Unfolding of the regulatory subunit of cAMP-dependent protein kinase I.


Abstract

The unfolding of the recombinant regulatory subunit of cAMP-dependent protein kinase I was followed by monitoring the intrinsic protein fluorescence. Unfolding proceeds in at least two stages. First, the quenching of fluorescence due to cAMP binding is abolished at relatively low levels of urea (less than 2 M) and is observed as an increase in intensity at 340 nm. The high-affinity binding of cAMP is retained in 3 M urea even though the quenching is lost. The second stage of unfolding, presumably representing unfolding of the polypeptide chain, is seen as a shift in lambda max from 340 to 353 nm. The midpoint concentration, Cm, for this process is 5.0 M. Cyclic AMP binding activity is lost at a half-maximal urea concentration of 3.5 M and precedes the shift in lambda max. Unfolding of the protein in the presence of urea was fully reversible; furthermore, the presence of excess levels of cAMP stabilized the regulatory subunit. A free energy value (delta GDH2O) of 7.1 +/- 0.2 kcal/mol was calculated for the native form of the protein when denaturation was induced with either urea or guanidine hydrochloride. Iodide quenching of tryptophan fluorescence was used to elucidate the number of tryptophan residues accessible during various stages of the unfolding process. In the native cAMP-bound form of the regulatory subunit, only one of the three tryptophans in the regulatory subunit is quenched by iodide while more than two tryptophans can be quenched with iodide in the presence of 3 M urea. Study holds ProTherm entries: 4317, 4318 Extra Details: additive : EDTA(0.5 mM), cAMP binding; activity; tryptophan residues; regulatory subunit

Submission Details

ID: 5zEggzMV4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:25 p.m.

Version: 1

Publication Details
León DA;Dostmann WR;Taylor SS,Biochemistry (1991) Unfolding of the regulatory subunit of cAMP-dependent protein kinase I. PMID:1848784
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
4F9K 2012-06-06 2.8 Crystal Structure of human cAMP-dependent protein kinase type I-beta regulatory subunit (fragment 11-73), Northeast Structural Genomics Consortium (NESG) Target HR8613A
4DIN 2012-07-04 3.7 Novel Localization and Quaternary Structure of the PKA RI beta Holoenzyme

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
94.8 cAMP-dependent protein kinase type I-beta regulatory subunit P31321 KAP1_HUMAN
99.7 cAMP-dependent protein kinase type I-beta regulatory subunit P81377 KAP1_RAT
100.0 cAMP-dependent protein kinase type I-beta regulatory subunit P12849 KAP1_MOUSE