Analysis of conformational changes in native and mutants human transthyretin during the folding and aggregation processes

Transthyretin (TTR) is a homotetrameric protein that is one of 30 human proteins associated with systemic amyloidoses. In spite of its link to human pathology, an anti-amyloidogenic effect that prevents fibril formation of the amyloid β (Aβ) peptide associated with Alzheimer’s disease (AD) has been proposed for TTR. Although it has been shown that these two processes correlate with the ability of TTR to populate a monomeric state, a complete description of the conformational states populated in vitro by monomeric TTR at physiological pH is missing. We used an array of biochemical and biophysical methods and kinetic tests to investigate the folding and aggregation processes of monomeric TTR. Our results show that once monomers of transthyretin are released by the quaternary structure, the protein establishes an equilibrium between a set of conformational ensembles bearing different degrees of disorder. Thus, a molten globular state appears in equilibrium with the fully folded monomer, whereas an off-pathway species accumulates transiently during refolding of TTR. These two conformational ensembles are distinct in terms of structure, dynamics, kinetics and pathway of formation. Further subpopulations of the protein fold differently due to the occurrence of proline isomerism. We investigated the conformational states described above by exploiting an intramolecular Forster Resonance Energy Transfer (FRET) approach. As TTR possesses one single cysteine moiety, we labelled such residue (different mutants of transthyretin) with a probe which acts as acceptor of light emitted by tryptophan residues. This system reports on intramolecular distances and compaction of the different conformational states.

Keywords:

Transthyretin , Folding , Aggregation , FRET , Amyloidoses