Extremely high thermal stability of streptavidin and avidin upon biotin binding

The effect of biotin binding on the thermal stability of streptavidin (STV) and avidin (AVD) was evaluated using differential scanning calorimetry. Bi otin binding increases the midpoint of temperature T-m of thermally induced denaturation of STV and AVD in phosphate buffer from 75 and 83 degrees C t o 112 and 117 degrees C at full biotin saturation, respectively. This therm ostability is the highest reported for proteins coming from either mesophil ic or thermophilic organisms. In both proteins, biotin also increases the c alorimetric enthalpy and the cooperativity of the unfolding. Thermal stabil ity of STV was also evaluated in the presence of high concentrations of ure a or guanidinium hydrochloride (GuHCl). In 6 M GuHCl, STV remains as a tetr amer and the T-m of the STV-biotin complex is centered at 108 degrees C, a few degrees below the value obtained in phosphate buffer. On the contrary, STV under fully saturating condition remains mainly in its dimeric form in 8 M urea and the thermogram shows two endotherms. The main endotherm at a l ower temperature has been ascribed to the dimeric liganded state with a Tm of 87 degrees C, and the higher temperature endotherm to the tetrameric lig anded form with a T-m of 106 degrees C. As the thermostability of unligande d protein in the presence of urea is unchanged upon binding we related the extremely high thermal stability of this protein to both an increase in str uctural ordering and compactness with the preservation of the tetramer inte grity. (C) 1999 Published by Elsevier Science B.V. All rights reserved.