Why Knot? Studies Into the Knot of a Minimal Tied Trefoil
The Minimal Tied Trefoil from Thermotoga maritima (MTTTm) represents an ideal system for investigating knots in proteins. It is one of the smallest knotted proteins to contain a deep trefoil knot, and one of the smallest SPOUT methyltransferases. To date, however, little has been accomplished on this system probing into the knots purpose in the protein. The work presented here characterizes the role of the knot in MTTTm. There are two main facets for understanding the knot: its role in the native state, and its role in the denatured state. To probe these, the work following uses a myriad of biophysical techniques to further understand the knots role in both states. These experiments show that in the native state the knot helps to tether down what was expected to be a flexible loop, which if flexible would impact binding. As well, there exist a series of hydrophobic contacts beneath the knot that are critical for allostery, and would be disrupted if the knot were not there. In the denatured state, there appears to be a linkage between the knot and critical packing residues that would complicate refolding in vivo, but the knot helps to maintain those contacts to allow for easier refolding. The studies here give us more information on why the knot exists in MTTTm, and help us to further understand the role of knots in proteins as a whole.