Ultrafast Bioorthogonal Spin-Labeling and Distance Measurements in Mammalian Cells Using Small, Genetically Encoded Tetrazine Amino Acids

Abstract

Site-directed spin-labeling (SDSL)─in combination with double electron-electron resonance (DEER) spectroscopy─has emerged as a powerful technique for determining both the structural states and the conformational equilibria of biomacromolecules. DEER combined with in situ SDSL in live cells is challenging since current bioorthogonal labeling approaches are too slow to allow for complete labeling with low concentrations of spin label prior to loss of signal from cellular reduction. Here, we overcome this limitation by genetically encoding a novel family of small, tetrazine-bearing noncanonical amino acids (Tet-v4.0) at multiple sites in proteins expressed in Escherichia coli and in human HEK293T cells. We achieved specific and quantitative spin-labeling of Tet-v4.0-containing proteins by developing a series of strained trans-cyclooctene (sTCO)-functionalized nitroxides─including a gem-diethyl-substituted nitroxide with enhanced stability in cells─with rate constants that can exceed 10⁶ M^-1 s^-1. The remarkable speed of the Tet-v4.0/sTCO reaction allowed efficient spin-labeling of proteins in live cells within minutes, requiring only sub-micromolar concentrations of sTCO-nitroxide. DEER recorded from intact cells revealed distance distributions in good agreement with those measured from proteins purified and labeled in vitro. Furthermore, DEER was able to resolve the maltose-dependent conformational change of Tet-v4.0-incorporated and spin-labeled MBP in vitro and support assignment of the conformational state of an MBP mutant within HEK293T cells. We anticipate the exceptional reaction rates of this system, combined with the relatively short and rigid side chains of the resulting spin labels, will enable structure/function studies of proteins directly in cells, without any requirements for protein purification.

Figure 1.. Tet-v4.0 ncAAs and sTCO-nitroxides: Spin-label Stability, Genetic Encoding, and Reactivity.

(A) Tetrazine amino acid and sTCO-spin label structures. Rotatable side-chain dihedral angles preceding the tetrazine are illustrated for Tet-v3.0Bu and Tet-v4.0Me, as well as for sTCO-spin labels (B) Stability of sTCO-nitroxides (12 μM) in diluted HEK293T cytosolic extract at room temperature. EPR signal intensity was measured as the peak-to-trough amplitude of the X-band CW EPR spectrum (see supplementary Figure S29). Each trace is an individual time course from triplicate measurements. (C) Tet4 incorporation efficiency measured with GFP fluorescence of E. coli co-transformed with GFP150‒TAG and evolved Mb-Pyl-tRNA/RS pairs D4 or E1 in the presence and absence of Tet4 ncAAs (0.5 mM). (D) Reactivity of GFP150‒Tet4-Ph/Pyr monitored by ESI-Q-TOF mass spectrometry. Schematic representation of the IEDDA reaction between GFP‒Tet4 and sTCO-OH (top). Deconvoluted mass spectra of purified GFP150‒Tet4-Ph (bottom left) and GFP150‒Tet4-Pyr (bottom right) before (black) and after (red) reaction with sTCO-OH. Cal. Mass of GFP‒wt: 27827.02 Da (avg); GFP150‒Tet4-Ph (observed: 27941.5 Da, expected: 27940.1Da); GFP150‒Tet4-Ph + sTCO-OH (observed: 28065.5 Da, expected: 28064.1 Da); GFP150‒Tet4-Pyr (observed: 27941.3 Da, expected: 27941.1Da); GFP150‒Tet4-Pyr + sTCO-OH (observed: 28066.2 Da, expected: 28065.1 Da). Asterisks (*) mark peaks corresponding to the loss of N-terminal methionine. Low intensity peaks at higher mass are sodium and potassium adducts.

Figure 2.. Tet-v4.0 SDSL-EPR and DEER on Maltose Binding Protein.

(A) Surface rendering of maltose-bound MBP (1anf) with labeling sites indicated in color. For doubly-labeled constructs 211/295 (blue) and 278/322 (orange), the change in C_β distance between apo (dashed gray arrows) and holo (solid black arrows) conformations is indicated. (B) Room temperature X-band CW EPR spectra of purified MBP 211/295-Cys spin-labeled with MTSL (gray) and MBP 211/295-Tet4-Ph spin-labeled with sTCO-tM6 (black), -tM5 (red), or -tE5 (blue). (C‒F) Background-corrected DEER time traces and distance distributions for doubly spin-labeled MBP constructs 211/295 and 278/322. Maltose-free data are shown in black and data from samples recorded in the presence of 1 mM or 5 mM maltose are shown in red. Cysteine mutant constructs (C,E) were labeled with MTSL and Tet4-Ph constructs (D,F) were labeled with sTCO-tE5. The oscillation period of the long-distance components are indicated with triangles in (F) to highlight the shift in modulation frequency. Shaded distributions are predictions from in silico rotameric modeling based on available structures of apo (pdb 1omp; gray) and maltose-bound (pdb 1anf; pink) MBP.

Figure 3.. Encoding and reactivity of Tet-v4.0 in mammalian cells.

(A) Suppression efficiency of GFP150-TAG with Tet4-Ph and Tet4-Pyr amino acids measured by flow cytometry of cultured HEK293T cells, in comparison with Tet3-Bu. (B) Reactivity of GFP150‒Tet4 in whole-cell HEK293T lysate verified by mobility shift of GFP detected by SDS-PAGE upon incubation with sTCO-PEG5k. (C) In-cell labeling of GFP150‒Tet4-Ph/Pyr in HEK293T cells with sTCO-JF646 as monitored by fluorescent imaging of SDS-PAGE gel. (D,E) Quantitative analysis of GFP150‒Tet4-Ph (D) and GFP150‒Tet4-Pyr (E) labeling in live HEK293T cells with sTCO-JF646 using 2D single-cell fluorescent flow cytometry (red: 100 nM sTCO-JF646; black: 0.1% DMSO; 30 mins.). (F) Concentration dependence of in-cell spin-labeling of HEK293T cells expressing MBP322‒Tet4-Ph with sTCO-TEP assessed by pulse-chase with sTCO-JF646. MBP322‒Tet4-Ph molecules not labeled with sTCO-tE5 but subsequently labeled with excess sTCO-JF646 were quantified by in-gel fluorescence of whole-cell lysates. Points indicate individual experiments with mean JF646 fluorescence from triplicate experiments given by bars. (G,H) Live-HEK293T cell labeling of GFP150‒Tet4-Ph with sTCO-tE5 spin label. (G) Epifluorescence micrographs of HEK293T cells expressing GFP150‒Tet4-Ph at various times before and after perfusion with 1 μM sTCO-tE5. (H) Time course of GFP fluorescence increase of HEK293T cells expressing GFP150‒Tet4-Ph upon perfusion (at t = 0) with 1 μM sTCO-tE5 in DMEM + 10% FBS. Data are mean cell fluorescence divided by mean fluorescence at t = 0. Gray bars are mean ± standard error of the mean.

Figure 4.. CW EPR and DEER in HEK293 cells.

(A) Room temperature X-band EPR spectra of HEK293T cells transfected with GFP150-TAG and Tet4 Mb-PylRS/tRNA in the presence of 150 μM Tet4-Ph ncAA (top). Cells were spin-labeled with 200 nM sTCO-tE5 (10 min, r.t.) and spectra were recorded 1 h post-labeling. EPR spectra of labeled cells lacking either the Mb-PylRS/tRNA pair (middle) or Tet4-Ph ncAA (bottom) are displayed on identical scales. (B) Structural model of GFP150/222‒Tet4-Ph labeled with sTCO-tE5 displaying estimated rotameric distributions as mesh surface. Spin-labeled side chains were modeled with the chiLife package in Python. (C) 4-pulse DEER time traces (top) and distance distributions (bottom) for GFP150/222‒Tet4-Ph-tE5 recorded from in vitro-purified protein (black) and in intact HEK293T cells (blue). Experimental modulation depths (Δ) are indicated. In-cell DEER acquisition time was 224 h. Error bands on the distributions estimated from LongDistances are shown but are similar to the linewidth. The simulated distance distribution from rotameric modeling is shown in gray shade.

Figure 5.. Conformation of MBP W340A in mammalian cells by DEER.

(A) Structural model and schematic of the MBP construct used for in-cell DEER. Residues substituted with Tet4-Ph are indicated in blue. The maltose-binding pocket with Trp-340 highlighted is shown as zoomed inset. (B) Stability of the nitroxide radical in spin-labled MBP211/295‒Tet4-Ph-tE5 in HEK293T cells at room temperature determined by 9.8 GHz CW EPR (see supplementary Figure S29). A single-exponential fit to the data is shown as black solid line and timecourses for cell labeling and sample preparation are indicated. (C) DEER time-domain (left) and distance distributions (right) for MBP211/295‒Tet4-Ph-tE5 purified from E. coli and labeled in vitro (top), MBP(340A)211/295‒Tet4-Ph-tE5 purified from HEK293T cells and labeled in vitro (middle), and MBP(340A)211/295‒Tet4-Ph-tE5 within HEK293T cells (bottom). In-cell DEER acquisition time was 264 h. DEER time traces are background-corrected and scaled to the same modulation depth. Uncertainties in the distance distributions are shown as shaded error bands. Dashed lines centered at the maximum of the in vitro apo (black) and maltose-bound (red) distributions are shown for ease of comparison.