@misc{10481/31128,
year = {2011},
url = {http://hdl.handle.net/10481/31128},
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.},
organization = {This research was financed in part with grants from The Research Council of Norway and the Norwegian Cancer Society (to AM and SOD). ALP is supported by a Ramon y Cajal research contract from the Spanish Ministry of Sciences and Innovation (MICINN).},
publisher = {Public Library of Science (PLOS)},
keywords = {Atomic force microscopy},
keywords = {Biochemical simulations},
keywords = {Cricular dichroism spectroscopy},
keywords = {Crystal structure},
keywords = {Fluorescence},
keywords = {Fluorescence microscopy},
keywords = {Protein structure},
keywords = {Thermal stability},
title = {The Regulatory Subunit of PKA-I Remains Partially Structured and Undergoes β-Aggregation upon Thermal Denaturation},
author = {Dao, Khanh K. and Pey Rodríguez, Ángel Luis and Gjerde, Anja Underhaug and Teigen, Knut and Byeon, In-Ja L. and Døskeland, Stein O. and Gronenborn, Angela M. and Martínez, Aurora},
}