Graduate Studies
Date of this Version
Fall 12-10-2015
Citation
Xu, Bi, "Evolution of Non-Native Host-Guest Systems for Use in Living Cells" (2015). Student Research Projects, Dissertations, and Theses - Chemistry Department.
Abstract
Supramolecular chemistry allows for the controled assembly of molecular architectures, making this process a promising approach for controlling protein-protein interactions in biological systems. Synthetic supramolecular systems have been proven to be capable of modulating the assembling of proteins, however, the difficulty of delivering these synthetic components into living cells limits their applications in the cellular context. Herein, we asked whether it was possible to develop a host-guest pair by directed evolution that would be compatible with living systems. We evolved a series of miniprotein hosts from the tenth type III domain of fibronectin (Fn3), that display remarkable binding affinity towards a red-shifted environment-sensitive merocyanine derivative, termed sI-Pht. Importantly, the consensus binder isolated from directed evolution experiments (6.2.18) forms a higher order assembly in response to addition of sI-Pht, as assessed by analytical ultracentrifugation. sI-Pht-induced assembly of 6.2.18 results in a 570-fold increase in fluorescence compared to free dye. This property enables the direct visualization of host-guest assemblies by fluorescence microscopy. As a demonstration, we showed that supramolecular assembly of the 6.2.18-sI-Pht system can be visualized on the surface of living yeast cells. Furthermore, we then identified an improved host, termed 6.2.22, which produces a dramatic 750-fold increase in fluorescence upon supramolecular assembly with sI-Pht. Importantly, this new host displays an 4-fold increase in the EC50 for complexation compared to the 6.2.18-sI-Pht system. Taken together, these new host-guest pairs provide tools for the potential development of new materials as well as pathway engineering. In a broader context, this work details a new design paradigm for the discovery of host-guest systems that function in the context of living cells.
Advisor: Cliff I. Stains
Comments
A THESIS Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Master of Science, Major: Chemistry, Under the Supervision of Professor Cliff I. Stains. Lincoln, Nebraska: December 2015
Copyright (c) 2015 Bi Xu