Versatile Trityl Spin Labels for Nanometer Distance Measurements on Biomolecules In Vitro and within Cells

Angew Chem Int Ed Engl. 2017 Jan 2;56(1):177-181. doi: 10.1002/anie.201609085. Epub 2016 Dec 5.

Abstract

Structure determination of biomacromolecules under in-cell conditions is a relevant yet challenging task. Electron paramagnetic resonance (EPR) distance measurements in combination with site-directed spin labeling (SDSL) are a valuable tool in this endeavor but the usually used nitroxide spin labels are not well-suited for in-cell measurements. In contrast, triarylmethyl (trityl) radicals are highly persistent, exhibit a long relaxation time and a narrow spectral width. Here, the synthesis of a versatile collection of trityl spin labels and their application in in vitro and in-cell trityl-iron distance measurements on a cytochrome P450 protein are described. The trityl labels show similar labeling efficiencies and better signal-to-noise ratios (SNR) as compared to the popular methanethiosulfonate spin label (MTSSL) and enabled a successful in-cell measurement.

Keywords: EPR spectroscopy; distance measurements; in-cell spectroscopy; protein structures; spin labeling.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cytochrome P-450 Enzyme System / analysis*
  • Electron Spin Resonance Spectroscopy / methods*
  • Iron / analysis
  • Oocytes / enzymology
  • Pseudomonas putida / enzymology*
  • Signal-To-Noise Ratio
  • Spin Labels / chemical synthesis*
  • Trityl Compounds / chemical synthesis*
  • Xenopus Proteins / analysis*
  • Xenopus laevis* / metabolism

Substances

  • Spin Labels
  • Trityl Compounds
  • Xenopus Proteins
  • Cytochrome P-450 Enzyme System
  • Iron