Pulse dipolar EPR is widely used to study tertiary structure, dynamics and functional features of biomolecules. Nitroxides are commonly used spin labels. Trityl radicals or TAMs have appeared recently as an alternative source of spin labels using PDEPR. In this presentation we compared functional properties of spin labels based on TAMs and nitroxides.PDEPR in combination with MD were used to investigate the conformational changes in DNA with AP site and in complex with AP endonuclease1 (APE1). For this sake, triarylmethyl (TAM) based spin labels were attached to the 5′-ends of oligonucleotide duplex, and nitroxide spin labels were introduced into the APE1 site. In this way, we for the first time created the system that allowed to follow the conformational changes of the main APE1 substrate by EPR. The use of different (orthogonal) spin labels in the enzyme and in DNA substrate has crucial advantage and allows detailed investigation of local damages and conformational changes in AP-DNA alone, as well as in its complex with APE1. We use very hydrophilic OX063 with very-low toxicity and little tendency for aggregation as the basis for a spin label for human serum albumin (HSA). EPR spectra of HSA-OX063 have an intense, narrow line typical of TAM radicals in solution while HSA-FTAM showed extensive aggregation. In pulse EPR measurements, the measured Tm for HSA-OX063 is 6.3 μs at 50 K, the longest yet obtained with trityl based spin-labels. CW and PDEPR were used to study of the mechanism of penetration of TAM or nitroxide spin labeled intrinsically disordered protein into human cell.
This study was supported by Ministry of Science and Education of the RF(grant14.W03.31.0034).
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