Lasso peptides are an exciting class of natural products that are known to have diverse bioactivities. These ribosomally synthesized and post-translationally modified peptides (RiPPs) have great potential in the therapeutic world due to their interesting lariat knot-like shape, which endows them with impressive stability to proteases and extreme environments. Advances in bioinformatics have enabled the discovery and characterization of dozens of lasso peptides. However, our basic scientific understanding of how the biosynthetic proteins work remains limited, especially for the cyclase, which is responsible for tying the characteristic lasso knot. My research aims to answer long-standing questions about the substrate tolerance, substrate binding, and enzyme efficiency of the cyclase using a combination of bioinformatics, structural modeling, and quantitative enzymes assays. I hope to provide novel insights into how these enigmatic cyclase enzymes work and facilitate the engineering of new bioactive lasso peptides.