INTERACTION OF BIOTIN WITH STREPTAVIDIN - THERMOSTABILITY AND CONFORMATIONAL-CHANGES UPON BINDING

The effect of biotin binding on streptavidin (STV) structure and stabi lity was studied using differential scanning calorimetry, Fourier tran sform infrared spectroscopy (FT-IR), and fluorescence spectroscopy. Bi otin increases the midpoint temperature T-m, of thermally induced dena turation of STV from 75 degrees C in unliganded protein to 112 degrees C at full ligand saturation, The cooperativity of thermally induced u nfolding of STV changes substantially in presence of biotin, Unligande d STV monomer has' at least one domain that unfolds independently, The dimer bound to biotin undergoes a single coupled denaturation process , Simulations of thermogranls of STV denaturation that take into accou nt only the thermodynamic effects of the ligand with a K-alpha similar to 10(15) reproduce the behavior observed, but the estimated values o f T-m are 15-20 degrees C lower than those experimentally determined, This increased stability is attributed to an enhanced cooperativity of the thermal unfolding of STV. The increment in the cooperativity is a s consequence of a stronger intersubunit association and an increased structural order upon binding, FT-IR and fluorescence spectroscopy dat a reveal that unordered structure found in unliganded STV disappears u nder fully saturating conditions. The data provide a rationale for pre vious suggestions that biotin binding induces an increase in protein t ightness (structural cooperativity) leading, in turn, to a higher ther mostability.