Specific isotope labeling of transthyretin for nuclear magnetic resonance and mass spectrometry studies — Marcaje isotópico específico de transtiretina para estudios en resonancia magnética nuclear y espectrometría de masas

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 brain, 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 (wt-TTR) 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 at the moment that self association of monomeric subunits triggers the disease through tetramer dissociation, since stabilization of the quaternary structure suppresses aggregate formation. Recently, our group discovered that wt-TTR purified at 4°C is just as toxic to human neuroblastoma cells as the most toxic small molecular weight aggregates, while when purified at room temperature (22°C; RT) it is not. Strikingly, this cytotoxicity was exhibited by the protein as a tetramer. Biophysical studies revealed a slight rearrangement of the tertiary structure of the protein. It is of high interest whether this minor structural change can lead to the discovery of new cytotoxic epitopes. Herein, we explored the protein structure in more detail by biophysical methods to confirm the existence of this conformational change and attempt to resolve where it specifically takes place in the protein structure. Recombinant wt-TTR was expressed in Escherichia coli and purified by ion exchange chromatography and size exclusion chromatography at 4°C and RT. We studied the tetramer’s intrinsic tryptophan fluorescence to confirm that cold purified and stored wt-TTR has a lower activation barrier for dissociation into monomers compared to the protein purified and stored at RT. This was performed by submitting the protein to unfolding using a high concentration of urea (6 M) as the chaotropic agent. We also carried out a hydrogen/deuterium isotope exchange mass spectrometry (HXMS) viii experiment in order to study the local exchange of backbone amide hydrogens, which serve as probes for increased/decreased protection of different segments of the protein towards exchange, depending on the temperature. At last, nuclear magnetic resonance spectroscopy using the heteronuclear single quantum coherence (HSQC) experiment probing a N isotope labeled wt-TTR grown in ~99% deuterated water M9 medium, allowed us to track the two dimensional N-H J-coupling spectrum of the protein. Analysis of 2D spectra run at both temperatures revealed that some residues experience a clear change in chemical shift, indicating that changes in tertiary structure occurred. In conclusion, we determined that wt-TTR undergoes a conformational rearrangement when purified and stored at 4°C, although the exact location of this conformational change in the protein structure remains unclear.