Determining the Mechanistic Basis for Surface Interactions and Effects on Catalytic Efficiency in Tethered Enzyme
Abstract
Enzymes are the key component of superior chemical sensors for defense-related applications (such as for chemical warfare agents and signatures of nuclear weapon production) due to their unequaled binding specificity and catalytic activity. However, many detection platforms require that enzymes be immobilized or confined, impacting activity, stability, and specificity, creating significant practical challenges to development of robust technologies. It is clear that immobilization can perturb the structure and orientation of enzyme molecules, potentially impacting their chemical reactivity and the performance of a sensor. However, it is less well appreciated that immobilization necessarily introduces enzymes into a highly heterogeneous environment, so that individual enzymes can exist in a wide variety of states, making sensors less reproducible and quantitative. This is exacerbated by traditional non-specific methods of immobilization that tether random portions of the enzyme to the surface. Protein engineering can introduce unique chemical groups at desired locations on the enzyme, facilitating site-specific immobilization, but maintaining enzyme performance after immobilization is still an intensive process of trial-and-error due to a lack of fundamental understanding of the connection between immobilization and enzymatic activity. This project will develop and employ single-molecule (SM) fluorescence microscopy, ultra-stable fluorescent enzyme substrates, and protein-engineering methods to characterize, molecule-by-molecule, the structure and activity of immobilized enzymes that are directly relevant to detecting chemical signatures of weapons of mass destruction. We will develop SM tracking methods to quantitatively characterize the heterogeneous behavior of immobilized enzymes and use these methods to understand how immobilization strategies impact enzyme performance. This project will include high-throughput measurement of single-enzyme reaction rates using fluorescent enzyme substrates, SM studies of inhibitor binding dynamics, and SM measurements of enzyme conformational dynamics with high temporal and spatial resolution. The greatly improved understanding of immobilized enzyme behavior resulting from this research will enable the design of robust, stable, and highly sensitive next generation enzyme-based chemical sensors.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Jun 29, 2016
- Source ID
- HDTRA11610045
Entities
People
- Daniel K. Schwartz
Organizations
- Defense Threat Reduction Agency
- University of Colorado