Conformational transition accompanying the binding of calcium(2+) ion to the protein activator of 3',5'-cyclic adenosine monophosphate phosphodiesterase

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Abstract
The Ca2+-dependent protein activator of cyclic[c]AMP phosphodiesterase undergoes a conformational transition 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 2 tyrosyl residues have pK of 10.4 and 11.9 and are in different environments. The Ca2+-induced conformational changes is accompanied by an increased exposure to protons of the partially exposed tyrosine, as shown by a shift in its pK from 10.4-10.1. Increased solvation is also consistent with a negative difference spectrum at 287 and 279 nm as seen on 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 homogeneous protein activator and a direct and sensitive assay procedure for cAMP phosphodiesterase and its activator are also described.