Stratum corneum protein dynamics as evaluated by a spin-label maleimide derivative: Effect of urea

The stratum corneum (SC) protein dynamics in the sulfhydryl group regions w as studied by electron paramagnetic resonance (EPR) spectroscopy of a coval ently attached maleimide derivative spin label. A two-state model for the n itroxide described the coexistence of two spectral components in the EPR sp ectra. The so-called strongly immobilized component arises from a spin-labe l fraction with the nitroxide moiety hydrogen-bonded to protein (rigid stru cture) and the weakly immobilized component is provided by the spin labels with higher mobility (similar to 10 times greater) exposed to the aqueous e nvironment. The relative populations between these two states are in thermo dynamic equilibrium. The apparent energetic gain for the nitroxide to form a hydrogen bond with the backbone rather than to be dissolved in the local environment was similar to 10 kcal/mol in the temperature range of 2-30 deg reesC and similar to6 kcal/mol in the range of 30-70 degreesC. Urea treatme nt caused a drastic increase in the segmental motion of the polypeptide cha ins that was completely reversible by its removal. Our analyses also indica ted that the urea induced unfolding of the SC proteins opening the thiol gr oup cavities. This work can also be useful to improve the spectral analysis of site-directed spin-labeling, especially for a more quantitative descrip tion of the nitroxide side chain mobility.