Elucidation of folding pathways of knotted proteins.

Understanding the mechanisms by which proteins fold and thread into topologically knotted conformations has been challenging because of the apparent complexity associated with the folding and threading events. Nevertheless, many experimental and computational studies have provided insights into the folding pathways of knotted proteins and showed that most of the knotted proteins could spontaneously and reversibly fold into knotted topologies with highly populated intermediates and, at times, through multiple folding pathways. Our laboratory has reported the folding mechanisms of a variety of knotted proteins that have different knot types, ranging from the simplest trefoil 3 knot to the most complex Stevedore's 6 knot. Therefore, we focused on using multiplex thermodynamics and kinetics measurements to tease out unique information associated with different structural probes to obtain a more comprehensive overview of the folding mechanisms of the knotted proteins of interest. In this chapter, we shall discuss the use of different biophysical tools and analytical models to glean mechanistic insights into how intricate polypeptides attain knotted topologies.