CRYSTALLOGRAPHIC ANALYSIS OF THE PH-DEPENDENT BINDING OF IMINOBIOTIN BY STREPTAVIDIN

Streptavidin binds 2'-iminobiotin in a pH-dependent fashion-affinity d ecreases as the pH is lowered. This property makes the purification of compounds conjugated to streptavidin or iminobiotin possible under mi ld conditions by affinity chromatography. In order to understand the m olecular details of this pH-dependent binding, we analyzed the crystal structures of the complex of core streptavidin with 2'-iminobiotin at pH values 4.0 and 7.5. The two structures are very similar to each ot her even at their binding sites. Although the relative abundance of th e protonated species of the ligand is increased more than 3,000-fold o n going from pH 7.5 to pH 4.0, both structures contain only the nonpro tonated form of the ligand. Streptavidin selects the nonprotonated for m, which, at pH 4.0, is one part in 7.9 X 10(7).