Endogenous tryptophan residues of cAPK regulatory subunit type IIβ reveal local variations in environments and dynamics

The amino terminal dimerization/ docking domain and the two-tandem, carboxy- terminal cAMP-binding domains (A and B) of cAMP- dependent protein kinase regulatory (R) subunits are connected by a variable linker region. In addi- tion to providing a docking site for the catalytic subunit, the linker region is a major source of sequence diversity between the R-subunit isoforms. The RII isoform uniquely contains two endoge- nous tryptophan residues, one at position 58 in the linker region and the other at position 243 in cAMP- binding domain A, which can act as intrinsic re- porter groups of their dynamics and microenviron- ment. Two single-point mutations, W58F and W243F, allowed the local environment of each Trp to be probed using steady-state and time-resolved fluores- cence techniques. We report that: (a) the tryptophan fluorescence of the wild-type protein largely reflects Trp243 emission; (2) cAMP selectively quenches Trp243 and thus acts as a cAMP sensor; (3) Trp58 resides in a highly solvated, unstructured, and mo- bile region of the protein; and (4) Trp243 resides in a stable, folded domain and is relatively buried and rigid within the domain. The use of endogenous Trp residues presents a non-perturbing method for studying R-subunit subdomain characteristics in addition to providing the first biophysical data on the RII linker region. Proteins 2003;51:552-561.