Fibril-Droplet Relationship Through Liquid-Liquid Phase Separation; A BSA-MOF Case

Abstract

By providing a suitable platform that accelerates early-stage occurrences needed for triggering protein aggregation, liquid-liquid phase separation (LLPS) has the potential to promote this phenomenon. Among different proteins which their condensation propensity has been investigated, bovine serum albumin (BSA) has attracted attentions because of its globular and stable structure. BSA undergoes phase separation and phase transition in the presence of polyethylene glycol as a molecular crowder. The control of hydrophobicity, hydrogen bonding, and electrostatic forces as the main forces that conduct LLPS were provided by functionalizing a UiO-66 metal-organic framework (MOF) with -NH2 and -COOH functional groups. This work evaluated how the functionalized UiO-66 with -NH2 modulates the LLPS of BSA. Successful synthesis and functionalization of UiO-66 were confirmed using various physical and chemical analyses. Optical and fluorescence microscopy images correlated BSA LLPS droplet size with spectroscopic measurements of resultant BSA fibrils. UiO-66-NH2 was found to cause significant conformational changes in BSA, resulting in a decrease in its LLPS and aggregation rate, as demonstrated by various biophysical methods. This study suggested that more hydrophobic surrounding micro-environments caused by UiO-66-NH2 inhibited BSA LLPS, leading to decreased droplet size and number. The direct correlation between droplet size and fibril length also confirmed the role of LLPS as an important alternative pathway enabling fibril formation even in globular proteins.