The Regulatory Subunit of PKA-I Remains Partially Structured and Undergoes β-Aggregation upon Thermal Denaturation

Abstract

BackgroundThe 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 FindingsAs 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/SignificanceThermal 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.

Polyphosphate Stabilizes Protein Unfolding Intermediates as Soluble Amyloid-like Oligomers

“…In contrast, LDH incubated in the absence of polyP lacked much of its original signal (Figures 1B, C). Our finding that LDH undergoes an α-helical to β-sheet transition upon thermal denaturation was reminiscent of studies with the regulatory subunit of protein kinase PKA or ovalbumin, which both adopt soluble so-called β-aggregates during thermal unfolding [11, 12]. In contrast to PKA and ovalalbumin, however, soluble LDH intermediates were only populated in the presence of polyP.…”

“…Over the past years, a number of different non-amyloidogenic proteins have been shown to adopt β-cross sheet features under the appropriate unfolding conditions. Examples include apomyoglobin, a purely α-helical, compactly folded proteins, which undergoes cross β-sheet fibril formation upon incubation in boric acid at pH 9.0, 65°C [22] or the regulatory subunit of PKA, which is a mixed α-helix / β-sheet protein that forms soluble cross β-sheet oligomers upon thermal denaturation [11]. These results led to the proposal that many (if not most) polypeptides have an intrinsic propensity to adopt a cross β-sheet conformation [23].…”

Discovery of compounds that protect tyrosine hydroxylase activity through different mechanisms

“…We monitored the Thioflavin T (ThT) fluorescence with hTH1 in the presence of compounds 1 and 3 in order to evaluate whether the increased thermal stability was associated to cross-β-intermolecular aggregation leading to either fibrils or just to a more amorphous oligomerization, as has been observed for other proteins (see e.g. [44]). At least in the presence of compound 3 the increase in ThT fluorescence indicates that hTH1 undergoes intermolecular β-aggregation (Fig.…”

X-ray structure of the first `extremo-α-carbonic anhydrase', a dimeric enzyme from the thermophilic bacteriumSulfurihydrogenibium yellowstonenseYO3AOP1

“…Because of the very high thermal stability of SspCA, it was not possible to obtain a complete thermal unfolding curve as the instrumental setup was unable to work at temperatures higher than 378 K. Surprisingly, a midpoint transition was observed at a temperature of 360.5 K with an increased negative ellipticity, suggesting an increase in -structure, either by intra-subunit formation of -sheets or -turns or by the formation of aggregates via interchain cross-interactions ( Fig. 2; Dao et al, 2011). However, this event was reversible as SspCA recovered its native spectrum after 10 min at room temperature (data not shown), thus confirming its thermally resistant nature.…”