Spin labeling of oligonucleotides with nitroxides is hampered by their intrinsic instability under conditions of solid‐phase synthesis and enzymatic ligation. Although nitroxide decomposition can be avoided in some cases by postsynthetic introduction or by special reaction conditions, a more general solution would be reversible protection of the radical. We have recently developed such a method based on photolabile protection groups for DNA oligonucleotides and demonstrated their application in EPR spectroscopy. Here, we extend this method to RNA oligonucleotides. By improving the synthetic procedures, the yield of the coumarin‐protected phosphoramidite could be increased by a factor of 12. Effective recovery of the nitroxides on a duplex RNA enables pulsed EPR experiments to be performed directly after irradiation and air oxidation. Data at Q‐band frequency is shown and distances measured with PELDOR (pulsed electron‐electron double resonance) spectroscopy agree well with the calculated values.
We recently engineered encodable lanthanide binding tags (LBTs) into proteins and demonstrated their applicability in Nuclear Magnetic Resonance (NMR) spectroscopy, X-ray crystallography and luminescence studies. Here, we engineered two-loop-LBTs into the model protein interleukin-1b (IL1b) and measured 1 H, 15 Npseudocontact shifts (PCSs) by NMR spectroscopy. We determined the Dv-tensors associated with each Tm 3? -loaded loop-LBT and show that the experimental PCSs yield structural information at the interface between the two metal ion centers at atomic resolution. Such information is very valuable for the determination of the sites of interfaces in protein-protein-complexes. Combining the experimental PCSs of the two-loop-LBT construct IL1b-S2R2 and the respective single-loop-LBT constructs IL1b-S2, IL1b-R2 we additionally determined the distance between the metal ion centers. Further, we explore the use of two-loop LBTs loaded with Gd 3? as a novel tool for distance determination by Electron Paramagnetic Resonance spectroscopy and show the NMR-derived distances to be remarkably consistent with distances derived from Pulsed Electron-Electron Dipolar Resonance.
Nitroxide spin labels can be protected against critical conditions of DNA/RNA or peptide synthesis by reduction and alkylation with light‐sensitive groups such as nitrobenzyl‐ or aminocoumarins. High chemical stability qualifies tetraethylisoindoline 5 and, with some restrictions, (2,2,6,6‐tetramethylpiperidin‐1‐yl)oxy (TEMPO) precursor 4 as building blocks for spin‐labeled biopolymers. The yield of recovered nitroxides (80–95 %) is sufficient for PELDOR experiments. A protected TEMPO phosphoramidite was successfully used to synthesize a short spin‐labeled DNA with high yield and purity.
An isoindoline-nitroxide derivative of guanine (Ǵ, "G-spin") was shown to bind specifically and effectively to abasic sites in duplex RNAs. Distance measurements on a Ǵ-labeled duplex RNA with PELDOR (DEER) showed a strong orientation dependence. Thus, Ǵ is a readily synthesized, orientation-selective spin label for "mix and measure" PELDOR experiments.
The investigation of the structure and conformational dynamics of biomolecules under physiological conditions is challenging for structural biology. Although pulsed electron paramagnetic resonance (like PELDOR) techniques provide long-range distance and orientation information with high accuracy, such studies are usually performed at cryogenic temperatures. At room temperature (RT) PELDOR studies are seemingly impossible due to short electronic relaxation times and loss of dipolar interactions through rotational averaging. We incorporated the rigid nitroxide spin label Ç into a DNA duplex and immobilized the sample on a solid support to overcome this limitation. This enabled orientation-selective PELDOR measurements at RT. A comparison with data recorded at 50 K revealed averaging of internal dynamics, which occur on the ns time range at RT. Thus, our approach adds a new method to study structural and dynamical processes at physiological temperature in the <10 μs time range with atomistic resolution.
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