Andersen, Niels HGraham, Katherine Ann2019-02-222019-02-222018Graham_washington_0250E_19358.pdfhttp://hdl.handle.net/1773/43330Thesis (Ph.D.)--University of Washington, 2018The relationship between sequence, the folded structure, and the folding pathway by which that structure is reached presents a topic of tremendous importance in the understanding of proteins. However, the study of most proteins is difficult due to their size and inherent complexity. Smaller protein-like systems may offer a simpler alternative, making them useful model systems for their larger counterparts. At approximately 20 residues in length and with a folding time on the low-microsecond timescale, the Trp-cage miniprotein is an ideal system for characterizing the interactions that stabilize protein folding and exploring folding pathways. Since the development of the original sequence TC5b, the Trp-cage has been optimized through a series of stabilizing mutations. More recently, cyclized and circularly permuted versions of Trp-cage have been developed. These alternate versions of the miniprotein have been shown to adopt the same characteristic fold as the standard topology. The work described in this study seeks to characterize the stabilizing interactions and folding pathways of Trp-cage. The first part uses a series of point mutations to reverse the typical stability profile of Trp-cage under acidic conditions. The second part characterizes the stabilizing effect of key folded state interactions in the circularly permuted motif. Finally, dynamics studies explore the folding pathways of both the standard topology and circularly permuted series. Comparisons of the standard Trp-cage to its circularly permuted counterpart offer insights into how contact order influences the stabilizing effects of individual interactions as well as the order of those interactions in the folding pathway.application/pdfen-USnoneminiproteinprotein foldingTrp-cageBiophysicsChemistryThesis