The high-throughput characterization of solution binding equilibria is essential in biomedical research such as drug design as well as in material applications of synthetic systems in which reversible binding interactions play critical roles. Although isothermal titration calorimetry (ITC) has been widely employed for describing such binding events, factors such as speed, concentration, and sample complexity would principally favor a mass spectrometry approach. Here, we show a link between ITC and electrospray ionization mass spectrometry (ESI-MS) by incorporating solvation free energies in the study of the ternary complexes of the macrocyclic host cucurbit[8]uril (CB[8]). The binding affinities of 32 aromatic reference complexes were studied by ITC and ESI-MS and combined with solvation data of the guests from an implicit solvation model (SM8) to obtain a correlation between aqueous and gas-phase measurements. The data illustrates the critical importance of solvation on the binding strength in CB[8]'s ternary complexes. Finally, this treatment enabled us to predict association constants that were in excellent agreement with measured values, including several highly insoluble guest compounds.

The formation of modular, multi-component, host-guest complexes stable both in water and in the gas-phase is described; by modulating the stoichiometry of the different subunits in aqueous solution, quantitative self-assembly of the predicted and desired architecture is achieved, even allowing for ABA triblock copolymer assemblies stable in the gas-phase.

Transthyretin (TTR) amyloidosis is a fatal disease for which new therapeutic approaches are urgently needed. We have designed two palindromic ligands, 2,2'-(4,4'-(heptane-1,7-diylbis(oxy))bis (3,5-dichloro-4,1-phenylene)) bis(azanediyl)dibenzoic acid (mds84) and 2,2'-(4,4'-(undecane-1,11-diylbis(oxy))bis(3,5-dichloro-4,1-phenylene)) bis(azanediyl)dibenzoic acid (4ajm15), that are rapidly bound by native wild-type TTR in whole serum and even more avidly by amyloidogenic TTR variants. One to one stoichiometry, demonstrable in solution and by MS, was confirmed by X-ray crystallographic analysis showing simultaneous occupation of both T4 binding sites in each tetrameric TTR molecule by the pair of ligand head groups. Ligand binding by native TTR was irreversible under physiological conditions, and it stabilized the tetrameric assembly and inhibited amyloidogenic aggregation more potently than other known ligands. These superstabilizers are orally bioavailable and exhibit low inhibitory activity against cyclooxygenase (COX). They offer a promising platform for development of drugs to treat and prevent TTR amyloidosis.crystallography | mass spectrometry | protein structure | stabilization

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Transthyretin (TTR) is a plasma hormone carrier protein associated with hereditary and senile forms of systemic amyloid disease, wherein slow tetramer disassembly is thought to be an obligatory step. Plasma transport of retinol is carried out exclusively by the retinol-binding protein (RBP), through complexation with transthyretin. Using mass spectrometry to examine the subunit exchange dynamics, we find that retinol stabilizes the quaternary structure of transthyretin, through its interactions with RBP, reducing the rate of transthyretin disassembly ∼17-fold compared to apoTTR. In the absence of retinol but in the presence of RBP, transthyretin is only marginally stabilized with the rate of disassembly reduced ∼two-fold with respect to apoTTR. Surprisingly, we found two retinoids that stabilize transthyretin directly, in the absence of RBP, whereas retinol itself requires RBP in order to stabilize transthyretin. Our results demonstrate new roles for RBP and retinoids as stabilizers of transthyretin.