EPR spin labeling study of conformational transitions of β-glycosidase from the hyperthermophilic archaeon Sulfolobus solfataricus expressed in Escherichia coli

The conformational transitions of thermophilic β-glycosidase from Sulfolobus solfataricus and the mechanism of its thermal and chemical activation were studied by electron paramagnetic resonance (EPR) of nitroxide spin labels immobilized on the protein matrix. For this purpose, β-glycosidase was covalently modified by maleimide nitroxide spin label (MAR^̇) and iodoacetamide nitroxide spin label (IAR^̇), both specific for -SH groups. The degree of modification was found to be independent of the temperature as well as of the presence of two enzyme activators, sodium dodecyl sulphate (SDS) and butanol. In addition, a dansyl-piperidine nitroxide radical probe (DR^̇), which has an affinity to the hydrophobic surfaces of proteins, was used in this study. The noncovalent binding of DR^̇ results in immediate formation of a probe-enzyme complex. At room temperature, the rotation frequency of the immobilized labels decreases in order of IAR^̇ > MAR^̇ ≥ DR^̇. The temperature measurements of rotation correlation frequencies (v_c) display values ranging from 6·10⁷ to 2·10⁸ s^-1 and indicate a discontinuity with the inflection point at temperature T_in in a range from 312 to 313 K. The observed enthalpies (ΔH^‡) and entropies (ΔS^‡) of the activation of spin label rotation were derived from the Arrhenius plots. The activation parameters were found to be typical for rigid model systems. The addition of SDS and butanol produced a slight shift of the inflection point and changes of spin-label mobility. A correlation between conformational transitions and enzyme thermal activation was discussed.