Directed evolution mimics and accelerates natural evolution in the laboratory in order to create useful new biomolecules and to study evolutionary processes. Although methodologies for directed evolution are well- established in test tubes and in simple organisms like E. coli and yeast, there is still a major challenge. Specifi- cally, novel biomolecules derived from directed evolution campaigns in these platforms often fail to function when transferred to more complex cellular environments, such as that of human cells. To address this critical issue, our laboratory recently pioneered a directed evolution platform that can be used to repeatedly generate massive libraries of mutant biomolecules while continuously selecting and enriching the most functional vari- ants directly in the human cell environment. From a chemical biology perspective, we are also deeply engaged in studying functions of the proteostasis network – a vital and unique aspect of the human cellular environment that ensures proteins are correctly folded, processed and trafficked. We have developed an array of chemical genetic tools to modulate proteostasis, and we are now primed to integrate these tools with our directed evolu- tion platform to both evolve previously inaccessible biomolecule functions and gain a deeper understanding of how cells solve protein folding problems. Altogether, this NIGMS MIRA application seeks to combine two of my laboratory's primary interests: (1) Developing and applying next-generation, human cell-based directed evolution platforms to generate biomole- cules optimized for function in complex cells and (2) Integrating evolution with chemical modulation of proteo- stasis to gain new insights into fundamental principles of proteostasis network function. Here, we propose to integrate these research areas to deliver an array of biomolecules that reliably and robustly perform valuable new functions in the complex human cellular milieu. Examples include G-protein coupled receptors controlled by synthetic regulators for neuroscience applications, systems for incorporation of unnatural amino acids in proteins, and inhibitors of important signaling pathways related to disease. All of these targets have proven ex- ceedingly difficult to reliably evolve in lower organisms or test tubes. Beyond these practical advances, we will also integrate human cell-based directed evolution with proteostasis modulation to gain insights into how the network solves protein folding problems. For example, we will use our capacity to modulate proteostasis to test the hypothesis that chaperones can be used to “turbo-charge” directed evolution campaigns by providing ac- cess to otherwise biophysically unacceptable regions of the mutational landscape. Further, we will pursue an understanding of the roles of chaperones in human protein evolution, a process that is particularly important in the setting of tumorigenesis and in the development of drug resistance in oncogenes. Altogeth...