surface and ligands.
3.The phenylephrine-QD and prazosin-QD were used to observe their binding and anchoring status with the acceptor on the cell surface ofα_(1A)-AR HEK293A. Several methods have adopted to support the binding specificity of prazosin-QD and phenylephrine-QD toα_(1A)-AR respectively.Furthermore,we also observed the function difference when agonist-phenylephrine and antagonist-prazosin were respectively bound toα_(1A)-AR.
引文
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