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

The molecular dynamics in the vicinity of sulfhydryl groups of stratum corn eum (SC) proteins has been studied by electron paramagnetic resonance (EPR) spectroscopy of maleimide spin labels covalently bound to the proteins. Th e total amount of bound maleimide was around 4 nmol per mg of SC. We have i nterpreted 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 re lative populations among these two states, determined by spectral simulatio n, are in thermodynamic equilibrium. The calculated energetic gain for the nitroxide to form hydrogen bond with SC proteins rather than to be dissolve d in the buffer was similar to 12 kcal/mol in the temperature range of 2-30 degrees C and similar to 5 kcal/mol in the range of 30-86 degrees C. Tempe rature profiles of other EPR parameters related to the rotational diffusion of the probe also showed changes in the temperature interval of 26-42 degr ees C, suggesting alterations in the vibration modes of SC proteins which a re sensitive to higher motional freedom above 26-42 degrees C. We also comp ared samples of intact and lipid-depleted SC and we found that the delipidi zation process does not alter significantly the backbone mobility in the SH group regions, but the data suggest that the protein cavity is more open i n the case of the delipidized samples. These results contribute to the unde rstanding of the protein participation in the barrier function of SC, and c an be useful to improve the spectral analysis of site-directed spin labelin g, particularly for a more quantitative description of the dynamic modes of the nitroxide side chains. (C) 2000 Published by Elsevier Science B.V. All rights reserved.