Direct Comparison between Förster Resonance Energy Transfer and Light-Induced Triplet-Triplet Electron Resonance Spectroscopy

Abstract

To carry out reliable and comprehensive structural investigations, the exploitation of different complementary techniques is required. Here, we report that dual triplet-spin/fluorescent labels enable the first parallel distance measurements by electron spin resonance (ESR) and Förster resonance energy transfer (FRET) on exactly the same molecules with orthogonal chromophores, allowing for direct comparison. An improved light-induced triplet-triplet electron resonance method with 2-color excitation is used, improving the signal-to-noise ratio of the data and yielding a distance distribution that provides greater insight than the single distance resulting from FRET.

Figure 1

(a) LITTER pulse sequence: π/2 and π MW detection pulses (black) preceded by laser 1 (green) form a primary spin-echo (PE). The intensity is modulated by the formation of the second triplet by time-variant laser 2 (pump, red). (b) Amino acid sequences of [1] and [2], indicating the chromophore center-to-center distances determined by in vacuo DFT optimization (Figure S15) and chemical structures. Aib, α-aminoisobutyric acid; Eda, ethylenediamine. Other DFT optimized structures yielding higher energy local minima are presented in Figures S16 and S17 and Table S4.

Figure 2

(a) Room temperature optical absorption spectrum of [1] (black), with reference spectra of TPP (red) and ZnTPP (blue), normalized to the maximum of the Soret band. (b) Background-corrected and normalized 1-color (512/512 nm, τ = 960 ns, scans = 200, blue) and 2-color (512/556 nm, τ = 1730 ns, scans = 7580, red) LITTER traces of 40 μM [1] with modulation depths of 5% and 27%, acquired on the most intense feature of the TPP triplet spectrum and not orientationally averaged. The modulation-to-noise ratio (MNR) corrected for the number of scans for the two experiments is red:blue = 0.75:0.50.

Figure 3

(a) Room temperature absorption spectrum of [2] (black), TPP (red), and EB (blue). (b) Background-corrected and normalized 2-color (512/532 nm) LITTER traces at 40 μM (τ = 1730 ns, scans = 7410, blue) and 12 μM (τ = 1730 ns, scans = 4820, red) [2] with modulation depths of 8% and 23%, acquired on the most intense feature of the TPP triplet spectrum and not orientationally averaged. The modulation-to-noise ratio (MNR) corrected for the number of scans for the two experiments is red:blue = 0.55:0.40.

Figure 4

(a, b) 2-color LITTER traces averaged in orientation, background corrected, and normalized of [1] (a) (512/556 nm) and [2] (b) (512/532 nm) at 40 μM (red) with fits obtained using the Comparative DEER Analyzer in DEERAnalysis2022⁻ (black) with regularization parameter α of 0.22 and 0.31, respectively. (c, d) Concentration-normalized fluorescence spectra of [1] (c) and [2] (d) (black) and of singly labeled TPP peptide [3] (red), ZnTPP peptide [4] (c, blue), and EB peptide [5] (d, blue); see Table S1 for peptide sequences of [3], [4], and [5]. (e, f) Comparison of the distances determined for [1] (e) and [2] (f) by FRET (blue) with error bounds (cyan), LITTER (solid black line) with 95% confidence intervals (gray), which include analysis of the uncertainty in the background correction, and DFT (dotted black line).