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

Dao, KK and Pey, AL and Gjerde, AU and Teigen, K and Byeon, IJL and Døskeland, SO and Gronenborn, AM and Martinez, A (2011) The regulatory subunit of PKA-I remains partially structured and undergoes β-aggregation upon thermal denaturation. PLoS ONE, 6 (3).

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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 RIα and a truncated RIα(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 RIα proteins contain significant residual structure in the heat-denatured state. As evidenced by CD, the predominantly α-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 β-structure. A similar α→β 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-β-intermolecular aggregation and formation of non-fibrillar oligomers. Conclusions/Significance: Thermal denaturation of RIα 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 β-sandwiches in these domains favors inter-molecular β-aggregation, which is suppressed in the ligand-bound states of RIα under physiological conditions. Moreover, our results reveal that the CNB domains persist as structural cores through heat-denaturation. © 2011 Dao et al.

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Details

Item Type: Article
Status: Published
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Dao, KK
Pey, AL
Gjerde, AU
Teigen, K
Byeon, IJLilb6@pitt.eduILB6
Døskeland, SO
Gronenborn, AMamg100@pitt.eduAMG100
Martinez, A
Date: 11 March 2011
Date Type: Publication
Journal or Publication Title: PLoS ONE
Volume: 6
Number: 3
DOI or Unique Handle: 10.1371/journal.pone.0017602
Schools and Programs: School of Public Health > Biostatistics
Refereed: Yes
MeSH Headings: Calorimetry, Differential Scanning; Circular Dichroism; Cyclic AMP--pharmacology; Cyclic AMP-Dependent Protein Kinase RIalpha Subunit--chemistry; Cyclic AMP-Dependent Protein Kinase RIalpha Subunit--metabolism; Enzyme Stability--drug effects; Fluorescence; Humans; Light; Microscopy, Atomic Force; Molecular Dynamics Simulation; Protein Denaturation--drug effects; Protein Structure, Quaternary; Protein Structure, Secondary; Protein Unfolding--drug effects; Scattering, Radiation; Temperature; Thiazoles--metabolism
Other ID: NLM PMC3048872
PubMed Central ID: PMC3048872
PubMed ID: 21394209
Date Deposited: 07 Aug 2012 15:50
Last Modified: 29 Jan 2019 15:55
URI: http://d-scholarship.pitt.edu/id/eprint/13427

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