A two-step screening to optimize the signal response of an auto-fluorescent protein-based biosensor.
Shunsuke TajimaEiji NakataReiko SakaguchiMasayuki SaimuraYasuo MoriTakashi MoriiPublished in: RSC advances (2022)
Auto-fluorescent protein (AFP)-based biosensors transduce the structural change in their embedded recognition modules induced by recognition/reaction events to fluorescence signal changes of AFP. The lack of detailed structural information on the recognition module often makes it difficult to optimize AFP-based biosensors. To enhance the signal response derived from detecting the putative structural change in the nitric oxide (NO)-sensing segment of transient receptor potential canonical 5 (TRPC5) fused to enhanced green fluorescent protein (EGFP), EGFP-TRPC5, a facile two-step screening strategy, in silico first and in vitro second, was applied to variants of EGFP-TRPC5 deletion-mutated within the recognition module. In in silico screening, the structural changes of the recognition modules were evaluated as root-mean-square-deviation (RMSD) values, and 10 candidates were efficiently selected from 47 derivatives. Through in vitro screening, four mutants were identified that showed a larger change in signal response than the parent EGFP-TRPC5. One mutant in particular, 551-575, showed four times larger change upon reaction with NO and H 2 O 2 . Furthermore, mutant 551-575 also showed a signal response upon reaction with H 2 O 2 in mammalian HEK293 cells, indicating that the mutant has the potential to be applied as a biosensor for cell measurement. Therefore, this two-step screening method effectively allows the selection of AFP-based biosensors with sufficiently enhanced signal responses for application in mammalian cells.
Keyphrases
- quantum dots
- label free
- nitric oxide
- vascular smooth muscle cells
- sensitive detection
- gold nanoparticles
- living cells
- molecular docking
- binding protein
- stem cells
- single cell
- risk assessment
- healthcare
- cell proliferation
- oxidative stress
- molecular dynamics simulations
- network analysis
- cell death
- endoplasmic reticulum stress
- genome wide
- nitric oxide synthase