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The regulatory subunit of PKA-I remains partially structured and undergoes ?-aggregation upon thermal denaturation.

ABSTRACT: BACKGROUND: The regulatory subunit (R) of cAMP-dependent protein kinase (PKA) is a modular flexible protein that responds with large conformational changes to the binding of the effector cAMP. Considering its highly dynamic nature, the protein is rather stable. We studied the thermal denaturation of full-length RI? and a truncated RI?(92-381) that contains the tandem cyclic nucleotide binding (CNB) domains A and B. METHODOLOGY/PRINCIPAL FINDINGS: As revealed by circular dichroism (CD) and differential scanning calorimetry, both RI? proteins contain significant residual structure in the heat-denatured state. As evidenced by CD, the predominantly ?-helical spectrum at 25°C with double negative peaks at 209 and 222 nm changes to a spectrum with a single negative peak at 212-216 nm, characteristic of ?-structure. A similar ??? transition occurs at higher temperature in the presence of cAMP. Thioflavin T fluorescence and atomic force microscopy studies support the notion that the structural transition is associated with cross-?-intermolecular aggregation and formation of non-fibrillar oligomers. CONCLUSIONS/SIGNIFICANCE: Thermal denaturation of RI? leads to partial loss of native packing with exposure of aggregation-prone motifs, such as the B' helices in the phosphate-binding cassettes of both CNB domains. The topology of the ?-sandwiches in these domains favors inter-molecular ?-aggregation, which is suppressed in the ligand-bound states of RI? under physiological conditions. Moreover, our results reveal that the CNB domains persist as structural cores through heat-denaturation.

SUBMITTER: Dao KK

PROVIDER: S-EPMC3048872 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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The regulatory subunit of PKA-I remains partially structured and undergoes β-aggregation upon thermal denaturation.

Dao Khanh K KK Pey Angel L AL Gjerde Anja Underhaug AU Teigen Knut K Byeon In-Ja L IJ Døskeland Stein O SO Gronenborn Angela M AM Martinez Aurora A

PloS one 20110304 3

<h4>Background</h4>The regulatory subunit (R) of cAMP-dependent protein kinase (PKA) is a modular flexible protein that responds with large conformational changes to the binding of the effector cAMP. Considering its highly dynamic nature, the protein is rather stable. We studied the thermal denaturation of full-length RIα and a truncated RIα(92-381) that contains the tandem cyclic nucleotide binding (CNB) domains A and B.<h4>Methodology/principal findings</h4>As revealed by circular dichroism (C ...[more]

PMID: 21394209

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Project description:Some years ago, we showed that thermo-chemically denatured, partially-unfolded forms of Pyrococcus furiosus triosephosphateisomerase (PfuTIM) display cold-denaturation upon cooling, and heat-renaturation upon reheating, in proportion with the extent of initial partial unfolding achieved. This was the first time that cold-denaturation was demonstrated for a hyperthermophile protein, following unlocking of surface salt bridges. Here, we describe the behavior of another hyperthermophile protein, the small, monomeric, 53 residues-long rubredoxin from Pyrococcus furiosus (PfRd), which is one of the most thermostable proteins known to man. Like PfuTIM, PfRd too displays cold-denaturation after initial thermo-chemical perturbation, however, with two differences: (i) PfRd requires considerably higher temperatures as well as higher concentrations of guanidium hydrochloride (Gdm.HCl) than PfuTIM; (ii) PfRd's cold-denaturation behavior during cooling after thermo-chemical perturbation is incompletely reversible, unlike PfuTIM's, which was clearly reversible (from each different conformation generated). Differential cold-denaturation treatments allow PfRd to access multiple partially-unfolded states, each of which is clearly highly kinetically-stable. We refer to these as 'Trishanku' unfolding intermediates (or TUIs). Fascinatingly, refolding of TUIs through removal of Gdm.HCl generates multiple partially-refolded, monomeric, kinetically-trapped, non-native 'Trishanku' refolding intermediates (or TRIs), which differ from each other and from native PfRd and TUIs, in structural content and susceptibility to proteolysis. We find that the occurrence of cold denaturation and observations of TUI and TRI states is contingent on the oxidation status of iron, with redox agents managing to modulate the molecule's behavior upon gaining access to PfRd's iron atom. Mass spectrometric examination provides no evidence of the formation of disulfide bonds, but other experiments suggest that the oxidation status of iron (and its extent of burial) together determine whether or not PfRd shows cold denaturation, and also whether redox agents are able to modulate its behavior.

Dataset Information

The regulatory subunit of PKA-I remains partially structured and undergoes ?-aggregation upon thermal denaturation.

Publications

The regulatory subunit of PKA-I remains partially structured and undergoes β-aggregation upon thermal denaturation.

Similar Datasets

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