The N-terminal Helix Controls the Transition between the Soluble and Amyloid States of an FF Domain

Abstract

BackgroundProtein aggregation is linked to the onset of an increasing number of human nonneuropathic (either localized or systemic) and neurodegenerative disorders. In particular, misfolding of native α-helical structures and their self-assembly into nonnative intermolecular β-sheets has been proposed to trigger amyloid fibril formation in Alzheimer’s and Parkinson’s diseases.MethodsHere, we use a battery of biophysical techniques to elucidate the conformational conversion of native α-helices into amyloid fibrils using an all-α FF domain as a model system.ResultsWe show that under mild denaturing conditions at low pH this FF domain self-assembles into amyloid fibrils. Theoretical and experimental dissection of the secondary structure elements in this domain indicates that the helix 1 at the N-terminus has both the highest α-helical and amyloid propensities, controlling the transition between soluble and aggregated states of the protein.ConclusionsThe data illustrates the overlap between the propensity to form native α-helices and amyloid structures in protein segments.SignificanceThe results presented contribute to explain why proteins cannot avoid the presence of aggregation-prone regions and indeed use stable α-helices as a strategy to neutralize such potentially deleterious stretches.

Amyloid Formation by Human Carboxypeptidase D Transthyretin-like Domain under Physiological Conditions

“…The conversion of folded proteins into amyloid assemblies generally requires nonphysiological conditions, such as extreme pH values (30,31), organic co-solvents (32,33), or high temperatures (34,35). These destabilizing environments cause proteins to partially or fully unfold leading to the exposure of aggregation-prone regions, which are able to form intermolecular interactions, thus triggering aggregation (36).…”

Trifluoroethanol Modulates Amyloid Formation by the All α-Helical URN1 FF Domain

“…At pH 2.5 the URN1-FF forms an MG state that retains most of the α-helical secondary structure content characteristic of the protein native state [27]. Accordingly, in these conditions, the far-UV CD spectrum indicates that most of the polypeptide chain adopts an α-helical conformation and the spectra is similar to that of the native protein at pH 5.7 (Figure 2A).…”

“…This extent is confirmed by the observed increase in the fluorescence emission of the dye 4,4′-Dianilino-1,1′-Binaphthyl-5,5′-Disulfonic Acid (bis-ANS) and the blue-shift of its maximum in the presence of the protein at low pH, when compared with the spectrum of bis-ANS in the presence of the native protein (Figure 2C). Despite the fact that the acid induced URN1-FF MG does not exhibit any evidence of the population of β-sheet conformations, this state is aggregation competent [27], which contrasts with the native state, where the protein remains soluble for years. Two main reasons might account for the different solubility of the two states: (i) opening of the tertiary structure and associated exposition of previously hidden non-polar residues would promote the establishment of hydrophobic intermolecular contacts leading to the formation of aggregation nuclei; (ii) fluctuations of the helical secondary structure would result in the transient formation of disordered protein regions able to establish intermolecular backbone hydrogen bonds, leading to formation of β-sheet enriched oligomers.…”

“…The conformational properties of this domain are receiving increasing attention, since its folding reaction involves the transient population of an on-pathway intermediate whose structure has been has been solved at atomic resolution by NMR, providing thus high-quality data on the structural constraints driving the early stages of folding [25,26]. In a recent work, we have shown that the yeast URN1-FF domain (Figure 1) populates a molten globule (MG) state under mild denaturing conditions at low pH, which, despite being devoid of any detectable β-sheet element, self-assembles into highly ordered amyloid fibrils [27]. Here, we investigate the impact of TFE-induced structural rearrangements on the process of amyloid formation by this all α-helical protein.…”

Observation of a Quadrupole Surface Plasmon Mode for Au Nanorods: Effects of Surface Roughness and Crystal Facets

“…Aβ40 and Aβ42 cDNAs were cloned into a pET28a vector [17]. The Src homology 3 (SH3) domain was cloned into a pBat4 plasmid [36, 37], Ure2p cloned into pER26 [23], and the URN1 FF domain cloned into a pETM‐30 vector [38, 39]. The resulting plasmids were transformed into E. coli BL21 (DE3) cells.…”