Title: Investigating ligand binding and subunit exchange in transthyretin (TTR) complexes

The investigation of ligand binding and subunit exchange in transthyretin (TTR) complexes was discussed. Subunit exchange was followed by mixing equimolar quantities of L55P TTR uniformly labeled with 15N and unlabeled wild type WT to produce a solution with an overall concentration of 10 μM. It was observed that after incubation for 34 mins, separate peaks for the labeled and unlabeled tetramer were no longer visible. It was shown that in the presence of a 3-fold excess of thyroxine the rate of subunit exchange was reduced

L55P transthyretin accelerates subunit exchange and leads to rapid formation of hybrid tetramers

Transthyretin is a tetrameric protein associated with the commonest form of systemic amyloid disease. Using isotopically labeled proteins and mass spectrometry, we compared subunit exchange in wild-type transthyretin with that of the variant associated with the most aggressive form of the disease, L55P. Wild-type subunit exchange occurs via both monomers and dimers, whereas exchange via dimers is the dominant mechanism for the L55P variant. Because patients with the L55P mutation are heterozygous, expressing both proteins simultaneously, we also analyzed the subunit exchange reaction between wild-type and L55P tetramers. We found that hybrid tetramers containing two or three L55P subunits dominate in the early stages of the reaction. Surprisingly, we also found that, in the presence of L55P transthyretin, the rate of dissociation of wild-type transthyretin is increased. This implies interactions between the two proteins that accelerate the formation of hybrid tetramers, a result with important implications for transthyretin amyloidosis

Screening transthyretin amyloid fibril inhibitors: characterization of novel multiprotein, multiligand complexes by mass spectrometry.

Tetrameric transthyretin is involved in transport of thyroxine and, through its interactions with retinol binding protein, vitamin A. Dissociation of these structures is widely accepted as the first step in the formation of transthyretin amyloid fibrils. Using a mass spectrometric approach, we have examined a series of 18 ligands proposed as inhibitors of this process. The ligands were evaluated for their ability to bind to and stabilize the tetrameric structure, their cooperativity in binding, and their ability to compete with the natural ligand thyroxine. The observation of a novel ten-component complex containing six protein subunits, two vitamin molecules, and two synthetic ligands allows us to conclude that ligand binding does not inhibit association of transthyretin with holo retinol binding protein

Structural insights into the activity of enhancer-binding proteins.

Activators of bacterial sigma54-RNA polymerase holoenzyme are mechanochemical proteins that use adenosine triphosphate (ATP) hydrolysis to activate transcription. We have determined by cryogenic electron microscopy (cryo-EM) a 20 angstrom resolution structure of an activator, phage shock protein F [PspF(1-275)], which is bound to an ATP transition state analog in complex with its basal factor, sigma54. By fitting the crystal structure of PspF(1-275) at 1.75 angstroms into the EM map, we identified two loops involved in binding sigma54. Comparing enhancer-binding structures in different nucleotide states and mutational analysis led us to propose nucleotide-dependent conformational changes that free the loops for association with sigma54

Use of a microchip device coupled with mass spectrometry for ligand screening of a multi-protein target.

Nanoflow electrospray mass spectrometry has been applied previously to investigate noncovalent protein-protein and protein-ligand interactions. Here we evaluate a commercial microchip device for this application. We show that the microchip can be used to obtain mass spectra of the noncovalent tetramer transthyretin. The device showed a 10-fold increase in signal stability compared with a nanoflow capillary and a high level of nozzle-to-nozzle reproducibility. Binding of the natural ligand thyroxine was clearly observed, and a range of small molecules proposed as inhibitors of transthyretin amyloidosis were shown to be effective in stabilizing the tetramer. We propose that measuring the ability of small molecules to stabilize protein complexes using this automated microchip technology will enable high-throughput screening of multi-protein complexes by mass spectrometry