Rational design for high bioorthogonal fluorogenicity of tetrazine‐encoded green fluorescent proteins

Abstract

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. Key points 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.

Truncation-Free Genetic Code Expansion with Tetrazine Amino Acids for Quantitative Protein Ligations

“…34−37 A test expression with sfGFP yielded a fulllength protein as hoped (Figure 1C), but we suspected something was not correct as the protein was green, whereas reactive Tet-containing sfGFP 150 as derived from the DH10B cells was orange (Figure S1) due to quenching of the GFP fluorophore by the encoding of oxidized Tet ncAA. 38 Additionally, a gel mobility shift assay testing the protein's ability to react with an sTCO-PEG 5000 polymer confirmed that only a small fraction of the protein was reactive (Figure 1C). This result was unexpected and led to us evaluating the most plausible explanations for unreactive proteins.…”

“…Surprisingly, the majority of expressed protein was prematurely truncated (Figure B), consistent with RF1 outcompeting the suppression of UAG sites by aminoacylated Tet2-tRNA. To address this, we tested expression in a BL21­(DE3)-derived “truncation-free” RF1-knockout cell line, B95­(DE3) Δ A Δ fabR (subsequently referred to here as B95), which has shown robust growth and high GCE yields compared to other RF1-knockout strains. − In our hands, B95 has demonstrated robust ncAA encoding under a variety of expression conditions, so we chose it from among the various release-factor-deficient cell lines and cell-free expression systems that all have been able to minimize truncation and improve full-length protein production by increasing UAG readthrough. − A test expression with sfGFP yielded a full-length protein as hoped (Figure C), but we suspected something was not correct as the protein was green, whereas reactive Tet-containing sfGFP 150 as derived from the DH10B cells was orange (Figure S1) due to quenching of the GFP fluorophore by the encoding of oxidized Tet ncAA . Additionally, a gel mobility shift assay testing the protein’s ability to react with an sTCO-PEG 5000 polymer confirmed that only a small fraction of the protein was reactive (Figure C).…”

“…(A) Characterization of the “D12” Tet2RS with the original Tet2-Me and new Tet2-Et substrates (500 μM) using the SUMO 35 -sfGFP construct. SUMO 35 -sfGFP was used instead of sfGFP to avoid quenching that oxidized Tet will have on sfGFP fluorescence and improve the accuracy of expression measurements. , (B) TetraDuo active-site library overlaid on the Mj aaRS crystal structure (PDB ID: 1J1U). Amino acids are listed for the original Mj aaRS, the D12RS, and the E7RS.…”

Thiomethyltetrazines Are Reversible Covalent Cysteine Warheads Whose Dynamic Behavior can be “Switched Off” via Bioorthogonal Chemistry Inside Live Cells

“…Next, second-order rate constants (k 2 ) were determined for probes A, B, and C. Previous reports have shown that tetrazines have a unique ability to act as fluorescence quenchers when bound to fluorophores, 62 and as such, GFP fluorescence is quenched when bound to tetrazine. 63,64 Therefore, we predicted that thiomethyltetrazines can quench the fluorescence of GFP-SH when S N Ar chemistry has taken place on cysteine. Furthermore, we can determine k obs by monitoring the decrease in GFP-SH fluorescence over time at different tetrazine concentrations to calculate the biomolecular rate constant (k 2 ) of thiomethyltetrazine labeling on a purified protein.…”

Thiomethyltetrazines are Reversible Covalent Cysteine Warheads whose Dynamic Behavior can be "Switched off” via Bioorthogonal Chemistry Inside Live Cells

“…Next, second-order rate constants (k2) were determined for probes A, B, and C. Previous reports have shown that tetrazines have a unique ability to act as fluorescence quenchers when bound to fluorophores, 62 and as such, GFP fluorescence is quenched when bound to tetrazine. 63,64 Therefore, we predicted that thiomethyltetrazines can quench the fluorescence of GFP-SH when SNAr chemistry has taken place on cysteine. Furthermore, we can determine kobs by monitoring the decrease in GFP-SH fluorescence over time at different tetrazine concentrations to calculate the biomolecular rate constant (k2) of thiomethyltetrazine labeling on a purified protein.…”