Resolving the nanometer-scale structure of biomolecules in natural conditions still remains a challenging task. We report the first distance measurement in nucleic acid at physiological temperature using electron paramagnetic resonance (EPR). The model 10-mer DNA duplex has been labeled with reactive forms of triarylmethyl radicals and then immobilized on a sorbent in water solution and investigated by double quantum coherence EPR. We succeeded in development of optimal triarylmethyl-based labels, approach for site-directed spin labeling and efficient immobilization procedure that, working together, allowed us to measure as long distances as ~4.6 nm with high accuracy at 310 K (37 °C).
Tris(2,3,5,6-tetrathiaaryl)methyl cations, which were generated from the corresponding triarylmethanols in the presence of strong acids, underwent reaction with nucleophiles to give trityl radicals, as the product of a one-electron reduction of the carbocation. Depending on the nature of the nucleophile, the only byproducts were either diamagnetic quinone methides or asymmetrical monosubstituted trityl radicals. Herein, we report a protocol for the large-scale synthesis of the Finland trityl, which has the advantage of high overall yield and reproducibility.
The orchestrated interaction of transmembrane proteins with other molecules mediates several crucial biological processes. Detergent solubilization may significantly alter or even abolish such hetero-oligomeric interactions, which makes observing them at high resolution in their native environment technically challenging. Dipolar electron paramagnetic resonance (EPR) techniques such as pulsed electro-electron double resonance (PELDOR) can provide very precise distances within biomolecules. To concurrently determine the inter-subunit interaction and the intra-subunit conformational changes in hetero-oligomeric complexes, a combination of different spin labels is required. Orthogonal spin labeling using a triarylmethyl (TAM) label in combination with a nitroxide label is used to detect protein-ligand interactions in native lipid bilayers. This approach provides a higher sensitivity and total selectivity and will greatly facilitate the investigation of multimeric transmembrane complexes employing different spin labels in the native lipid environment.
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