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

The conformational transitions of thermophilic beta-glycosidase from Sulfol obus solfataricus and the mechanism of its thermal and chemical activation were studied by electron paramagnetic resonance (EPR) of nitroxide spin lab els immobilized on the protein matrix. For this purpose, beta-glycosidase w as covalently modified by maleimide nitroxide spin label (MAR(.)) and iodoa cetamide nitroxide spin label (IAR(.)), both specific for -SH groups. The d egree of modification was found to be independent of the temperature as wel l 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 formati on of a probe-enzyme complex. At room temperature, the rotation frequency o f the immobilized labels decreases in order of IAR(.) > MAR(.) greater than or equal to DR.. The temperature measurements of rotation correlation freq uencies (nu(c)) display values ranging from 6 . 10(7) to 2 . 10(8) s(-1) an d indicate a discontinuity with the inflection point at temperature T-in in a range from 312 to 313 K. The observed enthalpies (Delta H-not equal) and entropies (Delta S-not equal) of the activation of spin label rotation wer e derived from the Arrhenius plots. The activation parameters were found to be typical for rigid model systems. The addition of SDS and butanol produc ed a slight shift of the inflection point and changes of spin-label mobilit y. A correlation between conformational transitions and enzyme thermal acti vation was discussed.