Backbone flexibility of five sites on the catalytic subunit of cAMP-dependent protein kinase in the open and closed conformations.
To develop an alternative approach to measure peptidyl backbone flexibility and to expand our understanding of the segmental flexibility of cAMP-dependent protein kinase (cAPK), the effect of protein kinase inhibitor peptide, PKIalpha(5-24), and MgATP on the mobility of fluorescein selectively conjugated to five sites on the catalytic subunit of cAPK was examined. Specifically, five full-length, single-site catalytic subunit mutants (K16C, K81C, I244C, C199A, and N326C) were prepared, and fluorescein maleimide was selectively attached to the side chains of each substituted cysteine or, in the case of the C199A mutant, to the unprotected native C343. The time-resolved anisotropy decay profiles of the five fluorescein maleimide-conjugated mutants were well fit to a biexponential equation. The fast rotational correlation times of the fluorescein conjugates ranged between 1.9 and 2.8 ns and were inversely correlated (r = -0.87) to the averaged crystallographic main-chain atom B factors around each site of conjugation. The slow correlation times ranged between 25 and 28 ns and were about the same magnitude as the value of 21 ns estimated from the Stokes-Einstein equation. The presence of MgATP and PKIalpha(5-24), which induces the closed conformation of cAPK, was associated with a reduction of the fast rotational correlation time of the K81C conjugate, indicating that the peptidyl backbone around K81 is measurably less flexible when the C subunit is in the closed compared with the open conformation. The results suggest (i) that time-resolved fluorescence anisotropy can assess the nanosecond flexibility of short segments of the peptidyl backbone around each site of labeling and (ii) that the substrate/pseudosubstrate binding differentially affects the backbone flexibility of cAPK.
Gangal
M
M
Division of Biomedical Sciences, University of California, Riverside 92521-0121, Japan.
Cox
S
S
Lew
J
J
Clifford
T
T
Garrod
S M
SM
Aschbaher
M
M
Taylor
S S
SS
Johnson
D A
DA
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics; D005452:Fluoresceins; D005454:Fluorescence Polarization; D008239:Lysine / Q000235:genetics; D051379:Mice; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation
D005452:Fluoresceins; D010455:Peptides; C037041:fluorescein 5-maleimide; D017868:Cyclic AMP-Dependent Protein Kinases; D008239:Lysine; D003545:Cysteine