Intellectual Merit. This biomedical engineering project aims to create new cadherin molecules whose function can be reversibly switched with light (LiCads). Cadherins comprise one of the major cell-cell adhesion protein families and mediate intercellular contacts in metazoans by forming multimers in cellular junctions. Switchable LiCads would enable improved ways to study cell adhesion and its interplay with cell signaling and tissue development. Light control would also provide a new mechanism to manipulate adhesion in biomedical engineering applications. To achieve these objectives, it is proposed to create LiCads via engineering pairs of cysteine residues at computationally designed locations in the cadherin molecule, bridge these cysteines with an azobenzene-based, photoisomerizable chromophore, and switch cadherin function by triggering cadherin conformational changes through photoisomerization of the chromophore. The intellectual merit lies in the integration of life science and engineering principles for innovation in two areas: (i) a light-based approach to modulate cell-cell adhesion with improved spatial and temporal resolution (a novel application of existing azobenzene-based technologies), and (ii) an engineering methodology to design a new light-controlled protein conformational switch. The product from successful completion of this research, validated LiCads, would represent a considerable advance in the design of structure by engineering and characterizing a protein switch useful to probe complex cellular functions.
Broader Impact. There is great need for novel tools to characterize and control key biological processes in real time with high spatial resolution. This project aims to engineer and validate such a tool, LiCads, to control cell-cell interactions. Because of the importance of cadherin-mediated interactions in cell biology, tissue morphogenesis, tissue remodeling during development, wound healing and tumor invasiveness in cancer, LiCads can be expected to have considerable impact on many problems in basic and disease biology. Controlling cell assembly in three dimensions is also a key requirement of complex cellular engineering applications. Therefore, LiCads should be useful to both biologists and biological engineers. Students at the graduate, undergraduate and high school levels will participate in this research, building on a track record in mentoring students, and on existing partnerships with the UCSF Undergraduate Summer Research Program and the San Francisco Unified School District; these programs emphasize participation of groups underrepresented in science and engineering. Research under this grant will be integrated with education designed to foster collaboration between students from the life and physical/engineering sciences.
There is a great need for new tools to study important biological processes. One particularly promising approach is to reengineer macromolecules responsible for key biological functions so that they can be activated or deactivated with light. Given lightâ€™s advantages, namely high spatial and temporal precision, pulses of light can be used to start or stop a particular biological process at a given time and location in a cell or organism. The resulting changes can then be watched and studied at a temporal and spatial resolution higher than common alternatives. Our project aimed to develop new methods to reengineer proteins so that they can be controlled with light, utilizing computational prediction approaches based on the three-dimensional structures of proteins. We then constructed the new proteins in the laboratory and tested experimentally that their biological function is indeed light-switchable. One class of proteins we focused on were cadherins, which are important for forming cell-cell interactions in tissues and organs. Because of the role of cadherin-mediated interactions in cell biology, tissue formation, wound healing, and tumor invasiveness in cancer, the ability to control cadherins and their biological functions with light would have considerable impact for many research projects in both basic and disease biology. Controlling cell-cell assembly in three dimensions is also a key requirement of cellular engineering applications, yet only light offers the spatial precision required to control assembly in this fashion. Therefore, light-controlled cadherins are useful to both biologists and biological engineers. In the project supported by this grant, we were successful in engineering a functional light-triggered switch in the cell-cell adhesion protein E-cadherin using a new mechanism-based design strategy. Our work included a detailed examination of functional switching, and the results open avenues toward controllable tools that could be applied to many long-standing questions about cadherin's biological function in cell-cell adhesion and downstream signaling. The results of this project were reported in an open-access publication, and the engineered new cadherin tools are freely available to other researchers. We also tested whether our methods could be applied to other systems, to show broader utility. We were able to demonstrate that the strategy we developed for cadherin could be used to switch the function of another protein with light. Using the design principles developed under this grant, we showed that a large protein machine that resembles a barrel with a built-in lid that opens and closes could be engineered to switch between its two conformations with light. Moreover, the resulting nanocage structure could be used to capture and release different molecular cargos, opening avenues for using light-controlled proteins as controlled delivery systems. This work thus significantly broadened the impact of the methods developed under this grant and illustrates the generality of the underlying engineering principles. The award furthermore contributed to the training of undergraduate and graduate students and postdoctoral researchers in science and engineering through the integration of research and educational activities.