Investigation of Natural and Synthetic Aggregation Inhibitors using Microfluidic Applications

Abstract

Microfluidic approaches offer an extension of conventional biophysical techniques for the study of the intricate nature of biomolecule recognition. Recent microfluidic applications show the possibility of performing binding studies in the condensed phase without the need for extensive dilution, sample surface immobilization or changes to the molecular environment from the liquid to the gas phase. Capitalizing on such approaches, we used a microfluidic diffusion device to demonstrate a detailed characterisation of molecular chaperone activity in suppressing protein aggregation and the underlying binding mechanisms leading to specific inhibitory phenomena. In particular, we have studied in detail the entropic binding dynamics of the sHsp αB-crystallin to α-synuclein fibrils and have shown that changes in heat capacity are indicative of chaperone structural changes prior to the binding. For clusterin and Brichos, chaperone systems previously described in the context of Aβ(42) aggregation, binding studies and aggregation kinetics have been conducted and revealed a complex suppression system in which each chaperone specifically inhibits distinct microscopic aggregations steps without overlap. These results together with further binding kinetics of Brichos form a distinct structural picture of elongation sites on fibrillar ends and secondary nucleation sites along the fibrillar surface. As secondary nucleation seems to be the major inhibitory target desired in order to prevent the formation of toxic oligomeric species, mimicking nature's specific inhibition of aggregation for therapeutic applications seems like an ideal solution. However, we show that potential therapeutic antibodies for Alzheimer's, are not, on their own, sufficient for inhibiting secondary nucleation. Each antibody shows individual binding properties either to Aβ(42) monomers, fibrils or to both, resulting in a specific inhibition pattern. A full biophysical characterisation of potential therapeutics and all intermolecular interactions involved could thus increase the positive outcome in drug development.