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

威斯康星州麦迪逊大学获得了一个奖项，以使对单个含金属酶的测量成为可能。酶的显著催化活性常常来源于酶中金属原子的性质。通常，这些金属酶如何发挥作用的研究依赖于将酶捕获在不同的中间状态，这一过程并不总是能够揭示整个酶的机制。 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，金属酶不能通过最先进的单分子技术进行检测，因此需要开发新的仪器。使用光学微谐振器，将光限制在一个小的微体积的设备，将使时间分辨的行为的测量和推导出一个单独的工作金属酶的机制。这项工作的结果将是一种新的工具，能够提供关于生物和工业相关化学转化的广泛酶的独特信息。同时，参与学生的教育经验将是高度多学科的，结合光子学，仪器，纳米纤维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领域代表性不足的本科生的参与，以及PUI的本科生。 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。拟议的研究将有利于研究金属酶的大型科学界。 酶是支持生物有机体中几乎所有代谢活动的生物机器。这些机器中有近一半的核心是促进蛋白质功能的金属离子。这些金属酶经历多个单独的动力学步骤以执行其功能，包括电子转移事件和配体结合和解离。建立酶催化循环中这些个体事件的性质和时间尺度具有巨大的机理意义。单分子测量提供了构建酶执行其天然功能的“分子电影”的独特能力。这些测量对于更好地理解酶的功能非常强大，因为每个步骤都可以被观察到。然而，即使现代单分子技术已经使许多酶的关键细节被解开，目前用于对单个分子进行测量的方法未能提供有关金属酶中金属位点的有用信息，基本上对金属酶操作模式的最重要的机械细节保持盲目。这一建议涉及一种新技术的发展，允许单分子的金属酶的活性位点的调查。具体来说，我们将开发光学微谐振器作为高灵敏度的温度计，能够测量从单个光激发生物分子释放的热量。一旦这种热量被量化，人们就可以推断吸收了多少光，从而可以构建活性位点的电子吸收光谱。该光谱随着金属酶执行其功能而随时间变化，包含关于金属位点变化性质的大量信息，包括其氧化还原状态和配位环境。这项新技术将在单分子水平上开辟一个关键生物分子类别的大部分，为广大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.