Probing Macromolecular Interactions by Loss of Solvent Content in Protein Crystals: Application to the Amyloid‑β Peptide Binding to Transthyretin.

X-ray crystallography is commonly used to determine the structure of protein-protein complexes, revealing the atomic details of the interactions between macromolecules in the crystal. However, this technique has limited application in binary systems characterized by transient or weak interactions. Here, we demonstrate that protein crystals can still provide valuable information in such systems by assessing crystal solvent content. We applied this method to investigate the interaction between transthyretin (TTR) and the amyloid beta (Aβ) peptide, a system of interest due to transthyretin's proposed role in the clearance of Aβ peptide, whose accumulation in the brain is associated with Alzheimer's disease. Soaking TTR crystals separately with Aβ fragments results in distinct reductions of the crystal void volume and highlights the key sequence residues involved in binding to TTR. Our findings indicate that the middle Aβ12-28 fragment interacts more strongly with TTR than the N- and C-terminals. Analysis of the crystal packing and solvent content indicates an equimolar interaction between transthyretin and the Aβ12-28 peptide. This interaction likely involves surface-exposed regions of TTR, such as the thyroxine binding pocket or the dimer pocket.