Single-Molecule Optical Probes of Protein Biophysics

MoernerMCB 98169471。技术上，由于单分子光学检测和光谱学的成功，首先是在低温下，最近是在常温下，现在有一个特别的机会将这些方法应用于阐明理解蛋白质动力学和功能行为的重要突出问题。单分子方法完全消除了集合平均，从而允许直接观察通常被隐藏的异质性。可以测量被测量特性在集合成员上的真实分布，从而允许对复杂和不均匀的环境进行详细的描绘。对于依赖时间的过程，在整个酶循环中一次跟随一个单独的蛋白质，消除了整体同步的需要。最后，由于单分子极限是以前未被探索的区域，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闪烁和切换。这项研究是一项跨学科的工作，旨在利用光学单分子检测和光谱分析技术在体外研究蛋白质和酶的动力学和功能的几个方面。单分子体系的重要优点是，通常埋藏在大N(N=拷贝数)实验中的构象状态的特征将成为唯一可供研究的对象。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，运动蛋白马达蛋白的机械力化学循环，以及cAMP依赖的蛋白激酶的蛋白结合和酶行为。本研究利用衍射受限共聚焦、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的生物物理行为，特别是全面地表征和理解最近观察到的单拷贝绿色荧光蛋白意外闪烁和开关行为的机制，并应用单分子成像、偏振和能量转移方法来探索与运动超家族中运动蛋白的化学行为相关的蛋白质构象状态，并利用单分子光学光谱和显微镜来检测和表征cAMP依赖的蛋白激酶A的酶作用和蛋白质亚单位的结合和解离。光学专业知识将与几位有才华的合作者的分子、生物和生物化学专业知识相结合。归根结底，由于它的多学科组织，该计划的基础研究不仅将产生关于几个重要蛋白质系统的生物物理性质的新知识，而且，仪器方面的进展将为技术转移到其他相关学科提供新的基础，并且关于绿色荧光蛋白等荧光蛋白状态光驱动变化的知识的增加可能导致能够用作光记录和存储元件的突变体的工程。非技术性这项研究进入了一个新的领域：观察和理解单个蛋白质的单一拷贝。所使用的方法是光学的，即使用光来探测单个分子，这允许我们在每个蛋白质拷贝执行其作为酶的功能时，探测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并不容易。这意味着任何不均质性都被掩盖了。打个比方，假设有很多人在一个房间里，他们都在读同样的文本，比如《人权法案》，但他们并不是一起开始的。(在蛋白质世界中，这可能是在酶循环的不同阶段每个蛋白质的组装。)由于所有的个体都在向不同的鼓手行进，网络效应就像人们在拥挤的餐厅里听到的刺耳的声音。通过开发和使用单分子光谱学的新方法，人们能够有效地分别听取房间里的每个人的声音。然后就有可能辨别出不同个体可能说的不同方言，甚至可以分辨出他们中的一些人是否真的在阅读宪法的序言。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副本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。

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.