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 gxx 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.