Endogenous tryptophan residues of cAPK regulatory subunit type IIbeta 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 addition 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 RIIbeta isoform uniquely contains two endogenous 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 reporter groups of their dynamics and microenvironment. Two single-point mutations, W58F and W243F, allowed the local environment of each Trp to be probed using steady-state and time-resolved fluorescence 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 mobile 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 RIIbeta linker region.
Zawadzki
Kerri M
KM
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, USA.
Pan
Chia-Pin
CP
Barkley
Mary D
MD
Johnson
David
D
Taylor
Susan S
SS
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D002942:Circular Dichroism; D054757:Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004795:Enzyme Stability / Q000235:genetics; D005454:Fluorescence Polarization / Q000379:methods; D014644:Genetic Variation; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D014364:Tryptophan / Q000235:genetics* / Q000378:metabolism
D054757:Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit; D014364:Tryptophan; D017868:Cyclic AMP-Dependent Protein Kinases