Proteins are versatile molecules that play a variety of roles in maintaining the human body, e.g. transport of nutrients. Transthyretin (TTR) is a 55 kDa homotetrameric protein found in human plasma and in the cerebrospinal fluid, responsible for the transport of retinol (vitamin A) and T4 (thyroxine). This protein is probably not essential for life, since TTR knockout mice have normal fetal development and lifespan. TTR, like 25 other human proteins, has been associated to the deposition of amyloid aggregates. Previous research has shown that mutations considerably increase the propensity of the protein to form aggregates. However, the wild type protein also exhibits this ability to aggregate, giving rise to the senile systemic amyloidosis disease that affects 20% people over 80 years of age. It is well accepted that self-association of monomeric subunits triggers the disease through tetramer dissociation, since stabilization of the quaternary structure suppresses aggregate formation.
However, a detailed description of the self-assembly mechanism and fibril structure remains unresolved. Here, using a combination of primarily small -angle X-ray scattering (SAXS) and hydrogen exchange mass spectrometry analysis, we describe an unexpectedly dynamic TTR protofibril structure which exchanges protomers with highly unfolded monomers in solution. With SAXS, we reveal the continuous presence of a considerably unfolded TTR monomer throughout the fibrillation process, and show that a considerable fraction of the fibrillating protein remains in solution even at a late maturation state.
In our efforts to study both native and protofibrillar TTR, we realized the need for development of a fluorescent small molecule capable of binding native and protofibrillar TTR, providing distinguishable emission spectra. We used microwave heating for efficient synthesis and fluorescence spectral screening of compounds. We synthesized and tested 22 analogs displaying a variety of functional groups, most of them linked to a stilbene scaffold. We successfully developed two compounds that detect both TTR states at physiological concentrations. The compounds bound with nM-μM affinities and displayed very distinct emission maxima upon binding native or protofibrillar TTR (> 100 nm difference).
We expect these new findings regarding protofibril self-assembly mechanism, together with our novel molecules serve as important tools in future studies of TTR amyloid formation.
Linköping: Linköping University Electronic Press, 2015. , 37 p.