Hydrogen bonding to the nitroxide of protein bound spin labels

Abstract

Deuterium electron spin-echo envelope modulation spectroscopy (²H-ESEEM) (9.3 GHz, 0.33T) and continuous wave high-field (95 GHz, 3.4T) electron paramagnetic resonance (EPR) spectroscopy were applied to disentangle the sources of protons which are hydrogen bonded to nitroxides of protein bound spin labels. For a set of 12 colicin A mutants with nitroxide spin labels site-specifically introduced at residue positions 26, 33, 42, 62, 91, 105, 115, 154, 166, 169, 181 and 192 of the channel forming domain we analysed the heterogeneity in the g_xx region of EPR spectra detected at 95 GHz and quantified the fractions of nitroxide populations engaged in H-bonding to water and protein protons. The amplitudes of the narrow and broad components of ²H-ESEEM spectra arising from non-bonded and bonded water (D₂O) molecules or deuterated protein components reflect the local H-bonding equilibria. The combination of both methods allowed estimating the fraction of nitroxide populations hydrogen bonded to non-exchangeable protein protons. Hydrogen bonds are known to play a governing role in protein folding and function, and the presented approach extends the information obtainable from site-directed spin labelling in the investigation of complex biomolecular systems and their conformational changes.

Keywords:

• electron paramagnetic resonance
• ESEEM
• spin labelling
• nitroxide
• hydrogen bonding

Acknowledgements

The authors thank D. Klose and J. Klare for fruitful discussions and L.P. Pulagam for colicin A preparations. The authors gratefully acknowledge funding by the Deutsche Forschungsgemeinschaft (STE640-10 and SFB 944, P10).