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Endogenous tryptophan residues of cAPK regulatory subunit type IIβ reveal local variations in environments and dynamics
Author(s) -
Zawadzki Kerri M.,
Pan ChiaPin,
Barkley Mary D.,
Johnson David,
Taylor Susan S.
Publication year - 2003
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.10326
Subject(s) - linker , protein subunit , tryptophan , docking (animal) , biophysics , gene isoform , chemistry , binding site , biochemistry , biology , amino acid , gene , medicine , nursing , computer science , operating system
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 RIIβ 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 RIIβ linker region. Proteins 2003;51:552–561. © 2003 Wiley‐Liss, Inc.