Single-Molecule Optical Probes of Protein Biophysics

蛋白质生物物理学的单分子光学探针

• 批准号: 9816947
• 负责人: William Moerner
• 金额: $ 30万
• 依托单位: Stanford University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-01 至 2002-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9816947&HistoricalAwards=false
• 关键词: Single; Molecule; Optical; Probes; Protein

MoernerMCB 98169471. TechnicalAs a result of the success of single-molecule optical detection andspectroscopy, first at low temperatures, and more recently at roomtemperatures, a particular opportunity exists to apply these methods to theelucidation of important outstanding problems in the understanding ofprotein dynamical and functional behavior. Single-molecule methodscompletely remove ensemble averaging, thus allowing direct observation ofheterogeneity that is normally hidden. True distributions of measuredproperties over members of the ensemble may be measured, allowing adetailed picture of complex and inhomogeneous environments. Fortime-dependent processes, following individual proteins one at a timethrough the enzymatic cycle removes the need for synchronization of theensemble. Finally, since the single-molecule limit is a previouslyunexplored regime, new and unexpected behavior is likely to occur, such asthe blinking and switching of single copies of green fluorescent proteinmutants observed recently. This study is an interdisciplinary effort toinvetigate several aspects of protein and enzyme dynamics and functionusing optical single-molecule detection and spectroscopy with in vitrotechniques. The important advantage of the single-molecule regime is thatsignatures of the conformational state which are normally buried in large N(N = number of copies) experiments will become uniquely accessible tostudy. The principal biological problems of interest are the photophysicaldynamics of green fluorescent protein and its mutants, the mechanochemicalcycle of kinesin motor protein, and the protein association and enzymaticbehavior of cAMP-dependent protein kinase. The stuidy utilizesdiffraction-limited confocal, total internal reflection, and far-fieldoptical microscopy and spectroscopy to explore the biophysical behavior ofsingle copies of proteins and enzymes, specifically to fully characterizeand understand the mechanism for the unexpected blinking and switchingbehavior recently observed for single copies of green fluorescent protein,and to apply single-molecule imaging, polarization, and energy transfermethods to explore protein conformational states associated with themechanochemical behavior of motor proteins in the kinesin superfamily, andutilize single-molecule optical spectroscopy and microscopy to detect andcharacterize the enzymatic action and protein subunit association anddissociation for cAMP-dependent protein kinase A. To accomplish these ends,the PI's physical, chemical, and optical expertise will be combined withthe molecular biological and biochemical expertise of several talentedcollaborators. In the final analysis, owing to its multidisciplinaryorganization, the fundamental research in this program will not onlygenerate new knowledge about the biophysical properties of severalimportant protein systems, but, in addition the advances in instrumentationwill provide novel groundwork for technologytransfer to other relevant disciplines, and the increased knowledge aboutoptically driven changes in state of fluorescent proteins like greenfluorescent protein may lead to the engineering of mutants capable of useas optical recording and storage elements.2. Non-technicalThis study reaches into a new realm: that of observing and understandingindividual, single copies of proteins. The methods utilized are optical,i.e., using light to probe individual molecules, which allows us to probetime-dependent, dynamical events as each protein copy performs its functionas an enzyme. A key difficulty encountered in most conventional experimentswhich look at a huge number of protein copies at the same time, is thatsynchronization of the different copies is not easily possible. This meansthat any inhomogeneity is obscured. For an analogy, assume that manypeople are in a room, all reading the same text such as the Bill of Rights,but that they did not start together. (In the protein world, this might bean assembly of proteins each at different stages in the enzymatic cycle.)Since all the individuals are marching to a different drummer, the neteffect is something like the cacophony that one hears in a crowdedrestaurant. By developing and using the new methods of single-moleculespectroscopy, one is effectively able to listen to each person in the roomseparately. It will then be possible to discern different dialects thatmight be spoken by the different individuals, or even to tell if some ofthem are actually reading the Preamble to the Constitution instead.Returning to the protein world, this study is expected to learn importantdetails about the exact cycle the protein follows to perform a usefulfunction, and in particular, if some individual copies are intrinsicallydifferent due to the fold of the protein or to some other unforeseenmodification.

MoernerMCB 98169471。由于单分子光学检测和光谱学的成功，首先是在低温下，最近是在室温下，存在一个特殊的机会，应用这些方法来阐明在理解蛋白质动力学和功能行为方面的重要突出问题。单分子方法完全消除了集合平均，因此可以直接观察到通常隐藏的异质性。可以测量整体成员上被测量属性的真实分布，从而获得复杂和非均匀环境的详细图像。对于依赖时间的过程，通过酶循环一次跟踪单个蛋白质，消除了整体同步的需要。最后，由于单分子极限是一个以前未被探索过的机制，新的和意想不到的行为可能会发生，例如最近观察到的绿色荧光蛋白突变体的单拷贝的闪烁和切换。本研究是一个跨学科的努力，研究蛋白质和酶的动力学和功能的几个方面，使用光学单分子检测和光谱学与体外技术。单分子体系的重要优点是，通常隐藏在大N（N =拷贝数）实验中的构象状态的特征将变得独特地易于研究。主要的生物学问题是绿色荧光蛋白及其突变体的光物理动力学，动力蛋白的机械化学循环，以及camp依赖性蛋白激酶的蛋白质结合和酶行为。本研究利用衍射限制共聚焦、全内反射和远场光学显微镜和光谱学来探索蛋白质和酶的单拷贝生物物理行为，特别是全面表征和理解最近在绿色荧光蛋白单拷贝中观察到的意外闪烁和切换行为的机制，并应用单分子成像、极化、和能量转移方法来探索与运动蛋白超家族中运动蛋白的机械化学行为相关的蛋白质构象状态，并利用单分子光谱学和显微镜来检测和表征camp依赖性蛋白激酶a的酶促作用和蛋白质亚基结合和解离。光学专业知识将与几位有才华的合作者的分子生物学和生物化学专业知识相结合。在最后的分析中，由于其多学科组织，该计划的基础研究不仅将产生关于几种重要蛋白质系统的生物物理特性的新知识，而且，除了仪器的进步将为技术转移到其他相关学科提供新的基础。随着对荧光蛋白（如绿色荧光蛋白）的光驱动状态变化的了解的增加，可能会导致能够使用光记录和存储元件的突变体的工程。这项研究进入了一个新的领域：观察和理解单个的蛋白质拷贝。所使用的方法是光学的，即。，利用光探测单个分子，这使我们能够探测每个蛋白质拷贝执行其酶功能时依赖于时间的动态事件。在大多数同时观察大量蛋白质拷贝的传统实验中遇到的一个关键困难是，不同拷贝的同步是不容易实现的。这意味着任何不均匀性都被掩盖了。打个比方，假设很多人在一个房间里，都在阅读同样的文本，比如《权利法案》，但他们不是一起开始的。（在蛋白质世界，这可能是在酶循环的不同阶段的蛋白质组装。）由于所有的个体都是跟着不同的鼓点前进，其效果就像在拥挤的餐馆里听到的不和谐的声音。通过开发和使用单分子光谱的新方法，人们可以有效地分别倾听房间里的每个人。这样就有可能分辨出不同的人可能说的不同方言，甚至分辨出他们中的一些人是否实际上是在读宪法的序言。回到蛋白质世界，这项研究有望了解蛋白质执行有用功能所遵循的确切周期的重要细节，特别是，如果一些个体拷贝由于蛋白质的折叠或其他一些不可预见的修饰而本质上不同。