Biophysical Analysis of Enzyme Inhibitor Interactions

超出提供的空间。在分子水平上了解酶-抑制剂的相互作用是成功药物设计的基础。碳酸酐酶及其抑制剂为开发研究酶-配体相互作用的新方法提供了一个强大的原型系统。在这项研究中，我们将使用OFFEREE光谱技术在单分子水平上研究酶-抑制剂对碳酸酐酶的作用。我们的长期目标是检验提供光谱作为一系列酶及其抑制剂的分析工具的能力。其具体目标是：1.改进蛋白质在表面的固定和将抑制剂固定到悬臂探针上的策略。研究固定化表面上的生物特异性相互作用的一个基本要求是提供一个合适的表面，该表面能最大限度地减少非特异性附着，并具有较高的固定化催化活性酶的覆盖率。为此，本研究将考察几种通过特定控制酶的方向来固定化具有催化活性的碳酸氢酶的方法。缓蚀剂将通过柔性连接件连接到悬臂式探头上。2.用动态力谱分析酶与抑制剂的相互作用。我们将量化定向碳酸酐酶和固定在原子力显微镜悬臂上的磺胺抑制剂之间的相互作用。由于相互作用力取决于悬臂梁的负载率，因此负载率将改变以绘制酶-抑制物复合体的能量图景。3.确定竞争性相互作用如何影响酶-抑制物相互作用。我们计划通过引入其他竞争性抑制剂来阻断酶的活性部位，从而减少拴系抑制剂和酶之间的特定相互作用。这将提供一种获得抑制剂常数的方法，可以与其他技术进行比较。表演网站========================================Section End===========================================

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===========================================