Knots, Slipknots, and Disulfide Bonds in Protein Stability and Folding

For the engineer who would attempt to use proteins as materials for the development of novel therapeutics, understanding the mechanisms by which proteins fold to and maintain their native structures is crucial. My graduate work has focused on investigating complex topological features in proteins such as knots and slipknots, and the effects that they have on protein folding and stability. Chapter 1 of this dissertation consists of a review article that I coauthored on the use of topologically complex proteins as model systems for the study of protein folding and stability. Although several studies that have advanced our understanding of knotted proteins in particular have been published since this review was written, it still provides a concise and relevant introduction to the field. The introduction and discussion sections of chapters 4 and 5 provide updated summaries of recent advances. I discuss the discovery and characterization of a novel topological feature in proteins, the slipknot, in Chapter 2. This work suggested that slipknotted and knotted proteins may fold by similar mechanisms, an idea that has been supported by several recent studies involving molecular dynamics simulations of the folding of knotted proteins. Chapter 3 describes crystal structures of a protein that was targeted for structural studies due to the possibility that an intermolecular disulfide bond in the protein may have resulted in complex topological features. Although no such features were observed, the role played by the disulfide bond in the stability of the protein is discussed, as are the functional implications of the crystal structures. In Chapter 4 I describe the design of a novel knotted protein, and compare its biophysical properties to those of a similar, yet unknotted, control protein. This work represents a significant step forward in the field by allowing the effects of knots in proteins to be directly addressed in controlled experiments for the first time. The results of our initial characterization of the folding kinetics of the two proteins enable us to propose an evolutionary hypothesis for why knotted proteins, and, by extension, topologically complex proteins in general, are rare. Chapter 5 describes ongoing work attempting to uncover the extent to which natural selection has optimized the folding pathways of naturally knotted proteins. By computationally redesigning the sequences of naturally knotted proteins and attempting to design a novel knotted protein fold de novo, I aim to uncover whether specific, non-native interactions play a significant role in the folding pathways of naturally knotted proteins. Finally, in Chapter 6 I report on the current status of an ongoing project in the lab to produce and characterize highly knotted protein polymers. The preliminary results we have obtained suggests that the knotted topology of the polymers imparts to them a greater stability and/or a decreased tendency to aggregate under denaturing conditions.