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

Abstract

We present a novel pulsed electron paramagnetic resonance (EPR) spectroscopic ruler to test the performance of a recently developed spin-labeling method based on the photoexcited triplet state (S=1). Four-pulse electron double resonance (PELDOR) experiments are carried out on a series of helical peptides, labeled at the N-terminal end with the porphyrin moiety, which can be excited to the triplet state, and with the nitroxide at various sequence positions, spanning distances in the range 1.8-8 nm. The PELDOR traces provide accurate distance measurements for all the ruler series, showing deep envelope modulations at frequencies varying in a progressive way according to the increasing distance between the spin labels. The upper limit is evaluated and found to be around 8 nm. The PELDOR-derived distances are in excellent agreement with theoretical predictions. We demonstrate that high sensitivity is acquired using the triplet state as a spin label by comparison with Cu(II)-porphyrin analogues. The new labeling approach has a high potential for measuring nanometer distances in more complex biological systems due to the properties of the porphyrin triplet state.

Synthesis of μ2‐Oxo‐Bridged Iron(III) Tetraphenylporphyrin–Spacer–Nitroxide Dimers and their Structural and Dynamics Characterization by using EPR and MD Simulations

“…Both have a singlet ground spin state, but can be photoexcited to a paramagnetic triplet spin state . This allows such porphyrins to be studied by time‐resolved electron paramagnetic resonance (EPR) techniques and even to be considered for the role of spin labels for biomolecules . Furthermore, the porphyrin core can also host a number of different metal ions, such as Mg 2+ , Zn 2+ , Cu 2+ , Mn 2+ /Mn 3+ , Fe 2+ /Fe 3+ , or Co 2+ /Co 3+ .…”

“…[3] This allows such porphyrins to be studied by time-resolvede lectron paramagnetic resonance (EPR) techniques [4] and even to be considered for the role of spin labels for biomolecules. [5] Furthermore, the porphyrin core can also host an umber of different metal ions, such as Mg 2 + ,Z n 2 + , Cu 2 + ,M n 2 + /Mn 3 + ,F e 2 + /Fe 3 + ,o rC o 2 + /Co 3 + . [2] As several of these ions are paramagnetic, the corresponding metalloporphyrins can be studied by EPR.…”

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).…”