(22a) Controlling TIMP-MMP Protein-Protein Interaction By Photoactivation

Dronpa is an engineered green fluorescent protein with photoactivatable properties that enables researchers to control protein fluorescence in response to light. Upon exposure to specific wavelengths, typically in the ultraviolet and blue light ranges, Dronpa transitions between distinct fluorescent states. This property makes it a powerful tool for tracking the localization and dynamics of proteins in living cells. When activated by light, Dronpa emits bright fluorescence, allowing real-time visualization of protein behavior and providing valuable insights into cellular processes. In protein caging applications, Dronpa is used to physically mask or "cage" the function of a target protein, preventing it from interacting with other molecules or performing its biological activity until exposed to light. This light-controlled approach provides precise temporal control over protein function in live cells. In its dark state, Dronpa can be engineered to block or inhibit the protein’s active site or interaction domains, effectively preventing normal function. Upon light exposure, Dronpa undergoes a conformational change that either uncages the protein or releases it from inhibitory interactions, restoring its activity.

In one study, we used Dronpa-based photoswitchable protein fusion systems to control the interaction between tissue inhibitors of metalloproteinases (TIMP) and matrix metalloproteinases (MMPs). Dronpa was fused to either the N- or C-terminus of N-TIMP-1 (the N-terminal domain of TIMP-1) and expressed on the yeast surface as a fusion with Aga2 (a yeast surface protein), or in solution. Upon exposure to light, Dronpa oligomerization limited the binding of TIMP-1 to its target MMPs at both fusion sites. However, placing Dronpa at the N-terminus of N-TIMP-1 disrupted its inhibitory function, while the N-TIMP-Dronpa fusion was able to reversibly inhibit the MMP-3 catalytic domain. This was demonstrated through tight inhibitor assays using a plate reader. This light-controlled system offers a powerful method for studying dynamic cellular processes by enabling the activation or deactivation of specific protein functions with high spatial and temporal precision. Protein caging with Dronpa applies to various areas, such as signaling pathway modulation, drug delivery, bioimaging, and protein-protein interaction studies, thereby enhancing our understanding of complex biological networks.