Label-Free Analysis of Protein Aggregation and Phase Behavior.

Abstract

Phase transitions of protein molecules are central to biological function and malfunction. One such transition commonly encountered in nature is the conversion of soluble monomeric states into solid phases, which include crystals and amyloid fibrils, the latter of which are associated with the onset and development of neurodegenerative diseases. Monitoring aggregate formation and protein phase behavior is essential in gaining mechanistic insights into these fundamental processes. Fluorescence techniques have proven invaluable in observing biological molecules; yet, most such approaches rely on the use of an extrinsic fluorophore that binds to the molecule of interest, the installation of which can perturb the molecular systems under study. However, most proteins also possess aromatic amino acids within their peptide sequence and therefore exhibit intrinsic fluorescence. Here, we show that by measuring in space and time tryptophan autofluorescence for three proteins, reconstituted silk fibroin, β-lactoglobulin, and lysozyme, fibrillar self-assembly can be monitored accurately and without the need for extrinsic dyes. When fibrillar protein self-assembly takes place, hydrophobic burial occurs, resulting in the minimization of exposed tryptophan residues to the solvent and consequently leading to an increase in protein autofluorescence. Moreover, by employing a droplet-microfluidic approach to confine protein self-assembly in space, we demonstrate that intrinsic fluorescence can be used to image protein nanofibrils in a label-free manner and that the microstructural analysis obtained from intrinsic fluorescence microscopy correlates well with that from samples treated with extrinsic dyes. Finally, our results show that protein autofluorescence is not limited to the observation of β-sheet-rich structures, but can also be used to distinguish between different types of solid phases including spherulites and crystals, making this approach suitable for overall characterization of protein phase transition phenomena.