A Single Mutation Induces Amyloid Aggregation in the α-Spectrin SH3 Domain: Analysis of the Early Stages of Fibril Formation

Abstract

The Src-homology region 3 domain of chicken alpha-spectrin (Spc-SH3) is a small two-state folding protein, which has never been described to form amyloid fibrils under any condition investigated so far. We show here that the mutation of asparagine 47 to alanine at the distal loop, which destabilises similarly the native and folding transition states of the domain, induces the formation of amyloid fibrils under mild acid conditions. Amyloid aggregation of the mutant is enhanced by the increase in temperature, protein concentration and NaCl concentration. The early stages of amyloid formation have been monitored as a function of time and temperature using a variety of biophysical methods. Differential scanning calorimetry experiments under conditions of amyloid formation have allowed the identification of different thermal transitions corresponding to conformational and aggregation processes as well as to the high-temperature disaggregation and unfolding of the amyloid fibrils. Aggregation is preceded by a rapid conformational change in the monomeric domain involving about 40% of the global unfolding enthalpy, considerable change in secondary structure, large loss of tertiary structure and exposure of hydrophobic patches to the solvent. The conformational change is followed by formation of a majority of oligomeric species with apparent hydrodynamic radius between 2.5 nm and 10nm, depending on temperature, together with the appearance and progressive growth of protofibrillar aggregates. After these early aggregation stages, long and curved fibrils of up to several micrometers start to develop by elongation of the protofibrils. The calorimetric data indicate that the specific enthalpy of fibril disaggregation and unfolding is relatively low, suggesting a low density of interactions within the fibril structure as compared to the native protein and a main entropy contribution to the stability of the amyloid fibrils.

Application and use of differential scanning calorimetry in studies of thermal fluctuation associated with amyloid fibril formation

“…In remarkable contrast, the WL fibrils of β 2 -m show no effect of exothermic heat prior to the endothermic peak that represents the melting of the fibrils. Similar effects of heat have been observed in DSC measurements in other studies (Azuaga et al 2002;Razaei et al 2002;Baxa et al 2004;Morel et al 2006Morel et al , 2010. These contrasting results may be ascribed to the structural diversity of amyloid fibrils formed from a protein (Kreplak and Aebi 2006;Pedersen et al 2010) and the solution conditions used.…”

“…With a non-linear least-square fitting program, the calculated DSC curves were fitted to the observed curves so that a and b were determined (Sasahara et al 2005) Thermally induced melting of amyloid fibrils Recent calorimetric studies on protein aggregation are summarized in Table 1. It has been shown that aggregates of several proteins, including amyloid fibrils, can be melted and dissociated by heating at high temperatures, giving rise to characteristic endothermic transitions in DSC experiments (Azuaga et al 2002;Rezaei et al 2002;Baxa et al 2004;Morel et al 2006Morel et al , 2010. Conejero-Lara and colleagues have reported the results of detailed DSC analyses of amyloid fibrils formed from an N47A mutant of the α-spectrin SH3 domain (Morel et al 2010).…”

Crystallographic structure of the SH3 domain of the human c‐Yes tyrosine kinase: Loop flexibility and amyloid aggregation

“…Tests for amyloid formation including Congo red and Thioflavin T assays as well as far-UV circular dichroism and electron microscopy experiments were carried out as described in Morel et al [22].…”

Identification of a Conserved Aggregation-Prone Intermediate State in the Folding Pathways of Spc-SH3 Amyloidogenic Variants

“…[15][16][17] A series of recent reports has shown that such ability extends to other members of the SH3 family, namely, to the Spc-SH3 domain. [18][19][20] Thus, SH3 domains are also ideal model systems to explore the free energy landscape of proteins beyond the folding process and to learn about how proteins aggregate into amyloid fibrils.…”