Porphyrin Triplet State as a Potential Spin Label for Nanometer Distance Measurements by PELDOR Spectroscopy

Abstract

This work demonstrates, for the first time, the feasibility of applying pulsed electron-electron double resonance (PELDOR/DEER) to determine the interspin distance between a photoexcited porphyrin triplet state (S = 1) and a nitroxide spin label chemically incorporated into a small helical peptide. The PELDOR trace shows deep envelope modulation induced by electron-electron dipole interaction between the partners in the pair, providing an accurate distance measurement. This new labeling approach has a high potential for measuring nanometer distances in more complex biological systems due to the sensitivity acquired from the spin polarization of the photoexcited triplet state spectrum.

Light‐Induced Pulsed EPR Dipolar Spectroscopy on a Paradigmatic Hemeprotein

“…[19] Recently, Cu(II) and high spin Mn(II) tags have also been successfully employed. [25,26] The large electron spin polarization of the photoexcited triplet state, [27,28] and the consequently high sensitivity of the experiment, furthermore allowed light-induced PDS methodology to be applied to a photosynthetic protein, containing an endogenous carotenoid triplet state probe. [24] One important new development is the demonstration that the triplet state of porphyrin chromophores can be exploited to determine inter-spin distances.…”

“…[20][21][22][23] The search for alternative spin labels is an active area of research. [29] The dipolar measurements were performed with light-induced DEER (LiDEER), [25,26] a variation of the conventional 4-pulse DEER sequence where a laser pulse is used to generate the triplet state before the application of dichromatic microwave pulses: the detection frequency is resonant with the photo-induced porphyrin triplet and the pump is resonant with the stable nitroxide radical. The first work in this area was conducted on a peptide-based molecular ruler containing a nitroxide probe and porphyrin moiety.…”

“…For this purpose we employed an α-helix peptide, used in previous studies, [25,26,30] labeled with a tetra-phenylporphyrin moiety and with the unnatural amino acid TOAC (4-amino-1-oxyl-2,2,6,6-tetra-methylpiperidine-4-carboxylic acid). For this purpose we employed an α-helix peptide, used in previous studies, [25,26,30] labeled with a tetra-phenylporphyrin moiety and with the unnatural amino acid TOAC (4-amino-1-oxyl-2,2,6,6-tetra-methylpiperidine-4-carboxylic acid).…”

Light‐Induced Porphyrin‐Based Spectroscopic Ruler for Nanometer Distance Measurements

“…Recently, we have demonstrated the feasibility of applying PELDOR to determine the interspin distance between a photoexcited porphyrin triplet state ( S =1) and a nitroxide spin label chemically incorporated into a small helical peptide . This new labeling approach provides high sensitivity due to the characteristics of the triplet state.…”

“…The nitroxide label was inserted at various sequence positions, spanning distances in the range of 1.8–8.0 nm (for details see Figure ), replacing the original Aib amino acid with the structurally comparable 4‐amino‐1‐oxyl‐2,2,6,6‐tetramethyl‐piperidine‐4‐carboxylic acid (TOAC) residue, in which the nitroxyl radical is fixed on a rigid heterocyclic structure. Aib residues are strong promoters of helical conformations and consequently ensure a well‐defined geometry in terms of distance, relative orientation, and restricted conformational flexibility, as already demonstrated for the parent compound …”