Flexibilities of isoindoline-derived spin labels for nucleic acids by orientation selective PELDOR

Abstract

Pulsed electron electron double resonance experiments with rigid spin labels can reveal very detailed information about the structure and conformational flexibility of nucleic acid molecules. On the other hand, the analysis of such data is more involved the distance and orientation information encoded in the time domain data need to be extracted and separated. In this respect studies with different spin labels with variable internal mobility are interesting and can help to unambiguously interpret the EPR data. Here orientation selective multi-frequency/multi-field 4-pulse PELDOR/DEER experiments with three recently presented semi-rigid or conformationally unambiguous isoindoline-derived spin labels were performed and simulated quantitatively by taking the spin label dynamics into account. PELDOR measurements were performed for a 20-mer dsDNA with two spin labels attached to two defined uridine derivatives. Measurements were recorded for different spin label positions within the double helical strand and for different magnetic field strengths. The experimental data sets were compared with simulations, taking into account the previously described dsDNA dynamics and the internal motions of the spin label itself, which had shown distinct differences between the three spin labels used. The (ExIm)U spin label shows a free rotation around a single bond, which averages out orientation effects, without influencing the distance distribution as it can occur in other spin labels. The (Im)U and (Ox)U spin label, on the other hand, show distinct orientation behaviour with minimal intrinsic motion. We could quantitatively determine this internal motion and demonstrate that the conformational dynamics of the nucleic acid and the spin label can be well separated by this approach.

The structural flexibility of the human copper chaperone Atox1: Insights from combined pulsed EPR studies and computations

“…EPR spectroscopy has emerged as an excellent tool for investigating such systems, since it does not require crystallization, and its performance is independent of protein size . EPR requires paramagnetic probes; for diamagnetic systems, site‐directed spin labeling (SDSL) is currently widely used in biophysical research .…”

Nucleotide‐Independent Copper(II)‐Based Distance Measurements in DNA by Pulsed ESR Spectroscopy

“…Furthermore, owing to limited spectral excitation, multiple experiments are often required to resolve a single distance. There has also been a large effort to develop spin labels that require less post‐synthesis modification ,,. However, these labels position the nitroxide away from the duplex, further complicating data analysis.…”

Effects of Fluorophore Attachment on Protein Conformation and Dynamics Studied by spFRET and NMR Spectroscopy

“…While potential problems associated with the use of large fluorescent labels, such as GFP mutants have been discussed [11–13] , the conjugation of small organic fluorophores is usually thought to have negligible effects. A few studies have already shown the effect of fluorophore attachment to nucleic acids [8–10,14,15] and some deal with the effects of dyes on certain protein characteristics [14,16,17] . For example, Dallmann et al modified DNA oligomers with 2-aminopurine and investigated their structural changes by 2D NMR spectroscopy.…”

“…This problem is not unique to labeling of proteins with fluorescent dyes, but extends to other types of protein tagging methods as well. For example, in NMR and EPR studies, paramagnetic tagging with spin labels (e.g., IPSL, MTSSL), lanthanide chelating tags (e.g., DOTA, DTPA, EDTA), or lanthanide binding tags (LBTs), are regularly used for investigation of structure, dynamics, and function of proteins and protein–ligand interactions. To date, fluorescent proteins are frequently used in fusion constructs with proteins of interest to be studied due to the straightforward genetic encoding for expression in cells.…”

“…Though potential problems associated with the use of large fluorescent labels, such as mutants of the green fluorescence protein (GFP) have been discussed, the conjugation of small organic fluorophores is usually thought to have negligible effects. A few studies have investigated the effect of fluorophore attachment to nucleic acids and some deal with the effects of dyes on certain protein characteristics . For example, Dallmann et al.…”