IDBR: TYPE A; Optical Microresonators as Platforms for Probing Single Metalloproteins in Action

威斯康星大学麦迪逊分校被授予一项奖项，以实现对单个含有金属的酶的测量。酶的显著催化活性通常来自于酶中金属原子的性质。通常，对这些金属酶如何发挥作用的研究依赖于捕获处于不同中间状态的酶，这一过程并不总是揭示整个酶机制。单分子测量，即一次对一个分子进行的研究，是理解功能的强大工具，因为它们允许人们获得化学行为的“分子电影”。然而，金属酶不能用最先进的单分子技术进行检测，这就需要开发一种新的仪器。光学微谐振器是一种将光限制在小体积内的装置，它的使用将使时间分辨行为的测量成为可能，并推断单个工作金属酶的机制。这项工作的结果将是一种新的工具，能够提供关于对生物和工业相关的化学转化至关重要的广泛酶的独特信息。同时，参与的学生的教育体验将是高度多学科的，融合了光子学、仪器仪表、纳米制造和生物无机化学的元素。该项目还将包括生物光子学教学模块的开发，STEM领域中代表性不足的本科生的参与，以及PPI本科生的参与。本项目的目的是开发一种能够测量单个生物分子的时间分辨电子吸收光谱的光谱仪。这项拟议的研究将使研究金属酶的大科学界受益。酶是支持生物有机体中几乎所有代谢活动的生物机器。在这些机器中，近一半的核心是一种促进蛋白质功能的金属离子。这些金属酶经历了多个单独的动力学步骤来实现它们的功能，包括电子转移事件和配体的结合和解离。在酶的催化循环中确定这些个别事件的性质和时间尺度具有极大的机械意义。单分子测量提供了构建酶执行其自然功能的“分子电影”的独特能力。这些测量非常强大，可以更好地了解酶的功能，因为每个步骤都有可能被观察到。然而，尽管现代单分子技术已经使许多酶的关键细节得以解开，但目前对单个分子进行测量的方法无法提供有关金属酶中金属位置的有用信息，基本上对金属酶工作模式的最重要机制细节视而不见。这项建议涉及一种新技术的开发，以允许单分子研究金属酶的活性部位。具体地说，我们将开发光学微谐振器作为高灵敏度的温度计，能够测量单个光激发生物分子释放的热量。一旦热被量化，人们就可以推断出有多少光被吸收，从而可以构建活性中心的电子吸收光谱。随着金属酶履行其功能，这一光谱随时间变化，包含了关于金属位置变化性质的大量信息，包括其氧化还原状态和配位环境。这项新技术将打开一个关键的生物分子类的相当大一部分在单分子水平上进行探测，为大量研究人员提供新的机会。

An award is made to the University of Wisconsin Madison to enable measurements on individual metal-containing enzymes. The remarkable catalytic activities of enzymes frequently derive from the properties of a metal atom within the enzyme. Typically, studies of how these metalloenzymes function rely on trapping the enzyme in different intermediate states, a process that cannot always reveal the entire enzyme mechanism. Single-molecule measurements, i.e. studies performed on one molecule at a time, are powerful tools for understanding function because they allow one to acquire "molecular movies" of chemical behavior. However, metalloenzymes cannot be examined by state-of-the-art single-molecule techniques, necessitating the development of a new instrument. The use of optical microresonators, devices that confine light to a small microvolume, will enable the measurement of time-resolved behavior and deduce the mechanism of an individual working metalloenzyme. The results of this work will be a new tool capable of providing unique information on a broad range of enzymes critical for biological and industrially relevant chemical transformations. Simultaneously, the educational experience for participating students will be highly multidisciplinary, incorporating elements of photonics, instrumentation, nanofabrication, and bioinorganic chemistry. This project will also include the development of biophotonics teaching modules, participation of undergraduate students under-represented in STEM fields, and undergraduate students at a PUI. This purpose of this project is to develop a spectrometer capable of measuring time-resolved electronic absorption spectra of individual biomolecules. The proposed research will benefit the large scientific community studying metalloenzymes. Enzymes are the biological machines that support nearly all metabolic activity in biological organisms. At the heart of nearly half of these machines is a metal ion that facilitates protein function. These metalloenzymes go through multiple individual kinetic steps in order to carry out their function, including electron transfer events and ligand binding and dissociation. It is of immense mechanistic interest to establish the nature and timescale of these individual events in the enzyme's catalytic cycle. Single-molecule measurements offer the unique ability to construct 'molecular movies' of enzymes performing their natural functions. These measurements are extremely powerful for enabling a greater understanding of how enzymes function since each step can potentially be observed. However, even as modern single-molecule techniques have enabled critical details of many enzymes to be unraveled, current methods for performing measurements on individual molecules fail to provide useful information about the metal site in metalloenzymes, essentially remaining blind to the most important mechanistic details of metalloenzyme mode of operation. This proposal concerns the development of a new technique to allow single-molecule investigation of the active sites of metalloenzymes. Specifically, we will develop optical microresonators as highly sensitive thermometers capable of measuring the heat released from a single photoexcited biomolecule. Once this heat is quantified, one can infer how much light was absorbed, allowing the construction of the electronic absorption spectrum of the active site. This spectrum, which varies as a function of time as the metalloenzyme carries out its function, contains a tremendous amount of information about the changing nature of the metal site, including its redox state and coordination environment. This new technique will open up a substantial fraction of a critical biomolecule class to be probed at the single molecule level, enabling new opportunities for a large community of researchers.