Methods To Study Protein Folding And Evolution In Vivo

Living systems have developed complex gene networks consisting of chaperones and quality control factors which maintain protein homeostasis (proteostasis) by actively monitoring protein folding processes in an organelle-specific and dealing with protein misfolding in a stress-responsive manner. While we know that these "proteostasis networks" are capable of influencing protein folding, we lack molecular details regarding how particular components of proteostasis networks work in concert to deal with protein misfolding. Unfortunately, this gap in knowledge also prevents us from understanding the consequences of proteostasis regulation on higher order biological processes, such as the impact chaperone and quality control factors have on protein evolution. Furthermore, we are not able to comment on how dysfunctional proteostasis networks contribute to prominent disease states, including neurodegeneration, cancer, and even pathogenic infections. Thus, an overarching interest in the Shoulders lab at MIT is to fill the aforementioned knowledge gaps by studying how living metazoan systems handle protein folding problems. The lack of available methods for controlling the activity of proteostasis network components has significantly limited the study of proteostasis in metazoans. In this thesis, I present work that has focused on addressing the limitation in chemical biology tools for studying proteostasis by developing chemical genetic methods tune the level of proteostasis components. Similarly, the inability to conveniently explore protein folding and fitness landscapes on the laboratory timescale has hindered the study of evolution in higher eukaryotes. Thus, my later work sought to overcome this limitation by creating new evolution platforms. Though the inspiration for my work stemmed from a desire to study proteostasis and evolution in metazoans, the methods I developed have allowed other scientists to overcome technical limitations in their own work and progressed the study of many other biological processes beyond proteostasis.