HyPer7 is a genetically encoded biosensor for cellular H2O2. HyPer7 comprises an H2O2-sensing domain linked to a fluorescent protein, providing the readout. HyPer7 exhibits high ratiometric pH stability in the physiological pH range and is a bright, ultrafast and ultrasensitive biosensor for H2O2 compared to other H2O2 biosensors, and it is functional in mammalian cells. Numerous attempts to obtain well-diffracting crystals for HyPer7 have been unsuccessful thus far. Knowledge of the crystal structure of HyPer7 will provide in-depth insights into the mechanistic details of the functioning of HyPer7 and help guide the design of other biosensors in the future. Another issue with HyPer7 is its cleavage observed in mammalian cells, yeast, and E. coli. This results in two populations which may impede the production of well-diffracting crystals. Finally, HyPer7 is a dimer, whereas having it in a monomeric form could further improve its functionality. Here, I tried to produce crystals with a good diffraction pattern, solve the HyPer7 cleavage issue, create monomeric HyPer7, and characterize the most promising mutants for their responses to H2O2 using numerous biochemical and biophysical issues. I obtained diffracting crystals at 2.8 Å resolution with a HyPer7 mutant. This mutant exhibits the same properties as HyPer7 regarding pH stability, H2O2 sensitivity, secondary structure, and oligomeric state. While HyPer7 mutants designed to prevent cleavage exhibit promising results, they are still subject to cleavage, albeit considerably less than the original version, and demonstrate a reduced response to H2O2 compared to the HyPer7 original version. Moreover, I was not able to make a monomeric version. Based on the results obtained, I present several suggestions for other mutations to prevent cleavage and make HyPer7 a monomer while keeping its superior H2O2 sensing properties.
|Date of Award
|30 Jun 2023