Conformational transition accompanying the binding of Ca2+ to the protein activator of 3',5'-cyclic adenosine monophosphate phosphodiesterase.

The Ca2+-dependent protein activator of 3':5'-cyclic adenosine monophosphate phosphodiesterase is shown to undergo a conformational transition upon binding of 2 mol of Ca2+/mol of activator. Circular dichroic studies indicate that Ca2+ induces an increase of 5-8% in alpha-helix content with a concomitant decrease in the amount of random coil. In the absence of Ca2+ and in the presence of [ethylenebis(oxoethylenenitrilo)]tetraacetic acid (EGTA), the protein contains 30-35% alpha helix, 50% random coil, and 15-20% beta-pleated sheat. Spectrophotometric titration indicates that the two tyrosyl residues have pK's of 10.4 and 11.9 and are therefore in different environments. The Ca2+-induced conformational change is accompanied by an increased exposure to protons of the partially exposed tyrosine, as shown by a shift in its pK from 10.4 to 10.). Increased solvation is also consistent with a negative difference spectrum at 287 and 279 nm as seen upon Ca2+ binding. Modification in the environment of all or some of the phenylalanine residues also is part of the conformational change accompanying Ca2+ binding. A new and rapid purification procedure which yields large amounts (25-30% yields) of homogenous protein activator and a direct and sensitive assay procedure for cAMP phosphodiesterase and its activator are also described.