Mechanical Features of Transthyretin: Probing Folding Intermediates of an Amyloid Forming Protein by Single Molecule Force Spectroscopy

Abstract

Formation of amyloid aggregates by transthyretin (TTR) is associated with severe neurological disorders. Over 80 naturally-occurring mutations have been reported to promote the misfolding of TTR leading to an enhanced aggregation propensity. Yet, such dramatic decrease in stability is generally not apparent from analysis of their corresponding X-ray crystal structures.To better understand the folding transitions in TTR we undertook single molecule force spectroscopy measurements of wild type (WT) TTR and two mutants (Y78F and L55P). All three TTR variants exhibit different susceptibilities to form amyloid aggregates in the pH range of 3.6 - 7.4 at which measurements were carried out. The resulting force-extension curves were characterized by closely spaced transitions presumably arising from the successive unfolding of secondary structure elements along a single polypeptide chain. Each of these transitions corresponds therefore to an unfolding intermediate. We observed that the frequencies with which these intermediates occurred depended on the experimental conditions. In addition, mutations in the TTR sequence greatly affect the stability of these intermediates as demonstrated by differences intermediate unfolding-force in a manner that may explain their ability to form amyloid aggregates at different pHs.