Latching Orthogonal Cage Key Proteins (LOCKR) uses protein switches with bioactive peptides that can modulate gene expression, alter signaling pathways, control protein-protein interactions, or degrade proteins in cells. One component of this system is the Latch, which contains the bioactive peptide that is integrated into the Cage. In this state, the bioactive sequence is locked in the “off” state. Upon the addition of a Key protein, the Key unlocks the Latch from the Cage thus turning “on” its bioactive peptide for biological function. Because the Key-Cage interaction determines biological function, understanding what strengthens or weakens this interaction is critical. One factor that influences this interaction is the length of the toehold, which shortens the Latch relative to the Cage. Our research goal is to generate Latches with different toeholds and to identify which toehold variants bind Key best. To do this, we used molecular biology to create different Latch toeholds, expressed these proteins, and tagged them with a fluorescent molecule. We hypothesize that small toeholds will always be “off”, while larger toeholds will always be “on.” Our preliminary experiments show differences in Key binding based on Latch toehold length; further research will reveal the optimal toehold length for LOCKR.