Iron is an essential cofactor for symbiotic nitrogen fixation, required by many of the enzymes involved, including signal transduction proteins, O2homeostasis systems, and nitrogenase itself. Consequently, host plants have developed a transport network to deliver essential iron to nitrogen‐fixing nodule cells. Ferroportin family members in model legume These data indicate that MtFPN2 plays a primary role in iron delivery to nitrogen‐fixing bacteroids in
The development of bioorthogonal fluorogenic probes constitutes a vital force to advance life sciences. Tetrazine‐encoded green fluorescent proteins (GFPs) show high bioorthogonal reaction rate and genetic encodability but suffer from low fluorogenicity. Here, we unveil the real‐time fluorescence mechanisms by investigating two site‐specific tetrazine‐modified superfolder GFPs via ultrafast spectroscopy and theoretical calculations. Förster resonance energy transfer is quantitatively modeled and revealed to govern the fluorescence quenching; for GFP150‐Tet with a fluorescence turn‐on ratio of ∼9, it contains trimodal subpopulations with good (P1), random (P2), and poor (P3) alignments between the transition dipole moments of protein chromophore (donor) and tetrazine tag (Tet‐v2.0, acceptor). By rationally designing a more free/tight environment, we created new mutants Y200A/S202Y to introduce more P2/P1 populations and improve the turn‐on ratios to ∼14/31, making the fluorogenicity of GFP150‐Tet‐S202Y the highest among all up‐to‐date tetrazine‐encoded GFPs. In live eukaryotic cells, the GFP150‐Tet‐v3.0‐S202Y mutant demonstrates notably increased fluorogenicity, substantiating our generalizable design strategy.
Ultrafast spectroscopy reveals FRET in action and inhomogeneous populations with different transition dipole moment alignments. Rational protein design of two new superfolder GFP mutants with record‐high fluorogenicity. Bioimaging application of the designed bioorthogonal protein mutant in live eukaryotic cells.
- NSF-PAR ID:
- 10373306
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Natural Sciences
- Volume:
- 2
- Issue:
- 4
- ISSN:
- 2698-6248
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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