Application of commercially available fluorophores as triplet spin probes in EPR spectroscopy

Site-directed attachment of photoexcitable spin labels for light-induced pulsed dipolar spectroscopy

“…S9, ESI †) were derived. The values for RB-MA deviate slightly from those for unmodified RB 24 (D and E decrease by B130 and 80 MHz, respectively), while the results for the EO derivatives and AT12-MA are in good agreement with EO disodium salt 31 and with AT12 bearing a carboxylic acid residue in place of a maleimide. 24 Consequently, the chemical modification appears to have a limited effect on the triplet parameters.…”

“…[20][21][22] Serrer et al proposed dyes that are used in photodynamic therapy or ground state depletion microscopy, as they reach high triplet quantum yields F T and, unlike other compounds with a photoexcited triplet state such as fullerenes, 23 are water-soluble. 24 In this work, we set out to expand the applicability of LiPDS and present a set of photoexcitable spin labels for site-directed attachment to proteins. Together with a characterization of their light-excited triplet state, we show successful distance determinations by LaserIMD spectroscopy between a nitroxide spin marker and these labels, site-selectively attached to the oxidoreductase thioredoxin (TRX).…”

“…The latter is a well-established model system in SDSL owing to its rigid three-dimensional structure, the availability of X-ray structural data and the possibility to remove the native cysteines by site-directed mutagenesis without perturbing structural integrity. 25 Based on the work of Serrer et al, 24 we selected Rose Bengal (RB), 26 Eosin Y (EO) 26 and the microscopy dye Atto Thio12 (AT12) 27 as label candidates; all bear heavy atom substitutions promoting intersystem crossing to the triplet state through increased spin-orbit coupling. 28 For the orthogonal, siteselective dual labeling of TRX, we opted for a combination of maleimide-cysteine conjugation and the copper-catalyzed azido-alkyne cycloaddition (CuAAC) reaction between azidefunctionalized labels and an alkyne-bearing non-canonical amino acid (ncAA).…”

Light-Induced Triplet–Triplet Electron Resonance Spectroscopy

“… 29 These limitations could be overcome by a two-color version of LITTER, replacing one of the porphyrins by a suitable optically orthogonal photoexcitable moiety. 30 …”

“…29 These limitations could be overcome by a two-color version of LITTER, replacing one of the porphyrins by a suitable optically orthogonal photoexcitable moiety. 30 Conversely, theability to measure the dipolar interaction and thereby structural information between two chromophores of the same type is useful for systems in which orthogonal labeling is difficult, for example, homodimeric systems. In such systems, Forster resonance energy transfer (FRET) between two chromophores of the same type is possible only by observing fluorescence depolarization.…”