A directly regulated, single protein design offers simplicity, tight regulation, and targeted control. Several attempts have been made to create the protein logic gates, but most involve either indirect control or are multi-protein systems 8, 9, 10, 11. Robust input/output control has been reported in various systems using protein engineering approaches 2, 5, 6, 7, but to create a program, logic gates must be incorporated into the circuit. The challenge in this case is that protein 3D structure and dynamics need to be reprogrammed.
Alternatively, we can directly control the function of a protein by “rewiring” its structure without the overhead of gene expression machinery. Such simplicity comes with the disadvantage of a “heavy” program: reprogramming requires that many kilobases of nucleotides be re-written or added in order to control activity of a single protein. The advantage of using DNA as a “programming language” is its simplicity: control is established by regulating expression of a particular gene, and, hence, the activation of the encoded protein. Most of the developments in the field of synthetic biology have focused on reprogramming DNA sequence 4. Cellular programming offers unparalleled opportunities in biotechnology and medicine, and its most intriguing features are the abilities to piggyback on natural processes and to incorporate evolutionary pressure into the syntax. The process hijacks one or more components and alters their function using external (e.g., organic or inorganic molecules or light) 2 or internal controls 3. This work provides proof-of-principle for fine multimodal control of protein function and paves the way for construction of complex nanoscale computing agents.Ĭellular programming involves embedding of instructions within natural cellular constituents (e.g., DNA, RNA, or protein) to control phenotype 1. We demonstrate that dynamic FAK activation profoundly increased cell multiaxial complexity in the fibrous extracellular matrix microenvironment and decreased cell motility. Orthogonal regulation of protein function was possible using the chemo- and optogenetic switches. In the engineered FAK, all of FAK domain architecture is retained and key intramolecular interactions between the kinase and the FERM domains are externally controlled through a rapamycin-inducible uniRapR module in the kinase domain and a light-inducible LOV2 module in the FERM domain. Our system is based on chemo- and optogenetic regulation of focal adhesion kinase. Here, in an important step toward this goal, we report an engineered, single protein design that is allosterically regulated to function as a ‘two-input logic OR gate’. Combining these nanoscale computing agents into larger molecules and molecular complexes will allow us to write and execute “code”.
Advances in protein design have brought us within reach of developing a nanoscale programming language, in which molecules serve as operands and their conformational states function as logic gates with precise input and output behaviors.
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