Stratum corneum protein mobility as evaluated by a spin label maleimide derivative

The molecular dynamics in the vicinity of sulfhydryl groups of stratum corneum (SC) proteins has been studied by electron paramagnetic resonance (EPR) spectroscopy of maleimide spin labels covalently bound to the proteins. The total amount of bound maleimide was around 4 nmol per mg of SC. We have interpreted the coexistence of two spectral components in the EPR spectra by a two-state model with a fraction of label hydrogen bonded to proteins and another fraction exposed to the aqueous environment. We showed that the relative populations among these two states, determined by spectral simulation, are in thermodynamic equilibrium. The calculated energetic gain for the nitroxide to form hydrogen bond with SC proteins rather than to be dissolved in the buffer was approximately 12 kcal/mol in the temperature range of 2-30 degrees C and approximately 5 kcal/mol in the range of 30-86 degrees C. Temperature profiles of other EPR parameters related to the rotational diffusion of the probe also showed changes in the temperature interval of 26-42 degrees C, suggesting alterations in the vibration modes of SC proteins which are sensitive to higher motional freedom above 26-42 degrees C. We also compared samples of intact and lipid-depleted SC and we found that the delipidization process does not alter significantly the backbone mobility in the SH group regions, but the data suggest that the protein cavity is more open in the case of the delipidized samples. These results contribute to the understanding of the protein participation in the barrier function of SC, and can be useful to improve the spectral analysis of site-directed spin labeling, particularly for a more quantitative description of the dynamic modes of the nitroxide side chains.