| This dissertation describes the development of a novel technique that used lysozyme as a model system and featured individual copies of the protein attached to a single-walled carbon nanotube-field effect transistor (SWNT-FET). By correlating fluctuations in SWNT-FET conductance with protein conformational motion, a single enzyme could be monitored for extended periods of time (>30 min). The following three observations with this system demonstrates the power of the approach to uncover key insights into enzyme function. First, lysozyme was found to be a processive enzyme. Second, lysozyme exhibits single-step hinge closure and multi-step opening. Third, the basis for the enzyme's pH-dependence was shown to be due to increased time spent in a nonproductive binding state or an inactive state at non-optimal pH values.;The SWNT-FET method offers investigators a new approach for monitoring interactions between binding partners (e.g., enzyme-substrate, enzyme-inhibitor, protein-protein, and others). Future nanocircuit experiments performed with enzyme variants associated with disease states could offer new insight into the catalytic mechanism of the attached protein. In addition, nanocircuit experiments performed with enzyme inhibitors could illuminate mechanisms of inhibition by different antagonists. The use of nanocircuit devices for improving enzymes and their agonists and antagonists offers a new and exciting frontier for biochemical research.;This dissertation also describes the design and selection of an HIV integrase inhibitor. Phage display selections applied a library of variants based on the C-terminal domain of the eye lens protein human gammaS-crystallin. A crystallin variant from the library, termed Integrase-Binding Protein-10 (IBP-10), inhibits the three reactions catalyzed by integrase with nanomolar Ki values. Enzymatic assays performed with truncated integrase variants demonstrate that IBP-10 inhibits catalytic activities through binding to the C-terminal domain of integrase. The results demonstrate the applicability of the crystallin scaffold for the discovery of binding partners and enzyme inhibitors, including proteins from pathogenic viruses. |
