Probing the multidomain structure of the type I regulatory subunit of cAMP-dependent protein kinase using mutational analysis: Role and environment of endogenous tryptophans

The regulatory R-subunit of cAMP-dependent protein kinase (cAPK) is a therm ostable multidomain protein. It contains a dimerization domain at the N-ter minus followed by an inhibitor site that binds the catalytic C-subunit and two tandem cAMP-binding domains (A and B). Two of the three tryptophans in the RI alpha subunit, Trp188 and Trp222, lie in cAMP-binding domain A while Trp260 lies at the junction between domains A and B. The unfolding of wild -type RIa (wt-RI), monitored by intrinsic fluorescence, was described previ ously [Leon, D. A., Dostmann, W. R. G., and Taylor, S. S. (1991) Biochemist ry 30, 3035 (1)]. To determine the environment of each tryptophan and the r ole of the adjacent domain in folding and stabilization of domain A, three point mutations, W188Y, W222Y, and W260Y, were introduced. The secondary st ructure of wt-RI and the point mutants has been studied by far-UV circular dichroism spectropolarimetry (CD), The CD spectra of wt-RI and the three po int mutants are practically identical, and the thermal unfolding behavior i s very similar. Intrinsic fluorescence and iodide quenching in the presence of increasing urea established that: (a) Trp222 is the most buried, wherea s Trp188 is the most exposed to solvent; (b) Trp260 accounts for the quench ing of fluorescence when cAMP is bound; and (c) Trp222 contributes most to the intrinsic fluorescence of the wt-RI-subunit, while Trp188 contributes l east. For wt-RI, rR(W188Y), and rR(W260Y), removal of cAMP causes a destabi lization, while excess cAMP stabilizes these three proteins. In contrast, r R(W222Y) was not stabilized by excess cAMP.