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

University Of Wisconsin-Madison, Madison WI

Investigators

Abstract

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.