Thursday, May 15, 2025
5/15/2025
BIOPHYSICAL ANALYSIS OF ENZYME INHIBITOR INTERACTIONS - EXCEED THE SPACE PROVIDED. Understanding enzyme-inhibitor interactions at the molecular level is fundamental to successful drug design. Carbonic anhydrase and its inhibitors provide a robust and prototypic system for developing new methods to study enzyme-ligand interactions. In this study, the technique offeree spectroscopy will be used to investigate enzyme-inhibitor interactions on carbonic anhydrase at the single molecule level. Our long term goal is to examine the capabilities offeree spectroscopy as an analytical tool for a range of enzymes and their inhibitors. The specific aims are designed to: 1. Improve strategies for protein immobilization on surfaces and attaching inhibitors to cantilever probes. An essential requirement for studying biospecific interactions on immobilized surfaces is to present a suitable surface that minimizes non-specific attachments and has a high coverage of the immobilized catalytically active enzyme. To this end, this study will examine several methods for immobilizing the catalytically active carbonic anhydrase with specific control over the orientation of the enzyme. The inhibitor will be attached to a cantilever probe via a flexible linker. 2. Analyze the enzyme-inhibitor interactions by dynamic force spectroscopy. We will quantify the interactions between an oriented carbonic anhydrase enzyme and a sulfonamide inhibitor tethered to an atomic force microscope cantilever. Since the interaction forces are dependent on the loading rate of the cantilever, the loading rate will be varied to map the energy landscape of the enzyme-inhibitor complex. 3. Determine how competitive interactions affect enzyme-inhibitor interactions. We plan to block the active site of the enzyme by introducing other competitive inhibitors, and thereby reduce the specific interactions between the tethered inhibitor and the enzyme. This will provide a method for obtaining inhibitor constants which can be compared to other techniques. PERFORMANCE SITE ========================================Section End===========================================
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